Hormone Receptors on the Plasma Membrane

Characteristics of Receptors in GeneralFive Groups of Membrane-Bound ReceptorsThe G Protein-Coupled Receptor Superfamily

Signal Transduction through Cyclic AMPSignal Transduction through Phospholipase C

Role of CalciumRole of Protein Kinase C

General Characteristics of Receptors

• Receptors bind hormones, resulting in a biological response

• All receptors exhibit general characteristics:

- Specific Binding (structural and steric specificity)

- High Affinity (at physiological concentrations)

- Saturation (limited, finite # of binding sites)

- Signal Transduction (early chem event must occur)

- Cell Specificity (in accordance with target organ specificity).

Specific Binding• A receptor will only bind (recognize) a certain

hormone, or closely related hormones.

LH

hCG

FSH

LH Receptors

LH

hCG

FSH

Receptors Have High Affinity• In the bloodstream, there are thousands of different

peptides.• Hormones are present in very small quantities

(nanogram or picogram). • Receptors must therefore be very sensitive to the

presence of a hormone (they must be able to bind the hormone even if it is present in low amounts).

• Thus, they have high affinity (ability to bind at low hormone concentrations).

Analysis of Receptor Binding Sites

Na- I + hCG125

-hCGI 125

TRACER

TESTIS

SeminiferousTubules

Leydig/InterstitialCells

RT O/N

WASH, PBS

Centrifuge

lactoperoxidasemethod

Count PelletCPM

• Receptor must possessReceptor must possess structural and steric specificitystructural and steric specificity for a for a hormone and for its close analogs as well.hormone and for its close analogs as well.

• Receptors are Receptors are saturable and limitedsaturable and limited (i.e. there is a finite number of (i.e. there is a finite number of binding sites). binding sites).

• Hormone-receptor binding is Hormone-receptor binding is cell specificcell specific in accordance with target in accordance with target organ specificity.organ specificity.

• Receptor must possess aReceptor must possess a high affinity for the hormone at high affinity for the hormone at physiological concentrationsphysiological concentrations. .

•Once a hormone binds to the receptor, some recognizable Once a hormone binds to the receptor, some recognizable early early chemical eventchemical event must occur. must occur.

Criteria for hormone-mediated eventsCriteria for hormone-mediated events

• Affinity: The tenacity by which a drug binds to its receptor.– Discussion: a very lipid soluble drug may have irreversible effects;

is this high-affinity or merely a non-specific effect?

• Intrinsic activity: Relative maximal effect of a drug in a particular tissue preparation when compared to the natural, endogenous ligand.– Full agonist – IA = 1 (*equal to the endogenous ligand)– Antagonist – IA = 0– Partial agonist – IA = 0~1 (*produces less than the maximal

response, but with maximal binding to receptors.)

• Intrinsic efficacy: a drugs ability to bind a receptor and elicit a functional response– A measure of the formation of a drug-receptor complex.

• Potency: ability of a drug to cause a measured functional change.

Receptors have two major properties: Recognition and Transduction Recognition: The receptor protein must exist in a conformational state that allows for recognition and binding of a compound and must satisfy the following criteria:

•Saturability – receptors exists in finite numbers.

•Reversibility – binding must occur non-covalently due to weak intermolecular forces (H-bonding, van der Waal forces).

•Stereoselectivity – receptors should recognize only one of the naturally occurring optical isomers (+ or -, d or l, or S or R).

•Agonist specificity – structurally related drugs should bind well, while physically dissimilar compounds should bind poorly.

•Tissue specificity – binding should occur in tissues known to be sensitive to the endogenous ligand. Binding should occur at physiologically relevant concentrations.

The failure of a drug to satisfy any of these conditions indicates non-specific binding to proteins or

phospholipids in places like blood or plasma membrane components.

Receptors have two major properties: Recognition and Transduction Transduction: The second property of a receptor is that the binding of an agonist must be transduced into some kind of functional response (biological or physiological).

Different receptor types are linked to effector systems either directly or through simple or more-complex intermediate signal amplification systems. Some examples are:

• Ligand-gated ion channels – nicotinic Ach receptors• Single-transmembrane receptors – RTKs like insulin or EGF receptors • 7-transmembrane GPCRs – opioid receptors• Soluble steroid hormones – estrogen receptor

Predicting whether a drug will cause a response in a particular tissue

Factors involving the equilibrium of a drug at a receptor.• Limited diffusion• Metabolism• Entrapment in proteins, fat, or blood.Response depends of what the receptor is connected to.• Effector type• Need for any allosteric co-factors – THB on tyrosine hydroxylase. • Direct receptor modification – phosphorylation

Receptor theory and receptor binding.

Must obey the Law of Mass Action and follow basic laws of thermodynamics.• Primary assumption – a single ligand is binding to a homogeneous population of receptors

NH+3

COO-

• kon = # of binding events/time (Rate of association) = [ligand] [receptor] kon = M-1 min-1

• koff = # of dissociation events/time (Rate of dissociation) = [ligand receptor] koff = min-1

• Binding occurs when ligand and receptor collide with the proper orientation and energy.

• Interaction is reversible.• Rate of formation [L] + [R] or dissociation [LR] depends

solely on the number of receptors, the concentration of ligand, and the rate constants kon and koff.

  kon/k1

[ligand] + [receptor] [ligand receptor]

koff/k2

•At equilibrium, the rate of formation equals that of dissociation so that:

[L] [R] kon = [LR] koff

KD = k2/k1 = [L][R] [LR]

*this ratio is the equilibrium dissociation constant or KD.

KD is expressed in molar units (M/L) and expresses the affinity of a drug for a particular receptor.• KD is an inverse measure of receptor affinity.• KD = [L] which produces 50% receptor occupancy

• Once bound, ligand and receptor remain bound for a random time interval.

• The probability of dissociation is the same at any point after association.

• Once dissociated, ligand and receptor should be unchanged.

• If either is physically modified, the law of mass action does not apply (receptor phosphorylation)

• Ligands should be recyclable.

Receptor occupancy, activation of target cell Receptor occupancy, activation of target cell responses, kinetics of bindingresponses, kinetics of binding

•Activation of membrane receptors and Activation of membrane receptors and target cell responses is target cell responses is proportional to the proportional to the degree of receptor occupancy.degree of receptor occupancy.

•However, the hormone concentration at However, the hormone concentration at which which half of the receptorshalf of the receptors is occupied by a is occupied by a ligand (Kligand (K

dd) is often lower than the ) is often lower than the

concentration required to elicit a concentration required to elicit a half-half-maximal biological responsemaximal biological response (ED(ED

5050))

Receptor Fractional Occupancy

F.O. = [LR]____ = [LR]___ *now substitute the KD equation. [Total Receptor] [Rf] + [LR]

[R] = KD • [LR] F.O. = [Ligand]

[L] [Ligand] + KD

Use the following numbers: [L] = KD= 50% F.O.

[L] = 0.5 KD = 30% F.O.

[L] = 10x KD = 90%+ F.O.

[L] = 0= 0% F.O.

100

50

0 Ligand Concentration

Fra

ctio

nal

Occ

upan

cy

Assumptions of the law of mass action.

• All receptors are equally accessible to ligand. • No partial binding occurs; receptors are either

free of ligand or bound with ligand.• Ligand is nor altered by binding• Binding is reversible• Different affinity states?????

Studies of receptor number and function• We can directly measure the number (or density) of receptors in the LR complex.• Ligand is radiolabeled (125I, 35S. or 3H). Selection of proper radioligand:

– Agonist vs. antagonist (sodium insensitive)– Higher affinity for antagonists– Longer to steady state binding

• Saturation binding curve-occurs at steady state conditions (equilibrium is theoretical only).

• Demonstrates the importance of saturability for any selective ligand.• Provides information on receptor density and ligand affinity and selectivity.

Scatchard transformation

• Y-axis is Bound/Free (total radioligand-bound)• X-axis Bound (pmol/mg protein)• Straight lines are easier to interpret.

• The amount of drug bound at any time is solely determined by:– the number of receptors– the concentration of ligand added– the affinity of the drug for its receptor.

• Binding of drug to receptor is essentially the same as drug to enzyme as defined by the Michelis-Menten Equation.

Thus, to reiterate…,Calculating Affinity• Take a cell which has the receptor on it (ie, granulosa cells

with FSH receptor).• Prepare membrane homogenate.• Incubate membranes with increasing amounts of labeled

hormone.• Determine how much binding of hormone occurs at each

dose.• Dissociation constant (Kd) is dose where 50% of maximal

binding occurs.

% b

indi

ng

Dose of Hormone10 30 100 300 1000

100

50Kd

0

Thus, to reiterate…,Saturation• There is a finite limit to the numbers of receptors

which can be on a cell.• Therefore, there’s a maximum amount of binding

which can occur (all receptors are saturated)

% b

indi

ng

Dose of Hormone10 30 100 300 1000

100

50

0

saturation

Biological Response to Ligand Binding

• A receptor not only binds hormone; there must also be a biological response from the cell (e.g., increased transcription, phosphorylation, etc.)

• This is also called “signal transduction”.• The biological response can result from:

- the ligand itself (e.g., Fe, LDL)- the receptor (e.g., increased cyclic AMP, transcription, phosphorylation)

Determinants of Biological Response

• The strength of the response of the cell to the hormone depends upon three factors:1) the amount of hormone present to bind to the receptors2) the numbers of receptors on the cell3) the affinity of the receptor for the hormone (how much hormone do you need to get receptor binding?)

Regulation of Receptor Number:the Phenomenon of Spare Receptors

• We know that cells typically have about 20 times more receptors than is needed for a maximal biological response.

• A complete biological response occurs after binding to only 5% of the receptors on a cell.

• This remaining 95% are called “spare receptors”.• Why have spare receptors?

BiologicalResponse(% max)

Receptor Occupancy (%)

100

50

0

0 25 50 75 100

Effect of Decreasing Receptor Number in a Cell Which Does Not Have Spare Receptors

• No change in affinity.• Decrease in maximal biological response.

Hormone Concentration (M)

% o

f re

cept

ors

occu

pied

% maximalresponse

100

75

50

25

0

100

75

50

25

010-11 10-10 10-9

Kd

Effect of Decreasing Receptor Number in a Cell Which Has Spare Receptors

• In this example, assume you need 5000 receptors occupied for maximal biological response.

• If you start w/ 20,000 receptors occupied, decreasing receptor number does not change receptor affinity (Kd).

Number of receptors bound

20,000

15,000

10,000

5,000

0

0

50

75

% R

eduction in Receptor N

umber

Kd

# Receptorsfor maximalbiological response

Hormone Concentration (M)

Effect of Decreasing Receptor Number in a Cell Which Has Spare Receptors

• No change in maximal biological response (unless you go below 5000 receptors/cell).

• Requires higher dose of hormone to obtain maximal response.

Hormone Concentration (M)

10-11 10-10 10-9 10-8 10-7

% MaximalBiological Response

100

80

60

40

20

0

20,000 R/cell

10,000 5000

2500

Hormones, Agonists, and Antagonists

• Substances other than a receptors normal hormone may exist (or be made).

• Each substance that binds to a receptor has an intrinsic activity (related to the resulting biological response).

Substance Intrinsic Activityhormone 100% (by definition)superagonist >100%partial agonist <100%antagonist 0%

Antagonists

• If a substance binds to a receptor but does not cause a biological response, it blocks the natural hormone from binding to it.

• Example: RU486 binds to the progesterone receptor, but does not cause a response.

hormone bindingdomain

transcriptional domain

PR

RU486

progesterone

DNA

Regulation of Biological Response at the Receptor Level

• The strength of signal transduction can be regulated at the level of the receptor by several mechanisms:1) change the affinity of the receptor (make it bind more difficult or easier to bind hormone). This usually doesn’t happen.2) change the numbers of receptors on the cell (common)

- internalization and degradation of receptors- occupancy of receptors (prevents hormone binding)- gene expression/synthesis

3) change the signal transducing ability of the receptor (usually for rapid regulation)

- phosphorylation (usually inhibits receptor activity)- G protein uncoupling (stay tuned)

Plasma Membrane-Bound Receptors

• Recall that peptide hormones are polar, and cannot readily cross the cell membrane.

• Therefore, their receptors must be on the outside surface of the cell.

Types of Plasma Membrane Receptors

There are five basic types of membrane bound receptors (grouped by signal transduction method):

• tyrosine kinase receptors• receptors that are closely linked to tyrosine kinases• receptors with guanylyl cyclase activity• receptors that serve as transporters• G protein-coupled receptors

Signal Transduction by Plasma Membrane Receptors

1) Receptors with intrinsic tyrosine kinase activity. Binding of hormone to the receptor induces the phosphorylating activity of the receptor.Example: Insulin receptor

plasma membrane

extracellular domains(ligand binding)

tyrosine phosphorylase domains

phosphorylated enzyme (altered activity)

Signal Transduction by Plasma Membrane Receptors

2) Receptors that are closely linked to tyrosine kinases. These activate cytoplasmic tyrosine kinase enzymes.

Example: Growth Hormone Receptor

associatedtyrosinekinase

phosphorylated enzyme

Signal Transduction by Plasma Membrane Receptors

3) Receptors with Guanylyl Cyclase Activity. Binding to the receptor activates guanylate cyclase region of the receptor, causing conversion of GTP to cyclic GMP.Example: Atrial Natriuretic Peptide Receptor

guanylate cyclaseGTP

cyclic GMP protein kinase Gion channels

phosphodiesterase levels

Signal Transduction by Plasma Membrane Receptors

4) Receptors that serve as transporters. These move the ligand inside the cell, where they have an effect. (Not typical for hormones).

Example: Iron, transported by transferrin receptor

iron

transferrin

Signal Transduction by Plasma Membrane Receptors

5) G Protein-coupled receptors. (The largest group!) These receptors are coupled with guanine nucleotide-binding proteins (G proteins), which activate various signaling pathways.

Examples: Receptors for LH, FSH, TSH, GnRH, dopamine, serotonin, glutamine, parathyroid hormone, interleukins, etc.

G Protein-Coupled Receptor Superfamily• Common structural features:

- an amino terminus hormone-binding domain- seven hydrophobic transmembrane domains- a carboxyl terminus, intracellular domain

-NH2

COOH-

G Protein-Coupled Receptor Superfamily

• Common functional features:- binding to the receptor activates a G protein- each receptor is associated with a specific type of G protein- each G protein type has different functions:

- Gs: stimulates cyclic AMP- Gi: inhibits cyclic AMP- Go activates phospholipase C

How G Proteins Work

• G proteins are composed of three subunits: alpha (), beta (), and gamma ().

• In the inactive state, the three subunits are associated with the receptor at the plasma membrane. The alpha subunit has a guanosine diphosphate attached.

GDP

NH2

COOH

How G Proteins Work (cont.)• When hormone binds, the GDP leaves the alpha

subunit, and is replaced by a GTP. • The alpha subunit then goes off to activate signaling

pathways.

GDP

NH2

COOH

hormone

GTP

NH2

COOH

hormone

GTP

signal pathways

How G Proteins Work (cont.)• After activating the signal pathway, the GTP is

hydrolyzed into GDP, and the alpha subunit returns to the beta and gamma subunits at the membrane.

GTP

P

GDP

NH2

COOH

G Protein Stimulation of Cyclic AMP

• Binding of many hormones to their receptors results in the stimulation of the second messenger, cyclic AMP.

• G Protein involved: Gs

G Protein Stimulation of Cyclic AMP

GTP

a

b

ATP

cAMP

a

b

GTP GTPase

GDP

AC LHR

Gs

GTPGDP

a

bLH

Receptor-G protein InteractionsHow are receptor-G protein interactions measured?

• Ligand-binding assays:

High-affinity Low- affinityRG(GDP)

GDP GTPγS

R + G(GTP-δ-S)

Without GTP, both high- and low-affinity states are measured.With GTP and Mg2+, only low-affinity state is measured, becauseAgonist binding rapidly induces change from high- to low-affinity.

How Else Is Cyclic AMP Regulated?

• In addition to regulating the production of cyclic AMP, there is also regulation of its degradation.

• Degradation of cyclic AMP is by phosphodiesterases, which break down cyclic AMP into 5’-AMP

• Inhibitors of phosphodiesterases prolong activation of the cyclic AMP system.

Regulation of cAMP Levels by Phosphodiesterases

a

b

GTP

ATP

cAMP

5’-AMP

PDEs PDE InhibitorX (-)

Protein Kinase A Pathway• Increased cyclic AMP activates protein kinase A.• Protein kinase A (PKA) is composed of two regulatory

subunits and two catalytic subunits.• Binding of cyclic AMP to the regulatory subunits frees the

catalytic subunits, which have kinase activity.• PKA uses ATP to phosphorylate specific enzymes in the

cell, influencing their activity.

regulatory

catalytic

cyclic AMP

Effects of Cyclic AMP-dependent PKA on Gene Transcription

• Cyclic AMP regulates the transcription of many genes by increasing PKA and causing the phosphorylation of the Cyclic AMP Response Element Binding Protein (CREB).

• CREB is a transcription factor which binds to a consensus cyclic AMP-response element (CRE) on the 5’-flanking region of many genes.

intron

exon

CRE ERE TATA BOX

CAT

5’-flanking region

CREB• The CRE has a palindromic consensus sequence:

5’-TGACGTCA-3’3’-ACTGCAGT-5’

• Removing the CRE from cyclic AMP-responsive genes causes a loss of regulation by cyclic AMP.

• Adding a CRE to non-cyclic AMP-responsive genes confers responsiveness to cyclic AMP.

• CREB binds to the CRE as a dimer. • Phosphorylation increases the dimerization of CREB,

resulting in increased transcriptional activity.• Cells deficient in PKA cannot transcribe genes via the

CRE (phosphorylation of CREB is required)

Terminology: CRE(cyclic AMP response element); CREB: CRE binding protein; CBP: CREB binding protein

CREM• More recently, Cyclic AMP-Response Element

Modulators (CREMs) has been identified.• Structurally related to CREB.• Four isoforms exist, all the product of a single gene.• Three isoforms block cyclic AMP-dependent gene

transcription.• One isoform is an agonist for the CRE.• Relative expression of isoforms is regulated in the

testis:- immature sperm cells express antagonist form- maturing sperm cells express agonist form

Summary of Cyclic-AMP Signaling

hormone binds receptor

CREB initiates transcription

Gs activates adenylyl cyclase

adenylyl cyclase produces cyclic AMP

cyclic AMP activates PKA

PKA phosphorylates CREB

cAMP-GEFs

Ras/Rap

PKB/SgK

Why Make it So Complicated?

• Many steps = many places where regulation can take place

• In addition, at several steps the signal is AMPLIFIED. For example, activation of adenylyl cyclase produces several cyclic AMP molecules. Each activated PKA can phosphorylate many CREB molecules.

Influence of G Proteins on Phospholipase C

• Receptors coupled to Go activate phospholipase C, which hydrolyzes an inositol phospholipid into inositol triphosphate (IP3) and diacylglycerol (DAG).

• IP3 and DAG each activate separate signaling pathways:IP3 activates a Ca2+ pathway.

DAG activates the protein kinase C pathway.

Actions of IP3 and Ca2+

• IP3 causes release of intracellular Ca2+ stores (ER) and allows extracellular Ca2+ to enter the cell.

• Result: increased free cytoplasmic Ca2+.• Ca2+ can then bind to calmodulin, activating it.• Calmodulin influences the activity of other enzymes,

including kinases.

Go IP3 Ca2+ calmodulin

Gq signaling pathways, Ca2+, IP3, PKC

Actions of DAG and PKC

• DAG activates calcium-dependent Protein Kinase C (PKC), by increasing PKC’s affinity for calcium.

• PKC phosphorylates a number of enzymes at serine/threonine residues, influencing their activity.

• PKC activates the transcription factor AP-1:- jun/fos heterodimer- binds to AP-1 sites on the 5’-flanking region of

genes (similar to CRE, but different!)- binding influences gene transcription

Summary of Go Signaling

Go IP3 calcium calmodulin

DAG

PKC

AP-1

gene transcription

enzyme activity

enzyme activity

Next Lecture…..

Intracellular Hormone Receptors