IDENTIFICATION OF DISTINCT RESIDUES IN THE CARBOXYL TAIL REGULATING DESENSITIZATION AND INTERNALIZATION OF THE

Di DOPAMINE RECEPTOR

Michael Lamey

A thesis subrnitted in conformity with requirements for the degree of Master's of Science

Graduate Department of Pharmacology University of Toronto

@ Copyright by Michael Lamey (2000)

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ABSTRACT

Il)ENTIFICATION OF DISTINCT RESIDUES IN THE CARBOXYL TAU, REGULATING DESENSITIZATION AND INTERNALIZATION OF THE

Di DOPAMINE REXEPTOR

Michael Lamey

Degree of Master's of Science, 2000 Graduate Department of Pharmacology, University of Toronto

In order to investigate the mechanisms underlying agonist induced desensitization and

intemalization of the DI dopamine receptor, we used site-directed mutagenesis to remove

potential phosphorylation sites in the third intracellular loop and carboxyl tail. Three mutant

receptors with serinelthreonine residues changed to alanine in the distal carboxyl tail between

431-446 failed to intemalize, in cornparison to intemalization of 25% of the wild-type

receptors. As M e r serinelthreonine residues were mutated serially in the carboxyl tail, we

demonstrated that substitution of Thr360 in the proximd portion completely abolished

desensitization, compared to a 29% decrease in Vmax of the wild-type recepton. Since

Th360 is next to a glutamic acid residue, it represents a potential GRK2 phosphorylation

site. These resdts suggest that desensitization and intemalization are regulated by separate

and distinct residues within the carboxyl tail, and that GRK2 may be the predominant

regulator of rapid agonist induced desensitization of the Di dopamine receptor.

First and foremost, 1 would like to thank Dr. Susan George and Dr. Brian O'Dowd for

their wonderful support and guidance throughout my Masters program. 1 would especially

like to thank them for their support and understanding during my difficult times of illness 1

have encounrered in the iasr few years.

n i e successes of my research project would not have been possible without the

support of al1 the snidents and technical supervisors in the laboratory. 1 would like to

especially thank Hong Chi, Marek Sawzdargo and Miles Thompson for their technical

support, and for their contribution to this study.

Finally, I would like to thank and dedicate this thesis to my wife, Gina, and my

parents for their continuous love, support and encouragement that kept me going throughout

the tough times. 1 love you dl.

TABLE OF CONTENTS

TITLE PAGE rnSTRACT ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

SECTION 1 INTRODUCTION

1.1 G Protein-Coupled Receptors 1.2 CPCR Activation 1.3 Desensitization of GPCRs 1.4 G Protein-Coupled Receptor Kinases (GRKs) 1.5 htemdization of GPCRs 1.6 Dopamine Receptors 1.7 Desensitization/Intemalization of the Dopamine D 1 Receptor 1.8 Thesis Rationale

SECTION 2 METHODOLOGY

2.1 Generation of Mutant and Stable Ce11 Lines Expressing Wild Type or Mutant Receptors

2.2 Cell Cultures and Membrane Preparation 2.3 Radioligand Binding Assays 2.4 Intemakation Assays 2.5 Adenyly 1 Cyclase Assays 2.6 Data Analysis

SECTION 3 RESULTS

3.1 Generation and Expression of Mutant Dopamine Di Receptor Constnicts 3.2 Characterization of the DI Dopamine Receptor 3.3 Agonist induced Desensitization and Internaiization of the Dl Dopamine

Receptor

1 . . 11

iii iv vi vi i viii

Agonist induced Desensitization and Intemdization of Mutant Receptor A (243-268) Agonist induced Desensitization and Intemaiization of Mutant Receptor B (T446A) Agonist induced Desensitization and Intemdization of Mutant Receptor C (43 1-439) Agonist induced Desensitization and Intemaiization of Mutant Receptor D (428-439) Agonist induced Desensitization and Intemalization of Mutant Receptor E (372-446) Agonist induced Desensitization and Internalization of Mutant Receptor F (342-354,372446)

3.10 Agonist induced Desensitization and Intemdization of Mutant Receptor G (360,372-446)

3.1 1 Agonist induced Desensithion and Intemaiization of Mutant Receptor H (T360A)

3.12 Agonist induced Desensitization and Intemdization of Mutant Receptor I (S362A)

3.13 Sumrnary of Desensitization and Intemdization Results

SECTION 4 DISCUSSION, CONCLUSIONS, RECOMMENDATIONS

4.1 Discussion 4.2 Future Studies

REFERENCES

LIST OF TABLES

Table 1. [ 3 ~ ~ ~ ~ - 2 3 3 90 binding parameters for Di wild type and mutant receptors

LIST OF F I G W S

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Schematic 3D representation of GPCR54.

Activation of G protein-coupled receptors.

Schematic representation of desensitization and resensitization of the B2AR following agonist activation.

Structure of G protein-coupled kinases (GRKs).

Schematic 3 D representation of the human dopamine D receptor.

Schematic representation of intracellular residues of the human Di dopamine receptor.

Cornpetition binding of the Dl dopamine receptor.

Desensitization and intemalization of the Di dopamine receptor.

Desensitization and intemalization of mutant receptor A (243-268).

Figure L O. Desensitization and intemalization of mutant receptor B (T446A).

Figure I 1 . Desensitization and intemalization of mutant receptor C (43 1 - 4 3 ) .

Figure 12. Desensitization and internalization of mutant receptor D (428-439).

Figure 13. Desensitization and internakation of mutant receptor E (372-446).

Figure 14. Desensitization of mutant receptor F (342-354,372-446).

Figure 15. Desensitization of mutant receptor G (360,372446).

Figure 16. Desensitization and intemalbation of mutant receptor H (T360A).

Figure 17. Desensitization of mutant receptor I (S362A).

Figure 18. Summary of desensitization experiments.

Figure 19. Summary of intemalization experiments

vii

AC

P2AR

CHO

DMEM

GPCR

GRK

HEK

Ki

MAP

MGluR

M2mAC hR

PI

P U

PKC

PLC

adenylyl cylcase

Pz-adrenergic receptor

chinese hamster ovary

dulbeco's modified essential medium

G protein-coupled receptor

G protein-coupled receptor kinase

human embryonic kidney

dissociation constant

mitogen-activated protein

metabotropic glutamate receptor

m.2 muscarinic acetylcholine receptor

phosphatidy linositol

CAMP-dependent protein kinase

protein kinase C

phospholipase C

viii

Section 1

INTRODUCTION

1.1 G protein-Coupled Receptors

One of the largest systems of signal transduction consists of seven-transmembrane

receptors interacting with heterotrirnenc guanine nucleotide-binding proteins (G proteins) to

transduce signals to cellular effectors. These G protein-coupled receptors (GPCRs) are found

in an extremely wide variety of ce11 types and mediate responses to a diverse array of

signaling molecules, including peptide and glycoprotein hormones, small molecule

neurotransmitters, phospholipids, odorants and photons. Presently, the GPCR family consists

of over 1000 receptors and if the recent sequencing of the Caenorhabditis elegans genome is

any indication, up to 5% of the entire human genome may encode for these receptors

(Bargmann, 1998).

Al1 GPCRs share conserved stnictuml features, with seven transmembrane spanning

domains connected by three altemating extracellular and intracellular loops (Fig. 1). Based

on several key sequence motifs, GPCRs cm also be divided into 3 major subfarnilies.

Farnily A receptors are related to rhodopsin, family B receptors are related to the calcitonin

receptor, and fmily C recepton are related to the metabotropic receptors. Of these, the

rhodopsin subfamily is by far the largest (constituting approximately 90% of al1 GPCRs), the

most extensively investigated and will therefore be the main focus of this introduction. In

addition to rhodopsin and the B-adrenergic receptors, this subfamily includes receptors for

other small molecules such as the neurotransmitters dopamine and serotonin, for peptides

such as angiotensin and substance P, and glycoproteîns such as follicle-siimulating hormone.

Other common features of GPCRs include sites for post-translationai modifications.

AI1 the receptors cloned to date have at least one consensus sequence for N-linked

glycosylation in the extracellular amino tenninus or second extracellular loop. Although

glycosylation does not appear to be essentiai for ligand binding, it has been found to be

critical for normal expression of some GPCRs at the ce11 membrane (Karpa et al., 1999; Liu

et al., 1993). Most GPCRs also have a highiy conserved cysteine residue in the proximal

portion of the intracellular carboxyl terminus. These residues have been shown to be

palmitoylated in a nurnber of receptors, and aithough the function of the palmitoyl group is

not entirely clear, this modification may anchor the tail to the plasma membrane to form a

fourth intracellular loop (Jin et al., 1999; O'Dowd et al., 1989; Ovchinnikov et al., 1988). In

the Pz-adrenergic receptor &AR), palmitoylation is critical for normal G protein coupling

(O'Dowd et al., 1989), however, in other GPCRs, such as rhodopsin (Kamik et al., 1993) and

the dopamine Di receptor (Jin et al., 1997), the removal of palmitoylation sites does not

affect G protein interaction. GPCRs also contain other highly conserved cysteine residues in

the second and third extracellular loops. These cysteine residues are linked together in a

disulfide bond which appears to play an important role in structure and high afinity ligand

binding in many receptoa (Karnik et al., 1988; Savarese et al., 1992; Zhang et al., 1999).

There is also evidence that this disuifide bond is essential in the dimerization of

m3 muscarinic (Zeng and Wess, 1999) and metabotropic glutamate (mGluR) receptors

(Romano et al., 1996), and rnay have a role in ce11 surface receptor localization (Xie et al.,

unpublished observations). Finally, the carboxyl temiinus and third intracellular loop of

GPCRs contain several serine and threonine residues that are targets for phosphorylation by a

variety of regulatory kinases that act to desensitize the recepton (Le£kowitz, 1998).

The general structurai characterktics of GPCRs are shown in Figure 1.

Figure 1. Schematic 3D representation of GPCR54. S h o w is a 3D mode1 of orphan receptor

GPCR54, a GPCR with a typical structure of seven transmembrane spanning domains with three

altemating intracellular and extracellular loops. GPCRS4 possesses three potential N-glycosylation

sites in the arnino terminus, and three cysteines in the carboxyl terminus that represent putative

palmitoylation sites. Two cysteine residues in the second and dird extracellular loops are predicted

to form a disulfide bond that may function in stabilizing the receptor conformation. FinalIy, two P K A

consensus phosphorylation sites are found in the second and third intrace

several other serine and threonine residues in the carboxyl terminus

phosphorylation sites for other regulatory kinases.

'Ilular loops, dong with

representing potential

Mutations of GPCRs that prevent signahg or proper expression on the ce11 surface

have been discovered that relate to a wide variety of hereditary and somatic diseases and

disorders fiom diabetes to cancer (Spiegel, 1996). Therefore, these receptors represent a

major target for h g therapies, and it is estimated that GPCRs constitute approximately 45%

of al1 targets for phannaceutical therapies (Drews, 2000).

GPCR domains that are involved in ligand binding are generally dependent on the

chernical structures of the agonist and have been determined mainly through mutagenesis

studies. Small molecular weight ligands such as dopamine bind to sites within the

hydrophobic core formed by the transmembrane spanning a-helices (Dohlman et al., 199 1 ;

Hibert et al., 1993). For larger peptide ligands and hormones, the amino terminus and

extracellular loops as well as the transmembrane domains are involved in the formation of

the binding site (Strader et ai., 1994).

Until very recently, obtaining high resdution structural anaiysis of GPCRs through

3D crystal structures has been next to impossible due to their low natural abundance and the

dificulty in producing and puriQing significant amounts of protein. A low-resolution

structure of fiog rhodopsin detennined from cryo-electron rnicroscopy ha been very usefui

in predicting the structure and function of rhodopsin as well as other closely related farnily

A receptors (Unger et al., 1997). Howev-er, a significant breakthrough has just recently

emerged in the field of GPCRs with the detemination of the 3D crystal structure of

rhodopsin (Palczewski et ai., 2000). One of the many exciting discoveries that has emerged

fiom this study is that rhodopsin forms an elaborate multilayered plug made up fiom most of

the extracellular domain that prevents the activated ligand all-tram-retinal from exiting the

binding pocket. It will be interesthg to see if other receptors possess a similar regulatory

rnechanism of ligand binding and activation. Therefore, this new study has given further

insights into the structure and molecular mechanism of rhodopsin activation, and will

undoubtedly enhance and modi@ our understanding of rnany aspects of GPCR structure and

function as more receptors are visualized in this manner in the future.

1.2 GPCR Activation

G protein-coupled receptors mediate their intracellular actions via a pathway that

involves the activation of heterotrirneric guanine nucleotide binding proteins (G proteins).

Al1 G proteins consist of three tightly associated subunits, a, B, and y. In the basal or

inactive state, the G protein exists as the heterotrimer with GDP bound to the a subunit.

Agonist binding to a G protein-coupled receptor results in a conformational change that

ailows it to interact with the heterotrimeric G protein in the ce11 membrane, forming a high

affinity agonist receptor G protein complex. Activated receptor catalyzes release of GDP,

thereby facilitating GTP binding to the a subunit and dissociation of the G protein complex

into its a and By subunits. The Ga-GTP complex is an active conformation that is ultimately

dissociated from the receptor and dong with the py subunits are able to activate downstream

effector proteins and ion charnels, such as adenylyl cyclase, guanylyl cyclase,

phospholipases C and Az, ca2+and K' channels (Muller and Lohse, 1995). The activated

state of G a and py subunits persists until GTP is hydrolysed to GDP by the intrinsic GTPase

activity of Ga. Interestingly, the rate of GTP hydrolysis varies fiom one Ga subunit to

another, and certain effectors may even accelerate intrinsic GTPase activity (Bourne and

Stryer, 1992; Carty et al., 1990). Therefore, GTP hydrolysis functions to tum off the

G protein signaling and indirectly, may function as a mechanism contributing to the

desensitization of the receptor. Once Ga-GDP is formed, it reassociates tightly with the

py subunits, fonning an inactive heterotrimenc complex that is capable of receiving and

transducing a new signal (Fig. 2).

One of the most important and characteristic features of G protein-rnediated signal

transduction is the tremendous amplification that occurs in the response to extracellular

agonist. This process arises fiom the ability of the agonist activated GPCR to stimulate many

G protein molecules, the ability of an activated G protein to stimulate many effector

molecules, and of an effector molecule to undergo many catalytic cycles. Thus, signais

cornrnunicated by G proteins to elicit cellular responses can theoretically be achieved by a

single excited receptor molecule, demonstrating the extreme sensitivity of GPCR signal

transduction ( Wensel, 1999).

To date, there have been 23 distinct G a proteins identified in marnais, and they are

divided into four subfamilies based on their sequence similarity. The Gai farnily is the most

abundantly expressed and includes GailJJ, Ga,, Gatl2 (rod and cone G proteins), Ga,,,

(gustatory G protein) and Ga, which stimulate cGMP phosphodiesterase, inhibit adenylyl

cyclase (AC) and regulate ca2' and K+ channels. The Ga, farnily includes Ga, and Gaoif,

(olfactory G protein) which stimulate AC and regulate ca2+ and K' channels. The Ga,

family includes the Ga, and Gall, Gai4, Gais and Gais which activate phospholipase C

(PLC), and the Gai2 farnily includes the Galz and Gai3 which stimulate bIaf-Ht exchangers

(reviewed by Wensel, 1999).

Basal State

...- ........... e::?!!:--?:+f

Active State activation

Figure 2. Activation of G protein-coupled receptor. Upon binding to agonist (hormone in this

example), the receptor catalyzes release of GDP, facilitating GTP binding to the a subunit and

dissociation of the heterotrimer into its a and subunits. The Ga-GTP complex activates effector

proteins, such as enzymes, and the receptor remains in the activated state until GTP is hydrolysed to

GDP. Once Ga-GDP is fomed, it reassociates tightly with the subunits, forming an inactive,

basal state of the receptor once again. Figure modified fiom Wensel, 1999.

The P and y subunits are less diverse than a proteins, with four P subunits (p 1-4) and

eleven y subunits (y14 1) (Ray et al., 1995) discovered to date. The various subunits are

expressed in a wide range of tissues, with much overlap in their distribution (Hepler and

Gilman, 1992). Both the P and y subunits are tightly associated with one another in a

noncovalent complex throughout the signaling cycle. The py heterodimer is foremost

involved in catalyzing the high affinity interaction between ligand activated receptor and Ga-

GDP which initiates guanine nucleotide exchange. Once the py heterodimer is dissociated

From Ga-GDP following G protein activation, it may activate effector molecules such as

AC (Tang and Gilman, 199 1; Ueda et al., 1994), PLC-B (Boyer et al., 1994), and K' channels

(Logothetis et al., 1987). The py subunits have also been found to bind G protein-coupled

receptor kinases 2 and 3 ( G W , GRK3) and translocate them to the membrane where they

play an important role in homologous desensitization (Premont et al., 1995).

1.3 Desensitization of GPCRs

An important property of GPCRs is their ability to regulate their responsiveness in the

presence of continuous agonist exposure - a phenornenon that is termed desensitization.

Desensitization rnay serve to terminate the cellular response to an individual stimulus, or it

may cause a general reduction in responsiveness without being responsible for the actual

termination of a single signaling event. GPCR desensitization involves the contribution of a

series of three events: (1) the uncouphg of receptor fiom its G protein by phosphorylation

(within seconds to minutes); (2) intemalization of plasma membrane associated recepton

(minutes to hours); and (3) the down-regdation of the total cellular complement of recepton

as a result of reduced mRNA and protein synthesis, as well as increased lysosomal

degradation of preexisiting receptors (many hours) (Lohse, 1993).

There have been two major patterns of desensitization that have been characterized -

homologous and heterologous desensitization. Homologous desensitization is agonist

specific, thus only activated receptors are desensitized while other nonactivated receptors are

able to function normally. In contrast, in heterologous desensitization the activation of one

receptor can result in the reduced responses to activation of unrelated receptors, particularly

when there is an activation of signding pathway or effectors cornmon to the receptor types

affected. In both cases, phosphorylation of GPCRs has been strongly linked to

desensitization, however the specific mechanisrns appear to be distinct for different GPCRs.

There are two classes of serine/threonine protein kinases responsible for mediating

phosphorylation of GPCRs: the G protein-coupled receptor kinases (GRKs), and the second

messenger dependent kinases, which include CAMP-dependent protein kinase (PKA) and

protein kinase C (PKC). Second messenger dependent kinases have been shown to

phosphorylate and regulate many GPCRs, and are most commonly thought of to be involved

in heterologous desensitization. In contrast, GRK phosphorylation is exclusively involved in

homologous desensitization, since GRKs only phosphorylate agonist activated recepton.

GRK mediated phosphorylation additionally promotes the interaction of the receptor with

arrestin proteins, which recruit the receptors to clathrin coated pits for subsequent

intemalization and down-regulation. Second messenger dependent protein kinases generally

respond to desensitize GPCRs at a relatively slower rate than GRKs, (tic of 3 minutes,

compared to tin of 15 seconds) (Roth et al., 1991). From studies in the P2AR, it has been

discovered that PKA mediated phosphorylation desensitizes most effectively at low

concentrations of agonist (nanomolar), whereas the GRK phosphorylation occurs in the

presence of higher concentrations of agonist (micrornolar) (Hausdorff et al., 1989; Lohse et

al., 1990).

The process of desensitization has been most extensively studied in the P2AR because

it was the first nonvisual GPCR to be purified in significant quantities, and because it also

demonstrates very marked desensitization. Therefore, much of Our knowledge about

desensitization has corne fiom studies in the BtAR and it has served as an excellent mode1 for

other GPCRs. The events involved in desensitization of the PzAR are summarized in

Figure 3.

Interestingly, the P2AR has also been shown to be involved in an atypical method of

desensitization, involving the switching of coupling from Ga, to Ga i (Daaka et al., 1998). It

was discovered that upon agonist stimulation of the P2AR, mitogen-activated protein (MM)

kinase activity was also stimulated. Since MAP kinase is known to be stimulated through the

Gai subunit, experiments using various inhibiton of the MAP kinase pathway determined

that receptor coupling to Ga, and subsequent PKA mediated phosphorylation was required to

switch coupling and activation of Gai in the B2AR. Therefore, since coupling of P2AEb to

Gui leads to an inhibition of AC, this switching mechanism can be viewed as a general

mechanism to darnpen the Ga, stimulatory signal, and potentially represents an additional

mechanism of heterologous desensitization.

Figure 3 . Schematic representation of desensitization and resensitization of the bA.R following

agonist activation. Brîefly, upon agonist bindinp the receptor is phosphorylated by GRKs. which

promotes karrestin binding and subsequent intemalization through clathrin-coated vesicles. The

receptor is dephosphorylated in an endosomal cornpartment and is recycled back to the membrane for

Further activation (resensitization). A. agonist; a. P. and y. subunits of heterotnrneric G protein: E.

effector enzyme: GEW, G protein-coupled receptor kinase: @K. panestin: P. phosphate. The figure

is modified frorn Ferguson et al.. 1996.

1.4 G protein-coupled receptor kinases (GRKs)

GRKs were hitidly discovered fiom investigations of homologous desensitization in

the Pz-adrenergic receptor and rhodopsin. The B-adrenergic kinase (GRK2) and rhodopsin

kinase (GRKI) were subsequently cioned (Benovic et al., 1989; Lorenz et al., 1991) and a

few years later, the remaining four mernbers of the farnily identified to date were cloned:

GRK3 (B-adrenergic kinase 2) (Benovic et al., 199 l), GRK4 (Ambrose et al., 1992), GRK 5

(Kunapuli and Benovic, 1993; Premont et al., 1994) and GRK6 (Benovic and Gomez, 1993).

Whereas GRKl is almost exclusively expressed in the retina (Lorenz et al., 199 I), and GRK4

predominantiy found in the testis (Premont et al., 1996), al1 other GRKs are ubiquitously

expressed (Freedman and Le fkowitz, 1 996).

The GRK family can be divided into three subfamilies based on their Function and

sequence homology: (a) GRISI, (b) GRK2 and GRK3 (84% sequence homology), and (c)

GRK4, GRKS, and GRK6 (70% sequence homology) (Premont et al., 1995).

Stnicturally, GRKs posses a centrally located catalytic domain flanked by large

arnino and carboxyl terminal regdatory domains. The amino terminal domains share similar

size and homology, and are thus speculated to f i c t i on in receptor recognition. In contrast,

the carboxyl terminais are more diverse in length and structure and are thought to be

involved in membrane and receptor targeting (Fig. 4) (reviewed by Pitcher et al., 1998).

Figure 4. Structure of G protein-coupled kinases (GR&). The six known GRKs are represented

schematically. The solid area represents areas of divergent amino acid sequence within the GRK

farnily. The bifurcation in the carboxyl terminal domain represents the variable regions in the GRKs.

GRKI (rhodopsin kinase, RK) is famesylated G M , and GREO (PARK and (PARK2 respectively)

possess a binding domain, GRK4 and GRK6 are both palmitoylated, and GRKS contains a

polybasic phospholipid binding domain. Figure modified From Krupnick et al., 1998.

GRKl is post-translationally modified on the distal end of the carboxyl terminus that

promotes the association of a farnesyl group. The fmesylation modification is essential for

translocation of GRKl to the membrane in a light-dependent manner (Inglese et al., 1992).

Both GRIU and GRK3 are translocated to activated recepton upon binding of the by

subunits to specific py binding domains in the extended carboxyl terminal (Premont et al.,

1995). GRK4 and GRK6 are targeted to plasma membranes through palmitoylation in the

carboxyl terminal (Premont et al., 1996; Stoffel et al., 1994), while GRK5 contains a

polybasic carboxyl terminal domain that interacts electrostatically with phophoslipids on the

cytoplasmic face of the ce11 membrane (Fig. 4) (Premont et al., 1994).

GRKs have been shown to phosphorylate serine and threonine residues in the

carboxyl tail of receptors such as the P2AR (Fredericks et al., 1996) and rhodopsin (Ohpro

et al., 1995), or in the third intracellular loop of the m2 muscarinic acetylcholine

(m2mACh.R) (Pals-Rylaandarn and Hosey, 1997) and a2A-adrenergic (Eason et al., 1995).

However, unlike the second messenger dependent kinaes (PKA, PKC) that have

demonstrated distinct consensus sequences encompassing phosphorylation sites (Kennelly

and Krebs, 1991), GRKs have no strict consensus sequences identified in their receptor

substrates. However, peptide phosphorylation studies have deterrnined that both GRKl and

GR= are acidotropic kinases because they most actively phosphorylate serines and

threonines in close proximity to acidic amino acids (Onorato et al., 1991). GRIS2

preferentially phosphorylates serines and threonines with acidic amino acids on the arnino

terminal side, while GRKl recognizes serines and threonines with acidic residues localized

to the carbcxyl terminal side (Onorato et al., 1991). In contrast, peptide studies have

demonstrated that GRKS and GRIS6 preferentially phosphorylate serine residues containing

basic amino acids on the amino terminal side (Kunapuli et al., 1994; Loudon and Benovic,

1994).

Although in vitro studies have helped in determining substrate specificity of GRKs, it

is important to note that in vitro results have not always correlated well with functional

studies done in intact cells. Two exarnples are found in rhodopsin and the P2AR. Initial

in vitro studies of rhodopsin demonstrated that seven serine/threonine residues were

phosphorylated (Aton et al., 1984; Wilden and Kuhn, 1 W), whereas studies in vivo found

that only two of the residues were phosphorylated (Ohguro et al., 1995). In the P2AR, six

serine/threonine residues were identified as GRKî and GRK5 phosphorylation sites

(Fredericks et al., 1996) in peptide studies with the distal carboxyl tail. However, when these

residues were substituted with alanines, and mutant receptors were transfected in human

embryonic kidney (HEK) cells, no difference in agonist induced desensitization or

phosphorylation was observed compared to wild type (Seibold et al., 1998).

Therefore, although phosphorylation has been shown to be causally linked to

desensitization in many GPCRs, functionai desensitization studies are much more accurate in

detennining the phosphorylation sites responsible for desensitization, since it has observed in

many cases that serines and threonines that get phosphorylated are often not intirnately

involved in the mechanism of desensitization (Aton et al., 1984; Fredencks et al., 1996;

Seibold et al., 1998; Wilden and Kuhn, 1982)

GPCR intemalization is an agonist dependent process that induces the translocation of

activated receptors from the plasma membrane to an intracellular cornpartment. It has been

established in many receptors that GRK-catalyzed phosphorylation followed by p-arrestin

binding are crucial steps in the mechanism of intemaiization (Ferguson et al., 1996).

Arrestin binding sterically inhibits M e r interaction of the receptor its G protein, and binds

with high affiinity to clathrin chahs (Goodman et al., 1996). This interaction targets

receptors to clabin-coated pits, where vesicles are formed and pinched off from the ce11

membrane by the GTPase dynamin (Hinshaw and Schmid, 1995; Takei et al., 1995).

Recepton are either recycled back to the plasma membrane, or degraded within lysosomes.

The degradation of receptors has thought to be involved in a process of "down-regulation", in

which receptors are stimulated by agonist for prolonged penods of time (houn-days)

(Lefkowitz, 1998).

In the P2AR, it was revealed that intemalization plays a role in the resensitization of

the receptor (Freedman and Lefkowitz, 1996). It was proposed that the receptor is

dephosphorylated in the intemalization pathway (Pippig et al., 1995), and that an acid

phosphatase within the endosorne was responsible for this action (Knieger et al., 1997).

The arrestin family presently includes six distinct memben that can be broken down

into four subfamilies based on sequence homology and tissue distribution: (a) visual arrestin

(S antigen), (b) cone arrestin (C- or X-arrestin), (c) p-arrestini (arrestin2) and P-anestin2

(arrestin3), and (d) D- and E-arrestins. Both the visual and cone anestins are localized

primarily in the retina, where they primarily act in the desensitization process of light

activated rhodopsin. D- and E-arrestins are ubiquitously expressed, however they have yet to

be fblly characterized. The p-mestins are also ubiquitously expressed and are responsible

for the internalization and resensitization of numerous GPCRs.

Arrestins have been s h o w to have substrate specificity for receptors. For example.

visual arrestin binds preferentially to rhodopsin compared to the P2AR and rn2mAChR, while

both P-arrestinl and p-anestin2 bind the P2AR and m2mAChR in preference to rhodopsin

(Gurevich et al., 1995; Gurevich et al., 1993; Lohse et ai., 1992).

While there are general consensus sequences known for some of the kinases

regulating desensitization of GPCRs (Kemelly and Krebs, 199 1 ; Onorato et al., 199 1 ), there

are no similar consensus sequences identified for intemalization. M i l e there have been

expenments with tmcation and chimera studies that have identified the carboxyl tail as an

important region for regulating internalization (Faussner et al., 1998; Parent et al., 1999;

Tseng et al., 1995), a precise definition of these sites has not been established. A dileucine

motif conserved in the carboxyl tail of GPCRs has been s h o w to be important in the

intemalization of the p2AR (Gabilondo et al., 1997) and vasopressin Vla receptor (Preisser et

al., 1999), but not in the thromboxane A2 receptor (Parent et al., 1999). An NPXXY motif

found near the seventh transmembrane domains of many GPCRs has aiso been implicated in

the intemaiization of the B2AR ( B d et al., 1994), but it does so indirectly, by preventing

agonist induced activation of the receptor. In many receptors, substitution and tnincation of

s e ~ e and threonine residues has demonstnited a role in intemalization, implicating a

potential role of phosphorylation in the process (Benya et al., 1993; Maestes et al., 1999; Pak

et al., 1999; Pizard et al., 1999; Roth et ai., 1997), but again there does not seem to be a

universal structurai pattern detemiining these sites.

Although the function of intemalization had initially thought to contribute to

desensitization (since internalized receptors could not activate a response) evidence in most

receptors now suggests that both processes are distinct (Koenig and Edwardson, 1997). It

has been clearly established that internalization (minutes) occurs much slower than

desensitization (seconds). In some GPCRs, such as the p and 6 opioid receptors, mutations

of specific residues impairs both desensitization and internalization (Pak et al., 1999; Hasbi,

2000). In others such as the P2AR (Bouvier et al., 1988), m2mAChR (Pals-Rylaarsdam et

al., 1995) and N-formyl peptide (Maestes et al., 1999), and secretin (Holtmann et al., 1996)

recepton, the sites for desensitization and intemalization are distinct.

1.6 Dopamine Recepton

Although dopamine originally was considered only as a precusor to noradrenaline,

assays of regions in the central nervous system have revealed that distributions of dopamine

and noradrenaline are considerably distinct. Dopamine is now regarded as the major

catecholamine neurotransmitter in the central nervous system, where it is involved in the

regulation of many fùnctions, such as locomoter activity, emotion and neuroendocrine

secretion (Jaber et al., 1996). Peripherally, dopamine plays an important role in the control

of such processes as hormone secretion, rend function, and gastrointestinal motility (Missale

et al., 1998). There has been much interest in the dopaminergic system, primarily because it

has been discovered that diseases such as Parkinson's disease and schizophrenia were linked

to dysfunction of dopamine neurotransmission and that substances activating or blocking the

receptors could act as therapeutic agents (Strange, 1992). The fist evidence of central

dopamine receptors appeared in 1972, when it was discovered that dopamine stimulated

AC activity (Brown and Makrnan, 1972; Kebabian et al., 1972). Based on pharmacologicd

and biochemicai evidence, Spano et al. (Spano et al., 1978) proposed that there were two

distinct populations of dopamine receptors, one that coupled to AC and one that was

independent of the AC signahg pathway. In 1979, Kebabian and Caine (Kebabian and

Calne, 1979) sumrnarized Spano's earlier work and suggested the classification of hvo

subtypes of dopamine receptors: Dl and Dz. The DI receptor was defmed as the receptor that

stimulated AC, while the D2 receptor did not affect or inhibit AC. By the early 1980s, it was

concluded that D2 receptors were found to inhibit AC (Enjalbert and Bockaert, 1983;

Giannattasio et al., 198 1).

With the advent of gene cloning techniques in the late 1980s. understanding of the

divenity and complexity of the dopamine receptor system was revealed. The D2 receptor

was the first dopamine receptor cDNA to be cloned (Bumow et al., 1988), and shonly after,

splice vaxiants of the Dz were discovered (Dal Toso et al., 1989; Giros et al., 1989; Grandy et

al., 1989; Monsma et al., 1989; O'Dowd et al., 1990). Within a few yean, the D3 (Sokoloff

et al., 1 WO), the Di (Dearry et al., 1990; Monsma et al., 1990; Sunahara et al., 1990; Zhou et

al., 1990), the D4 (Van Tol et ai., 1991) and finally the D5 receptor (Sunahara et al., 1991)

were al1 identified. It was demonstrated that al1 five dopamine receptors belong to the GPCR

family, and that the initial "D i /Dc classification of the 1970s was still valid. The Dl and Ds

receptors belong to the Dl-Iike subfamily of dopamine receptor, because they share

considerable overall sequence homology (78%), and stimulate AC activity. Di-like receptors

have also been found to stimulate phosphatidylinositol (PI) hydrolysis to increase ca2+

current (Liu and Lasater, 1994), and inhibit a N~'/H' exchanger (Felder et al., 1993).

Meanwhile, the Dr, Di and D4 receptors belong to the D2-like subfamily, because they also

share high sequence homology with each other (46% for Dz and D3; 53% for Dz and D4), and

inhibit AC activity. Dl-like receptors have also been s h o w to inhibit intracellular ca2'

mobilization (Seabrook et al., 1994), potentiate the release of arachidonic acid (Piomelli et

al., 1991) and K' current (Castelletti et ai., 1989), and stimulate mitogenesis and ce11

differentiation (Lajiness et al., 1993; Swarzenski et al., 1994).

Genes for both Di and Ds receptors do not contain introns in their coding regions. In

contrast, genes encoding the D2, D3 and D4 receptors possess introns at similar locations, and

the presence of introns within the coding sequence has led to the generation and

identification of splice variants in the D2 (Da1 Toso et al., 1989; Giros et al., 1989; Grandy et

al., 1989; Monsma et al., 1989), the D3 (Fishburn et al., 1993; Giros et al., 1991; Snyder et

al., 1991), and polymorphic variations in the D4 receptors (Van Tol et al., 1992).

Stnicturally, although the dopamine receptors dl belong to the GPCR fmily, there

are similarities and dissimilarities between the Di-like and the D2-like subfamilies. There are

several conserved arnino acids found in the transmembrane domains of ail the dopamine

receptors that are thought to be involved in dopamine binding. These include an aspartic acid

residue in the third intracellular domain and two serine residues in the fi& transmembrane

domain (Mansour et al., 1992; Tomic et al., 1993). Al1 dopamine receptors also contain two

cysteine residues in the second and third extracellular loops, which are thought to form an

intermolecular disuEde bond to stabiiize the receptor structure (Dohlman et al., 1990).

The amino terminus of al1 subtypes contain a variable number of consensus N-

glycosylation sites. The Di-like receptors contain one site in the amino terminus and another

in the second extracellular loop. It has been discovered that N-linked glycosylation is

required for Ds, but not the Di receptor for fhctional ce11 surface expression in transfected

human embryonic kidney (HEK) cells (Karpa et al., 1999). Meanwhile, the D2 receptor has

four potentiai glycosylation sites, the D3 has three and the D4 only one. The Di-like receptors

possess a short third intracellular loop and a long carboxyl tail, characteristic of many

receptors coupled to Gs, while the D2-like recepton have a much longer third intracellular

loop and a short carboxyl terminus, features common to receptors coupling to Gi (Civelli et

ai., 1993; O'Dowd, 1993). Within the carboxyl terminus, the Di-like receptors have two

cysteines within a short distance Erom transmembrane domain seven, whereas the Dt-like

receptors each have one cysteine at the end of their short carboxyl terminus (Neve and Neve.

1997). These cysteines are conserved in most GPCRs and have been shown to be target sites

for palmitoylation in the Di (lin et al., 1999) and Dz receptors (Ng et al., 1994).

Dopaminergic neurons are localized in three major pathways in the brain. The

nigrostriata1 pathway is predominant and contains neurons originating in the substantia nigra

and terminating in the caudate nucleus. The mesolimbic pathway contains neurons from the

ventrai tegmental area and projects to the limbic system, nucleus accumbens and olfactory

tubercle. The tuberoinfundibular pathway contains neurons projecting from the

hypothalamus to the median erninence and pituitary gland (Jaber et al., 1996). The

distribution of dopamine receptor mRNAs in the brain has been accomplished mainly

through in situ hydridization techniques reviewed by (Missale et al., 1998). Of the five

dopamine receptors, the Di is the most widespread and highiy expressed in the brain (Dearry

et d., 1990; Fremeau et al., 1991). Dl rnRNA is found in the striaturn, the nucleus

accumbens, olfactory tubercle, limbic system, hypothalamus and thalamus. The Ds receptor

is expressed at a much Iower level in the brain than the Dt receptor and is found in the

hippocampus, the laterai mamillary nucleus and the parafascicular nucleus of the thalamus

(Meador-Woodniffet al., 1992; Tiberi et al., 1991). It is interesting to note that there are no

areas in which there are significant expression of both Dl and Ds receptors. D2 receptors

have been found in the striaturn, the olfactory tubercle, nucleus accumbens and pituitary.

The Di receptors have been localized to lirnbic areas such as the shell of the nucleus

accurnbens, olfactory tubercle, and islands of Calleja, with low expression in the striaturn.

D4 receptors are present in the frontal cortex, medulla, amygdala, hypothalamus and

mesencephalon, and are poorly expressed in the basal ganglia.

The human dopamine Di receptor is a 446 amino acid protein, and shares -60%

sequence homology with the D5 receptor, the other member of the Di-like receptor subfamily.

The dopamine Di receptor has two potential sites for N-linked glycosylation in the arnino

terminus and third extracellular loop. There are also multiple sites in the carboxyl terminus

that have been shown to be involved in palmitoylation of the receptor (Jin et al.. 1999). The

dopamine Di receptor has a reiatively short third intracellular loop and a long carboxyl

terminus, both which contain multiple serine and threonine residues that are potential sites

for PKA and GRK phosphorylation. The structure of the hurnan dopamine Di receptor is

s h o w in Figure 5.

- - - - - - -

Figure 5 . Schematic 3D representation of the human dopamine Di receptor. The human

dopamine Dl receptor is a 4.16 amino acid protein. The dopamine Dl receptor hns nvo putative

glycosylation sites in the amino terminus and the second extracellular loop. There are sevenl

intracellular serine and threonine residues that are putative sites for phosphorylation by PELA and

GRK. Specifically, two sites for P U phosphorylation are highlighted by an astetisk (+) at Thr268

ruid SerjsO in the human dopamine Dl receptor.

1.7 Desensitization / Intemalization of the Dopamine Dl Receptor

Prior to the inception of this project in 1998, there had been much work done to

charactenze and identiQ the regulatory rnechanisms of desensitization and intemalization in

the dopamine Dl receptor. However, there have been many confiicting results presented, and

the specific amino acid residues for each process have yet to be determined.

The very first characterization of dopamine receptor desensitization in a ce11 culture

system was carried out in NSZOY neuroblastoma cells endogenously expressing the

dopamine Di receptor (Barton and Sibley, 1990). The desensitization o f the dopamine

receptors was observed to be specifically homologous in nature, and Furthemore,

intemalization was found to be temporally distinct fiom the desensitization response. It was

then postulated that both PKA and GRK play significant roles in desensitization when

inhibitors of both kinases were not s h o w to attenuate receptor desensitization (Zhou et al.,

1991). Subsequent studies carried out by Bates et ai. (Bates et al., 1993) a few years later in

opossum kidney cells endogenously expressing the dopamine Dl receptor revealed similar

results to Barton et ai. The relative role of PKA in the mechanism of dopamine receptor

desensitization was exarnined in opossum kidney cells transfected with exogenous CAMP

phosphodiesterase, which provided a CAMP-resistent phenotype. CAMP accumulation (and

therefore PKA activation) was not necessary for desensitization, but was required for

intemalization - suggesting differential regulation of the two processes. Studies carried out

in our laboratory were the first to show that increases in phosphorylation upon agonist

treatment in the dopamine Di receptor correlated well with increased desensitization (Ng et

al., 1994). Our laboratory aiso demonstrated that desensitization and intemalization are

biochemically distinct mechanisms, as inhibitors of intemalization did not affect

desensitization of the dopamine Di receptor (Ng et al., 1995).

The first study to identiw specific residues involved in desensitization was carried out

by using a fusion protein of segment 372-446 of the carboxyi tail and examining the

phosphorylation by PKA and PKC (Zarnanillo et al., 1995). It was concluded that serine 380

was specifically phosphorylated by PKA, and that PKC was not involved in the

phosphorylation of the protein. However, this observation should be interpreted with

caution, because as observed in both rhodopsin and the P2AR, phosphorylation results Frorn

experiments carried out on receptor Fragments or peptide sequences have not correlated well

with functional desensitization of the intact receptor (Aton et al., 1984; Fredencks et al.,

1996; Ohguro et al., 1 995; Seibold et al., 1998).

The role of specific GR& in desensitization of the dopamine Di receptor was

addressed in a study by Tiberi et al. HEK cells (Tiberi et al., 1996). Overexpression of

GRK2, GRK3 and GRKS al1 enhanced desensitization, however GRK5 was found to be the

most efficient. In addition, phosphoamino analysis revealed that only serine residues were

phosphorylated in the receptor.

In the past few years that I have worked on this project, two more studies with

conflicting results have been presented. Lewis et al. (Lewis et al., 1998) examined PKA

rnediated desensitization of dopamine Dl receptor transfected in C-6 gliorna cells and

discovered that both stimulation of CAMP and treatment with three different PKA inhibitors

did not affect the desensitization response, suggesting that GRKs are exclusively responsible

in mediating desensitization of the dopamine Dl receptor. In contrast, a recent study

specifically investigating the role of PKA consensus sites in the rat dopamine Di receptor

expressed in C-6 glioma cells demonstrated that substitution of threonine 268 in the third

intracellular loop significantly impaired desensitization (Jiang and Sibley, 1999).

Furthemore, they also concluded that intemdization was not attenuated in this mutant,

suggesting that PKA was not involved in the intemalization response, in disagreement with a

previous study (Bates et al., 1993). Although this group was expressing the rat dopamine DI

receptor in their experiments, the PKA consensus site they recognized as being essential for

desensitization is conserved in al1 species of the receptor.

Therefore, there have been many conflicting results in past studies, mostly involving

the role of PKA and GRKs in desensitization. The one consensus result that has been

demonstrated repeatedly is that desensitization and intemalization are both temporally and

biochemicdly distinct mechanisms (Barton and Sibley, 1990; Bates et al., 1993; Ng et al.,

1995).

1.8 Thesis Rationale

Although there have been studies carried out in the dopamine Di receptor to identiQ

the sites and mechanisms of desensitization and internalization, there are still many

inconsistencies in their conclusions and no definitive site(s) of GRK action have been

identified. Serine and threonine residues in both the third intracellular loop as well as the

carboxyl tail have been implicated in both desensitization and internalization of the dopamine

Di receptor (Jiang and Sibley, 1999; Tiberi et ai., 1996). The major discrepancy that has

arisen between these studies has been in the relative roles of PKA and GRKs in the process

of desensitization. A possible explmation of the ciifferences seen in some of the experirnents

cornes from the fact that in many cases, different lengths of agonist exposure were examined

in the desensitization response. As a result, homologous and heterologous desensitization

were being compared, and not being clearly dissociated from each other. We have shown in

previous studies that agonist treatment of dopamine Di receptors with 10 pM dopamine for

15 minutes caused rapid homologous desensitization and intemalization (Ng et al., 1994; Ng

et al., 1995). Therefore, the purpose of the study was to investigate the mechanisms of

"rapid" agonist induced desensitization and intemalization of the dopamine Di receptor in

order to determine the specific residues involved in both processes.

Hypothesis: The human dopamine Di receptor contains serine and threonine residues in the

third intracellular loop and the carboxyl tail that are responsible for rnediating rapid agonist

induced desensitization and intemalization. In addition, we hypothesize that the sites

regulating both processes are distinct.

In order to achieve these goals,

(1) I proposed to identify the specific residues involved in rapid agonist induced

desensitization of the dopamine Di receptor by stably expressing wild type and serial

serine/threonine substitution mutants involving residues in the carboxyl tail and third

intracellular loop of the DI receptor.

(2) 1 proposed to identify the specific residues involved in rapid agonist induced

intemalization of the dopamine Di receptor by stably expressing wild type and serial

serine/threonine substitution mutants involving residues in the carboxyl tail and third

intracellular loop of the Di receptor.

Section 2

METHODOLOGY

2.1 Generation of Mutent and Stable Ce11 Lines Expressing Wild Type or Mutant

Receptors-The full length cDNA of the wild type dopamine Di receptor was cloned hto

the mammalian expression vector pRC/CMV (InVitrogen). This constmct became the

template for site directed mutagenesis using the Transformer Site-Directed Mutagenesis Kit

(Clontech). Various mutant dopamine Di constructs were made by substituting single or

multiple senne and/or threonine amino acid residues. Briefly, mutagenic primers were used

in concert with selection prirners designed to eliminate a unique ApaI restriction enzyme site

(located at the 3' region of the vector) or a unique NotI restiction site (located at the 5' of the

vector). The products fiom the T4 DNA polymerase - T4 DNA ligase reaction were digested

with the enzyme corresponding to the selection primer both before and afier transformation

into E. Coli. Clones lacking the unique restriction site were seiected for dideoxy sequencing

on both strands to confirm the incorporation of the desired nuceotide substitution. The

mutated dopamine Di receptor cDNAs were then subcloned into the pIresNeo (InVitrogen)

expression vector at the EcoRV site of the vector. The sequence of the hi11 length mutated

cDNA and its orientation in pIresNeo was confirmed by dideoxy sequencing. For stable

expression, the ce11 line CHO K-l (number CCL6I ; American Type Culture Collection) was

grown to approximately 60% coduency in a 60-mm dish and was transfected with wild type

or mutant dopamine Di receptor constnicts in the pIresNeo vector using a lipofectamine

trasfection kit (Life Technologies) according to the manufacturer's recommendations. Stable

transfectants were selected in I m g h l geneticin (Life Technologies Inc.) and clones with the

appropriate expression level were screened by the radioligand saturation binding assay.

Between 30-50 clones expressing varying numbers of receptors were screened to select those

with similar expression levels.

The construction of the various dopamine Di receptor mutants was performed by the

following members of our laboratory. Hong Chi constmcted the following substitution

mutants in the carboxyl tail: mutant receptor B (T446A), mutant receptor C (431-439), and

mutant receptor D (428-439). Marek Sawzdargo constmcted the substitution mutant

receptors A (243-268) and E (372-446), and Miles Thompson constructed the substitution

mutant receptors F (342-354, 372-446), G (360, 372-446), H (T360A) and 1 (S362A). The

various mutations are highlighted in figure 6.

2.2 Ce11 Cultures and Membrane Preparation-Chinese hamster ovary cells (CHO-KI,

no.CCL6 1 ; American Type Culture Collection) were maintained as monolayer culture in

Dulbeco's modified essential medium (DMEM) (GibcolBRL) supplemented with 10% fetal

bovine senun, 100 Ulm1 penicillin and 100 pdml streptomycin in an atmosphere of 95% air

and 5% CO2 at 37°C. Crlls were grown to full confiuency, washed twice with ice-cold

phosphate-buffered saline, scraped with a rubber policeman and centrifuged at 100 x g for 10

min. Cells were then lysed in hypotonic buffer (5 m M Tns-HCl, pH 7.8, 2 mM EDTA,

containing a protease inhibitor mixture (10 mg/ml leupeptin, 5 m g h l soybean trypsin

inhibitor and 5 mg/ml bern id ine) with a Polytron homogenizer (Bnnkman Instruments,

Westbury, NY) for two 30-s bursts at the 5.5 setting. The lysate was centrifuged at 80 x g for

10 min to pellet unbroken cells and nuclei. The supernatant was then centrifuged at 30,000 x

g for 20 min at 4'C and the resulting pellet was resuspended in buffer containing 50 m M

Tris-HCl, pH 7.8, 5 m M MgC12, ImM EGTA with the protease inhibitor mixture and used

irnmediately for radioligand binding or adenylyl cyclase assays. Protein concentrations were

determined by the method of Bradford (Bradford, 1976), (Bio-Rad), using bovine serum

albumin as a standard.

For agonist-induced desensitization and intemalization assays, cells grown to 80-90%

confiuence were incubated overnight in sem-fiee DMEM. Fresh sem-fiee medium was

again changed prior to the expenment and dopamine and ascorbic acid were added to a final

concentration of 10 FM and 100 pM respectively, for the indicated time. Control cells were

treated with serum free media containing 100 pM ascorbic acid only.

2.3 Radioligand Binding Assays-For radioligand saturation binding assays, ce11

membranes (20-30 pg of proteinhube) were incubated with increasing concentrations of

[ ' ~ ] ~ ~ ~ - 2 3 3 9 0 (specific activity 75.5 Ci/mmol; Mandel) in a total volume of 1 ml of

binding buffer (50 m M Tris-HCI, pH 7.4,5 m M EDTA, 1.5 mM CaC12, 5 rnM MgCl?, 5 mM

KCI and 120 mM NaCl). (+)-Butaclamol was added at the final concentration of 1 pM to

determine nonspecific binding. Competition experiments were performed using [ 3 ~ ~ ~ ~ -

23390 at approximately its Kd value (700 PM) and dopamine at concentrations ranging fiom

10-12 to 10" M. Afier the assay, tubes were incubated at room temperature for 90 min. and

bound ligand was isolated by rapid filtration through a 48-well ce11 harvester (Brandel,

Montreal, Quebec, Canada) onto GF/C Whatman filters. Filters were washed with 10 ml of

ice-cold 50 rnM Tris-HCI, pH 7.4, and incubated overnight in vials containing 5 ml of

scintillation fluid (Cytoscint; ICN, Costa, Mesa, CA). Tritium was counted using a Beckman

LS 6500 scintillation counter at a counting efficiency of 40%. ALI experiments were

performed in duplicate, and each experiment was repeated at least three times.

2.4 Internalization Assays-Cell membranes were prepared as above, however, the ce11

Iysate was Iayered on top of a 35% sucrose cushion and centnfùged at 150,000 X g for 90

min. at 4°C in order to separate the light vesicular and heavy fractions of the membrane. The

heavy fraction at the bottom of the sucrose cushion was resuspended in binding buffer and

used for radioligand saturation binding assays for analyzing the extent of receptor

sequestration. Internalization is expressed as the percentage decrease of the ce11 surface

receptors (specific binding) of the treated cells relative to the untreated cells.

2.5 Adenylyl Cyclase Assays-Adenylyl cyclase activity was determined essentially as

described by Salomon et al (Salomon et al., 1974). Membranes were prepared as descnbed

above. The assay mix contained 20 pl of membrane suspension (20-25 pg of protein), 12

FM ATP, 100 pM CAMP, 53 pM GTP, 2.7 mM phosphoenol-pyruvate, 0.2 units of pynivate

kinase, 1 unit of myokinase, 5 mM ascorbic acid and 0.1 3 pCi [ a - 3 3 ~ ] ~ ~ ~ in a final volume

of 50 pl. Enzyme activities were determined in duplicate in the absence of dopamine (basal

activity), or with increasing concentrations (1 mM to 10 nM) of dopamine for 20 min at

37°C. Reactions were stopped by the addition of 1 ml of an iceîold solution containing 0.4

m M ATP, 0.3 m M CAMP and [ 3 ~ c ~ ~ (25,000 cpm). [ 3 3 ~ ] c ~ ~ ~ and ?HICAMP were

isolated by sequential column chromatography using Dowex cation exchange resin and

aluminum oxide coltunns. The amount of ['H]CAMP was used to quanti@ individual column

recovery. Desensitization is expressed as the percentage decrease of the response of the

treated cells relative to the untreated cells. Al1 experirnents were performed in duplicate, and

each experirnent was repeated at least three times, udess otherwise noted.

2.6 Data Analysis-The data obtained fiom radioligand saturation and adenylyl cyclase

experiments were fitted by least squares nodinear regression using the cornputer program

Prism (GraphPad Software, San Diego, CA). Data fiom multiple experiments were averaged

and expressed as the means +_ S.E. The results were considered significantly different when

the probability of randomly obtaining a mean difference was ~0.05 using the paired Student's

t test.

Section 3

3.1 Generation and Expression of Mutant Dopamine Di Receptor Constructs-We

have shown in previous studies that agonist treatment of dopamine Di receptors with 10 pM

dopamine for 1 5 minutes caused rapid desensitization and internalization. (Ng et al., 1994;

Ng et al., 1995). Therefore, in order to identiQ the specific residues involved in the process

of desensitization and internalization, progressive substitution mutants were made in the

carboxyl tail and third intracellular loop, replacing serines and threonines with alanine

residues (Fig. 6). A mutant with al1 serine and threonine residues substituted to alanine in the

third intracellular loop between amino acids 243-268 was termed mutant A. Mutant

receptors B, C, D, E, F, G, H, and 1 were located in the carboxyl tail and consisted of

substitutions of serine and threonine residues with alanine between the following positions.

Mutant receptor B consisted of a single T446A substitution; mutant receptor C consisted of

substitutions between positions 431439; mutant receptor D consisted of substitutions

between 428-439; mutant receptor E consisted of substitutions between positions 372-446;

mutant receptor F consisted of substitutions between 342-354 and between 372-446; mutant

receptor G consisted of substitutions at position 360 and between residues 372-446. Mutant

receptors H and 1 consisted of single substitutions of T360A and S362A, respectively.

Arnong the serine and threonine residues substituted to alanine, there included the removal of

two consensus PKA sites encompassing -268 (mutant receptor A) in the third intracellular

loop and Ser380 (mutant recepton E, F and G) in the carboxyl tail (Fig. 6). Each of the

mutant and wild type Di receptors were stably expressed in CHO cells with levels of

expression ranging fiom approximately 0.3 to 2.0 pmoYmg of protein (Table 1). Cell lines of

two wild type Di receptors (Wt-a, Wt-b) were selected to control for the different levels of

expression in the various substitution mutants. In al1 of the receptor conscnicts, the Kd values

Fig. 6. Schematic representation of intracellular residues of the human DI dopamine receptor.

Alanine substitution mutations of residues in the third intracellular loop and carboxyl tail are

highlighted and nurnbered. The asterisk (*) denotes a PKA consensus recognition site. Mutants

made of the DI dopamine receptor were as followç: Mutant receptor A consisted of al1 serine and

threonine residues substituted to alanine in the third intrace1 lular loop between amino acids 243-268.

Mutant receptors B, C, D, E, F, G, H, and I were Iocated in the carboxyl tail and consisted of

substitutions of serine and threonine residues with alanine between the following positions. Mutant

receptor B consisted of a single T446A substitution; mutant receptor C consisted of substitutions

between positions 43 1-439; mutant receptor D consisted of substitutions behveen 428-439; mutant

receptor E consisted of substitutions between positions 372-446; mutant receptor F consisted of

substitutions between 342-354 and between 372-446; mutant receptor G consisted of substitutions at

position 360 and between residues 372-446. Mutant receptors H and I consisted of single

substitutions of T360A and S362A, respectively.

of [ 3 ~ ~ ~ ~ - 2 3 3 9 0 binding were sirnilar to wild type, at 0.43 + 0.06 M. We selected ce11

lines expressing mutant receptors with similar expression levels, and have s h o w that the

mutations had no effect on antagonist binding affinity (Table 1).

3.2 Characterization of DlWT recepton- Before performing any desensitization and

intemaiization assays, we wished to characterize our wild type dopamine Di receptor with a

competition binding assay to ensure that we were obtaining results typical of the dopamine

Di receptor and not another receptor (in addition to our sequencing data). The competition

assay involved the binding of increasing concentrations of dopamine (10'" to 10") in the

presence of -700 pM of the radiolabeled antagonist [ 3 ~ ] ~ ~ ~ - 7 " ,3390. Results showed a

typical biphasic curve with 72% of the recepton in the high affînity binding state. The ECSO

value for the high affinity binding site was 3.68 i 1.00 FM and 2.18 + 0.54 rnM for the low

binding site (Fig. 7).

3.3 Agonist induced Desensitization and Internalization of DlWT receptors-

Desensitization of wild type and mutant receptors was rneasured as a loss of AC response to

a range of dopamine concentrations (10 -'O to 10 M) after 20 min pretreatment with 10 pM

dopamine. Intemalization of wild type and mutant recepton was measured as the loss of ce11

surface receptors determined by specific [ 'H]S~~-23390 binding d e r a 20 min pretreatment

with 10 pM dopamine.

The rate of dopamine sbuiated maximai AC activity (Vmax) was decreased

similady in both ce11 lines expressing wild type receptor a and b by 28.6 f 5.1% d e r 20 min

dopamine pretreatment, and the ECSO was shified 1.5 fold to the right, consistent with

Figure 7. Cornpetition binding curve of the Dl Dopamine Receptor. CHO cells expressing wild

type dopamine DI receptor were incubated with -700 pM of ['HI SCH-23390 and increasing

concentrations of dopamine (16" to 1 ~ ~ ) . Experiments were conducted in duplicate and were

repeated three times.

agonist induced desensitization (Fig. 8a). EC50 values for the control membranes was 128 1

f 2 10 nM and 19 10 t 382 nM for the dopamine pretreated cells. The wild type recepton

demonstrated a 24.8 f 3.7% loss of ce11 surface receptors (Fig. 8b). This was observed in

both wild type a (Fig. 8b) and wild type b ce11 lines. The Kd values for the control and

dopamine pretreated wild type recepton were 0.46 k 0.06 and 0.27 t 0.04 nM respectively.

For al1 receptors, the EC50 value represents the concentration of dopamine that elicits a half-

maximal adenylyl cyclase response. The & value represents the concentration of

[)HI SCH-23 390 when half of the recepton are occupied.

3.4 Agonist induced Desensitization and Intemalkation of Mutant Receptor A

(243-268)- The first mutant receptor to be examined was mutant A consisting of al1 senne

and threonine residues substituted in the third intracellular loop (243-268). The rate of

dopamine stimulated maximal AC activity was decreased similar to wild type (32.3 + 4.2%)

(Fig. 9a) with a resulting 1.5 fold shiff in the EC50. EC50 values for the control membranes

was 13 18 I 202 nM and 1914 & 378 nM for the dopamine pretreated cells. Internalization of

mutant receptor A was dso similar to wild type, with a 18.8 + 4.2% loss of ce11 surface

receptors (Fig. 9b). The & values for the control and dopamine pretreated wild type

receptors were 0.28 I 0.04 and 0.19 f 0.05 nM respectively.

3.5 Agonist induced Desensitization and Internalization of Mutant Receptor B

(T446A)- The next mutant receptor to be examined was the single terminal threonine mutant

B (T446A). The rate of dopamine stimulated maximal AC activity was decreased similar to

wild type (24.3 2 4.8%) (Fig. 10a) with no shift in the EC50. EC50 values for the control

[DA] log M

Figure 8: Desensitization and Internalization of Dopamine Dl Wild Type Receptor.

A, Desensitization. CHO celIs expressing dopamine Di wiid type (Wt-a) receptor were incubated in

the absence (e) or presence (O) of 10pM dopamine at 37OC for 20 minutes, and subsequently the

ability of increasing concentrations of dopamine (10"' to IO" M) to stimulate CAMP accumulation

\vas tested. Data are presented as a percentage of maximal stîmulated AC activity of untreated cells.

B, Internalization. CHO celb expressing dopamine Di wild type (Wt-a) were incubated in the absence

(H) or presence (O) of IOpM dopamine at 37OC for 20 minutes, and saturation binding was estimated

using increasing concentrations of [ 3 ~ ~ ~ ~ - 2 3 3 9 0 . Data are presented as a percentage of maximal

specific binding of untreated cells. Data shown for al1 experiments are the means f S.E. of at least

three independent experhents performed in duplicate.

Mutant A (243-268)

75 '"i

[DA] log M B

125, MutantA

Figure 9: Desensitization and Intenialization of Mutant Receptor A (243-268).

A, Desensitization. CHO ceils expressing mutant receptor A were incubated in the absence (O) or

presence (O) of 10pM dopamine at 37OC for 20 minutes, and subsequently the ability of increasing

concentrations of dopamine (10 '~~ to lo4 M) to stimulate CAMP accumulation was tested. Data are

presented as a percentage of maximal stimulated AC activity of untreated cells. B, Intenialization.

CHO cells expressing mutant receptor A were incubated in the absence (m) or presence (Cl) of 10pM

dopamine at 37OC for 20 minutes, and saturation binding was estimated using increasing

concentrations of [ 3 w ~ ~ ~ - 2 3 3 9 0 . Data are presented as a percentage of maximal specific binding of

untreated cells. Data show for al1 expenments are the means k S.E. of at least three independent

experîrnents performed in duplicate.

Mutant B (T446A)

[DA] log M B

1251 Mutant B

Figure 10: Desensitization and Internalhtion of Mutant Receptor B (T446A).

A, Desensitization. CHO cells expressing mutant receptor B were incubated in the absence (@) or

presence (O) of 10pM dopamine at 37OC for 20 minutes, and subsequently the ability of increasing

concentrations of dopamine (10"' ta loJ M) to stimulate CAMP accumulation was tested. Data are

presented as a percentage of maximal stimulated AC activity of untreated cells. B, Internalization.

CHO cells expressing mutant receptor B were incubated in the absence (m) or presence (Cl) of 10pM

dopamine at 37OC for 20 minutes, and saturation binding was estimated using increasing

concentrations of [ 3 ~ ~ ~ ~ - 2 3 3 9 0 . Data are presented as a percentage of maximal specific binding of

untreated cells. Data show for al1 experiments are the means f S.E. of at least three independent

experiments perfonned in duplicate.

membranes was 1469 t 242 nM and 1348 f 297 nM for the dopamine pretreated cells.

Internalization of mutant receptor B was almost completely abolished with a 4.6 t 3.9% loss

of ce11 surface receptors, significantly different fiom the wild type (Fig. 1 Ob). The Kd values

for the control and dopamine pretreated wild type receptors were 0.30 t 0.02 and 0.35 t 0.04

nM respectively.

3.6 Agonist induced Desensitization and Internalization of Mutant Receptor C

(431-439)- Progressing dong the carboxyl tail, mutant receptor C (43 1-439) encompassing

two serinehhreonine residues substituted was exarnined. The rate of dopamine stimulated

maximal AC activity was decreased similar to wild type (33.1 + 5.2%) with no shift in the

EC50 (Fig. 1 la). EC50 values for the control membranes was 18 19 f 350 nM and 1399 f

332 nM for the dopamine pretreated cells. Internalization of mutant receptor C was almost

completely abolished with a 5.8 f 5.1% loss of ce11 surface receptors, significantly different

fiom the wild type (Fig. 1 l b). The & values for the control and dopamine pretreated mutant

receptors were 0.27 f 0.02 and 0.29 f 0.08 nM respectively.

3.7 Agonist induced Desensitization and Internalization of Mutant Receptor D

(428-439)- Mutant receptor D (428-439) consisted of three serine/threonine residues

substituted in the distal C-tail. A similar reduction in AC activity as wild type (32.0 I 3.3%)

was observed with no shift in the EC50 response to the nght (Fig. 12a). EC50 values were

1600 + 294 nM for the control membranes and 1452 i: 308 nM for the dopamuie pretreated

cells. Internalization of mutant receptor D was again almost completely abolished with a 6.3

+ 4.8% loss of ce11 surface receptors, significantly different fiom wild type (Fig. 12b).

Mutant C "O\ (431439) I/

[DA] log M B

Figure 11: Desensitization and Internalization of Mutant Receptor C (431439).

A, Desensitization. CHO cells expressing mutant receptor C were incubated in the absence (O) or

presence (O) of 10pM dopamine at 37OC for 20 minutes, and subsequently the ability of increasing

concentrations of dopamine ( 1 ~ " to 104 M) to stimulate CAMP accumulation was tested. Data are

presented as a percentage of maximal stimulated AC activity of untreated cells. B, Internalization.

CHO cells expressing mutant receptor C were incubated in the absence (a) or presence (O) of IOpM

dopamine at 37°C for 20 minutes, and saturation binding was estimated using increasing

concentrations of [ J H ] s c H - ~ ~ ~ ~ o . Data are presented as a percentage of maximal specific binding of

untreated cells. Data show for a11 experiments are the means * S.E. of at least three independent

experiments performed in duplicate.

Mutant D lo0i (428439)

[DA] log M B

Mutant D (428439)

Figure 12: Desensituation and Internalizrition of Mutant Receptor D (428-439).

A, Desensitization. CHO cells expressing mutant receptor D were incubated in the absence (@) or

presence (O) of lOpM dopamine at 37OC for 20 minutes, and subsequently the ability of increasing

concentrations of dopamine (1 0'" to 1 o4 M) to stimulate CAMP accumulation was tested. Data are

presented as a percentage of maximal stimulated AC activity of untreated cells. B, Intemalization.

CHO cells expressing mutant receptor D were incubated in the absence (H) or presence (O) of 10pM

dopamine at 37OC for 20 minutes, and saturation binding was estimated using increasing

concentrations of [ - ' ~ ] ~ ~ ~ - 2 3 3 9 0 . Data are presented as a percentage of maximal specific binding of

untreated cells. Data shown for al1 experirnents are the means f S.E. of at Ieast three independent

experirnents perfonned in duplicate.

The Kd values for the control and dopamine pretreated mutant receptors were 0.21 t 0.04 and

0.18 + 0.05 nM respectively.

3.8 Agonist induced Desensitization and Intemalization of Mutant Receptor E

(372-446)- Mutant receptor E (372-446) was constnicted with ~ e l v e serine/threonine

residues substituted in the carboxyl tail. Mutant receptor E also displayed a reduction in the

Vmax consistent with wild type DI receptor, at 36.5 t 5.8% with no shift in the EC50

(Fig. 13a). EC50 values were 1766 f 269 nM for the control membranes and 1205 f 132 nM

for the dopamine pretreated cells. Intemalization of mutant receptor E \vas again almost

completely abolished with a 7.3 + 4.9% loss of ce11 surface receptors, significantly different

from wild type (Fig. 13b). The & values for the control and dopamine pretreated mutant

receptors were 0.30 t 0.08 and 0.34 k 0.05 nM respectively.

3.9 Agonist induced Desensitization of Mutant Receptor F (3420354,372446)- As there

were only five senne/threonine residues left to test in the carboxyl tail, a mutant containing

the three most proximal serine/threonine residues (342-354) was combined with mutant E

(372-446) and was termed mutant F. The rate of dopamine stimulated maximal AC activity

was decreased similar to wild type (22.5 t 3.8%) with no shift in the EC50 (Fig. 14). EC50

values were 476 + 60 nM for the control membranes and 386 f 75 nM for the dopamine

pretreated cells. As sites for internalization were found to be localized in the distal portion of

the carboxyl tail, intemalization experiments were not carried out for this mutant.

1004 Mutant E

[DA] log M B

1251 Mutant E

Figure 13: Desensikation and Internalizntion of Mutant Receptor E (372-446).

A, Desensitization. CHO cells expressing mutant receptor E were incubated in the absence (m) or

presence (O) of IOpM dopamine at 37OC for 20 minutes, and subsequently the ability of increasing

concentrations of dopamine ( 1 ~ ' ~ to IO*-' M) to stimulate CAMP accumulation was tested. Data are

presented as a percentage of maximal stimuiated AC activity of untreated cells. B, Intemalization.

CHO cells expressing mutant receptor E were incubated in the absence (M) or presence (O) of 10pM

dopamine at 37OC for 20 minutes, and saturation binding was estimated using increasing

concentrations of [- 'KJs~~-23390. Data are presented as a percentage of maximal specific binding of

untreated cells. Data shown for al1 expenments are the rneans k S.E. of at least three independent

experiments pei-formed in duplicate.

[DA] log M

Figure 14: Desensitization of Mutant Receptor F (342-354, 372-446). CHO cells expressing

mutant receptor F were incubated in the absence (O) or presence (O) of 10pM dopamine at 37°C for

20 minutes, and subsequently the ability of increasing concentrations of dopamine ( 1 0'" to 1 o5 M) to

stimulate CAMP accumulation was tested. Data are presented as a percentage of maximal stimulated

AC activity of untreated cells. Data show are the means * S.E. of at least three independent

experirnents performed in duplicate.

3.10 Agonist induced Desensitization and Internalization of Mutant Receptor G

(360, 372446)- Mutant receptor G was constnicted containing the single threonine

substitution at residue 360, combined with mutant receptor E (372446). Mutant receptor G

completely abolished the desensitization response with vimially no change in Vmax (2.8 f

4.7%) or shift in the EC50 response (Fig. 15). ECSO values were 1086 + 27 nM for the

control membranes and 1 177 f 137 nM for the dopamine pretreated cells. Again, as sites for

intemalization were found to be localized in the distal portion of the carboxyl tail,

intemalization experiments were not c d e d out for this mutant.

3.11 Agonist induced Desensitization and Internalization of Mutant Receptor H

(T360A)- In view of the previous results, since mutant receptor E (372-446) was observed to

desensitize normally, we wished to specifically examine the role of residue Th360 in

desensitization. Mutant receptor H was thus constnicted substituting the single threonine

residue at 360. Mutant receptor H also completely abolished the desensitization response

with virtually no change in Vmax (3.0 f 4.2%) or shift in the ECSO response (Fig. 16a).

EC50 values were 1479 + 362 nM for the control membranes and 1702 f 340 nM for the

dopamine pretreated cells. We wished to test the intemalization of this mutant receptor in

order to see if the processes of desensitization and internalization in the dopamine Di

receptor were distinct. Exposure of mutant receptor H to a g o ~ s t resulted in internaiization

similar to wild type receptor (18.7 I 5.6%) (Fig. 16b). The Kd values for the control and

dopamine pretreated mutant receptors were 0.3 1 k 0.08 and 0.23 t 0.03 nM respectively.

[DA] log M

Figure 15: Desensitization of Mutant Receptor G (360, 372-446). CHO cells expressing mutant

receptor G were incubated in the absence (a) or presence (O) of 1OpM dopamine at 37OC for 20

minutes, and subsequently the abiiity of increasing concentrations of dopamine (16" to W3 M) to

stimulate CAMP accumulation was tested. Data are presented as a percentage of maximal stimulated

AC activity of untreated cells. Data show are the means 2 S.E. of at least three independent

experirnents perfonned in duplicate.

loo] Mutant H

[DA] log M B

12s1 Mutant H

Figure 16: Desensitization and Internalization of Mutant Receptor H (T360A).

A, Desensitization. CHO cells expressing mutant receptor H were incubated in the absence (a) or

presence (O) of 1OpM dopamine at 37OC for 20 minutes, and subsequently the ability of increasing

concentrations of dopamine (WiO to 104 M) to stimulate CAMP accumulation was tested. Data are

presented as a percentage of maximal stimulated AC activity of untreated cells. B, Internalization.

CHO cells expressing mutant receptor H were incubated in the absence (W) or presence (El) of t O p M

dopamine at 37OC for 20 minutes, and saturation binding was estimated using increasing

concentrations of [ 3 ~ ~ ~ ~ - 2 3 3 9 0 . Data are presented as a percentage of maximal specific binding of

untreated cells. Data s h o w for al! experiments are the means k S.E. of at least three independent

experiments perfomed in duplicate.

3.12 Agonist induced Desensitization of Mutant Receptor 1 (S362A)- Mutant receptor I

was constructed containhg the last serine substitution in the carboxyl terminus to be tested at

residue 362. The rate of dopamine stimulated maximal AC activity was decreased slightly

compared to wild type (20.0 f 4.8%) with a 1.2 shift in the EC50 (Fig. 17). EC50 values

were 1102 f 207 nM for the control membranes and 1383 t 240 nM for the dopamine

pretreated cells. (This experiment has only been repeated twice). Intemalization

experiments for this mutant have yet to be carried out.

3.13 Summary of desensitization and internalization results. A surnmary of

desensitization results from al1 mutant receptors is presented in figure 18. Results fiom al1

intemalization expenments are sumrnarized in figure 19.

100- Mutant I

75-

50-

25-

0 T I 1 1 I I 1 1 -11 -10 -9 -8 -7 -6 -5 -4 -3

[DA] log M

Figure 17: Desensitization of Mutant Receptor 1 (S362A). CHO cells expressing mutant receptor I

were incubated in the absence (a) or presence (O) of 1 O p M dopamine at 37OC for 20 minutes, and

subsequently the ability of increasing concentrations of dopamine ( 1 0 " ~ to 10" M) to stimulate CAMP

accumulation was tested. Data are presented as a percentage of maximal stimulated AC activity of

untreated cells. Data shown are the means f S.E. of two independent experiments performed in

duplicate.

s

Figure 18.

I 243-268 446 431439 428439 372-446 342-354, 360, 360 362

I 372-446 372446 I Wild I Type Receptor Mutants

Summary of desensitization experirnents. CHO cells expressing wild type or mutant receptors werz incubated in the absence and presence of 1Om dopamine for 20 minutes, and subsequently die ability of increasing concentrations of dopamine to siimulate CAMP was tested. nie percentages of agonist induced receptor desensitization are presented as the means i- S.E. of at least three independent experiments. Signilicant differences from wild type is denoted by an asterisk @ < 0.05)

I Receptor Mutants

F@re 19. Summnry of intemalkation experiments. CHO cells expressing wild type or mutant receptors were incubated in the absence and presence of IOY dopamine for 20 minutes, and saturation biiiding was estimated using increasing concentrations of [31.1]~~~-23390. The percentages of IOSS of cell surface receptors are presented as the rneans i S.E. of at least three independeut experiments. Significant differences from wild type is denoted by an asterisk @ < 0.05)

Section 4

DISCUSSION, CONCLUSIONS, RECOMMENDATIONS

4.1 Discussion

In this study, we show that both Th360 and Ser362 in the proximal segment of the

carboxyl tail are important regulators of rapid agonist induced desensitization in the

dopamine Di receptor. However, both sites do not appear to be equivalent in the

desensitization in the dopamine Di receptor. It appears that Thr360 is the primary site of

desensitization, whereas Ser362 has a lesser or secondary role. We have also identified a

cluster consisting of two threonines and one serine at the distal portion of the carboxyl tail

(Thr446, Thr439, Ser431) that is responsible for rapid agonist induced intemalization.

Furthemore, we have shown that both processes of desensitization and intemalization are

biochemically distinct mechanisms since the distinct mutations that were involved in

abolishing the desensitization and intemalization responses did not display a concomitant

effect on the other process.

In order to identiS, sites reguiating desensitization and intemalization in GPCRs,

strategies utilizing tnincation, deletion and substitution mutants of intracellular serine and

threonine residues have been employed. Domains in the carboxyl tail have been show to be

critical for desensitization in many GPCRs including the y opioid (Pak et ai., 1997), al^-

adrenergic (Diviani et al., 1997), rhodopsin (Ohguro et al., 1993, A3 and A2. adenosine

(Palmer et al., 1995: Palmer and Stiles, 1997), N-formyi peptide (Maestes et al., 1999), and

PtARs (Bouvier et al., 1988; Fredericks et al., 1996; Hausdoflet al.. 1991). In contrast, the

aw-adrenergic (Eason et ai., 1995) and rn2mAChR (Pals-Rylaarsdam and Hosey, 1997) are

mediated by phosphorylation and desensitization in the third intracellular loop, since their

carboxyl tails are relatively short and contain few or no serine/thrconine residues. nius,

where the sites regulating desensitization occur depends on the general structure of the

carboxyl terminus.

In our studies, we decided to examine substitution mutants of serine and threonine

residues in order to detemine the sites mediating desensitization and intemalization in the

dopamine ai reccptor rather than senal bucation mutations. Since the carboxyl tail

comprises approximately 25% of the dopamine Dl receptor, tmcation mutants may

drastically alter the general structure of the receptor and result in effects that do not occur in

the intact receptor. For example, a study in the p opioid receptor recently examined the

effect of truncations in the carboxyl tail on phosphorylation and desensitization (Deng et al.,

2000). Though Thr394 at the distal end of the receptor has been shown to regulate

desensitization of the p opioid receptor through receptor phosphorylation (Deng et al., 2000;

Pak et ai., 1997), a tnincation of the last 45 amino acids in the carboxyl tail actudly resulted

in a significant increase in phosphorylation (Deng et al., 2000). Therefore, truncation

mutants used commoniy to identify regions regulating phosphorylation and desensitization in

GPCRs may not accurately depict what normally occurs in full length receptors.

The relative roles of PKA and GRKs in rapid desensitization have not been clearly

established for the dopamine Di receptor. Recently, a study of the rat dopamine Dl receptor

identified that a single PKA phosphorylation site in the third intracellular loop (Thr268) that

was important in regulating rapid homologous desensitization (Jiang and Sibley, 1999).

These results diRered from two other studies, which have concluded from expenments with

specific P K A inhibitors (Lewis et al., 1998), and CAMP deficient ce11 lines (Bates et al.,

1993) that PKA does not play a role in agonist induced desensitization. A role for GRKs was

determined by Tiberi et al., (Tiben et al., 1996) when coexpression of GRKs 2, 3 and 5 were

al1 found to enhance phosphorylation and desensitization of the Di receptor, and the

discovery that GRK5 was the most efficient. Phosphoamino analysis also concluded that

only serines were phosphorylated in the receptor. Finally, Zhou et al. (Zhou et al., 1991)

found that inhibitors of both PKA and GRK attenuated desensitization, implying a possible

role for both pathways.

In our study, we have mutated two PKA sites, one in the third intracellular loop and

the other in the carboxyl tail and fond no difference in desensitization of the mutant

receptors compared to wild type. Thus, we can conclude that PKA rnediated rapid

desensitization does not play a significant role in the human dopamine Di receptor. Our

M e r mutagenesis studies discovered a single residue in the proximal carboxyl tail (Thr360)

that significantly attenuated the rapid agonist induced desensitization of the receptor, as well

as a serine residue (Ser362) that partially decreased desensitization compared to wild type.

Threonine 360 is particularly interesting because it is flanked on its amino terminal side by

the acidic amino acid, glutamic acid. Although no clear consensus sequences have been

defined for the various GRKs, both GRKl and G W are known as acidotropic kinases

because they most actively phosphorylate serines and threonines in close proximity to acidic

amino acids (Onorato et al., 1991). GRK2 preferentially phosphorylates serines and

threonines with acidic amino acids on the amino terminal side, while GRKl recognizes

serines and threonines with acidic residues localized to the carboxyl temiinal side (Onorato et

ai., 1991). In contrast, GRK5 and GRK6 preferentially phosphorylate serine residues

containing basic amino acids on the amino terminal side (Kunapuli et al., 1994; Loudon and

Benovic, 1994).

Although the dopamine Di receptor was s h o w to be a substrate for GRK5 in a study

conducted by Tiberi et al. (Tiberi et al., 1996), it is interesting to note that Thr360, the

residue we found to be primarily important in regulating desensitization, would not be

phosphorylated by GRKS because a basic residue is not found adjacent to this threonine. To

explain the results obtained by Tiberi et al. (Tiben et al., 1996), it is possible that the

overexpression of G U 5 induces phosphorylation at sites that are not used in the

physiological desensitization of the receptor. There are examples in rhodopsin and the PzAR

in which serine and threonine residues that do get phosphorylated are not necessarily

involved in the mechanism of desensitization (Aton et al., 1984; Fredericks et al., 1996;

Wilden and Kuhn, 1982).

Therefore, based on the fact that among the serines and threonines we substituted

included two consensus PKA recognition sites, and that we obsewed no change in rapid

desensitization with these mutants (unlike results reported for longer term desensitization in

the rat Di receptor (Jiang and Sibley, 1999)). we conclude that PKA mediated

phosphorylation does not appear to be involved in rapid desensitization of the Di receptor.

Furthemore, since residue Thr360 is situated beside a glutarnic acid, we propose that GRK2

may be the primary regulator of rapid agonist induced desensitization of the Di receptor.

Although Ser362 was only observed to partially reduce functional desensitization of

the Di receptor, it is still possible that it May play an important role in a complete

desensitization response of the receptor. Ser362 may require the phosphorylation at Thr360

in order to be phosphorylated itself, and thus the substitution of Thr360 would completely

abolish phosphorylation (and hence desensitization) of both residues. Meanwhile the

substitution of Ser362 would result in a substantial but incomplete phosphorylation and

desensitization due to kinase activity at the single residue Thr360. A similar mechanism of

phosphorylation has been well characterized in rhodopsin, with rhodopsin kinase (GRK1)

preferentially phosphorylating Ser338, and subsequently phosphorylating Ser343 and Th336

(Ohguro et al., 1993).

While there are general consensus sequences known for kinases regulating

desensitization of GPCRs (Kemeliy and Krebs, 1991; Onorato et al., 1991), there are no

sirnilar consensus sequences designated for intemalization. A dileucine motif conserved in

the carboxyl tail of GPCRs has been s h o w to be important in the intemalization of the PzAR

(Gabilondo et al., 1997) and vasopressin V 1 a receptor (Preisser et al., 1999), but not in the

thromboxane A2 receptor (Parent et al., 1999). An NPXXY motif found near the seventh

trammembrane domains of many GPCRs has also been irnplicated in the internaiization of

the PÎAR (Barak et al., 1994), but activation of the receptor was also compromised. In many

GPCRs, substitution and truncation of senne and threonine residues have demonstrated a role

in intemalization such as for the gastrin releasing peptide (Benya et al., 1993), n t

somatostatin subtype 3 (Maestes et al., 1999), N-formyl peptide (Pizard et al., 1999) and B2

bradykinin receptors (Roth et al., 1997).

The mechanism and putative sites of the residues mediating intemalization have aiso

been inconsistent in studies in the dopamine Di receptor. An early study of the Di receptor

expressed in endogenous opossum kidney cells suggested that PKA activity was required for

intemalization (Bates et al., 1993). However, in a recent study, when al1 PKA sites were

substituted to alanine, intemaiization was found to be unaffected in the rat Dl receptor (Jiang

and Sibley, 1999). We have determined that three residues in the distal portion of the

carboxyl tail (Thr446, Thr439, and Ser431) are critical for intemaiization of the human

dopamine Di receptor. Since these residues do not represent PKA phosphorylation sites, we

also conclude that P U phosphorylation does not appear to be important in regulating

internalization of the dopamine Di receptor.

Previous evidence has suggested that desensitization and internalization of the

dopamine Di receptor are temporally distinct mechanisms (Barton and Sibley, 1990; Bates et

al., 1993; Ng et al., 1995). In addition, both processes were shown to be biochemically

distinct mechanisms as various inhibitors of intemalization did not affect desensitization (Ng

et al., 1995). In this study, we have determined that the sites regulating desensitization and

intemalization are separate and distinct in the carboxyl tail, and that both mechanisms can act

independent of each other. In some GPCRs, such as the p and 6 receptors, desensitization

and intemalization are mediated by cornmon sites in the carboxyl tail (Pak et al., 1999;

Hasbi, 2000). However, in GPCRs such as the B2AR (Bouvier et al., l988), m2mAChR

(Pals-Rylaarsdam and Hosey, 1997), N-formyl peptide (Maestes et al.. 1999), and CB 1

cannabinoid (Jin et al., 1999) receptors, sites for desensitization and intemalization are

distinct.

In summary, the resuits presented here have identified two separate and distinct sites

in the carboxyl tail consisting of specific serine and threonine residues that mediate

desensitization and intemalization of the human dopamine Dl receptor. We also provide

evidence that PKA phosphorylation does not likely play a significant role in rapid agonist

induced desensitization or intemalization, and that ORK2 may be the predominant regulator

of rapid agonist induced desensitization.

4.2 Future Studies

1. Role of phosphorylation in desensitization and intemalization of the dopamine Di receptor.

In the light of results obtained by Tiberi et al (Tiben et al., 1996), it would be

interesting to study the correlation of phosphorylation in the process of desensitization and

intemalization in the dopamine Di receptor. Mthough we have s h o w in a previous study

that phosphorylation was associated with desensitization (Ng et al., 1994), expenments

conducted with our desensitization and intemalization deficient mutants would M e r

characterize the role of phosphorylation in both regulatory processes. Specifically,

phosphoamino analysis studies examining phosphorylation of senne and threonine residues

in our mutant receptors would be helpful to darifi the role of phosphorylation of Thr360 in

the process of desensitization. Furthemore, phosphorylation studies rnay help in claribing

our hypothesis that sequentiai phosphorylation may occur on residues Thr360 and Ser362.

2. Role of GRK2 in desensitization of the dopamine Di receptor.

Since the residues that we have identified as being essential in the desensitization

response (Thr360, and possibly Ser362) are localized on the carboxyl side of an acidic

residue, there is a strong possibility that GRK2 rnay be the kinase responsible for mediating

homologous desensitization in the dopamine Di receptor. In order to test this hypothesis, a

substitution mutant replacing the glutamic acid residue at position 359 with alanine would

remove the GRK? binding site, and thus the role of GRIU may be clearly assessed.

Aitematively, the dominant negative mutant of GRK2 could be coexpressed with the Dl WT

constmct in CHO celis to characterize the role of GRKî in homologous desensitization.

3. Role of PKA in desensitization of the dopamine Di receptor.

In a few studies exarnining desensitization of the dopamine Di receptor, PKA has

been determhed to play an important role in heterologous desensitization (Jiang and Sibley,

1999; Zhou et al., 1991). Since our studies thus far have only been concemed with rapid

agonist induced homologous desensitization, it would be interesting to examine the effects of

longer pretreatment times (1 to 24 hours) on wild type and mutant receptors to elucidate the

possible role of PKA mediated heterologous desensitization and to characterize and

differentiate the sites involved in both homologous and heterologous desensitintion in the

dopamine D 1 receptor.

4. Mechanism of desensitization and intemalization of dopamine Ds receptor.

The dopamine Di and Ds receptors both belong to the same "Di-like" subfamiliy of

dopamine receptors since they share similar functional and structural characteristics (-60%

sequence homology). However, the majority of research has been focused on the dopamine

Di receptor, and presently not much is known about the sites involved in the regulatory

processes of desensitization and intemalization in the dopamine D5 receptor. The two sites

and surrounding arnino acid residues that we have found to be essential in regulating

homologous desensitization of the dopamine D 1 receptor (EDSIN) are almost perfectl y

conserved in the same region of the carboxyl tail of the dopamine Ds receptor (ETVISN).

Therefore, it would be interesting to see if the same residues involved in desensitization of

the dopamine Di receptor are conserved in the dopamine Ds receptor. Similarly, two of the

three residues that we have identified in the distal carboxyl tail regulating intemalization of

the dopamine Di receptor (Thr439 and Ser43 1) are conserved in the dopamine D5 receptor.

Thus, it would be interesting to test whether sites for intemalization are aiso conserved

between the D i-like receptors.

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