Evolution of the complexity of our perception of GPCR-mediated signaling from a pathway (A) to matrices (B) to networks (C). Receptors (R) interact with one or more G proteins (G) linked to one or more effectors (E), which then engage in effector cross-talk and regulation of cellular function at the surface membrane, within the cytosol, and among multiple cellular compartments, including the nucleus. Molecular Interventions 4:326-336, (2004)

3. Receptor activation…

Diversity…

Models of the Integration ofcomplex GPCRsignallingnetworks

The three subfamilies of GPCRs are depicted with examples of their endogenous agonists. The binding modes of the orthosteric ligands for each receptor type are depicted by a green rectangle. The GPCR signals either by coupling to heterotrimeric G-proteins consisting of   and     subunits (which trigger a wide range of metabolic cascades and ion channel activities) or by direct association with effector molecules. AC, adenylyl cyclase; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; PLC, phospholipase C; IP3,

inositol-3,4,5-tris-phosphate; DAG, diacylglycerol.

4. Functional regions within GPCRs: 4.1 G-protein interacting domains4.2 ligand binding domain

4.Functional regions within GPCRs

4.1 G-protein coupling domain

Using GPCR chimeras to identify Functional domains within receptors

Mol Pharmacol. 2004 65:1323-32.

Two different dopaminereceptors bind sameagonist but they couple to different signallingcascade

Binding properties of tritiated d[Cha4]AVP. Membranes from COS cells, expressing human V1b receptors (1-2 µg

protein/assay), were incubated for 1 h at 37 °C with increasing amounts of tritiated d[Cha4]AVP (a) or with 1-2 nm of the radioligand with, or without (control), increasing amounts of unlabelled selective vasopressin antagonists (SR149415, SR49059, SR121463) (b). Journal of Neuroendocrinology  16, 356-361

4. Functional regions within GPCRs

Pharmacological characterization of GPCRs

(A) Saturation isotherms:Determine the number of high affinity binding sites on the cell surface

(B) Displacement analysis:Determine the affinity of the GPCR for different agonists and antagonists

Structure, ligand binding properties, and functional profile of wild type and mutant V1 /V2 vasopressin receptors. [ H]AVP saturation binding studies were carried out. Km and Bmax values are given as means ± S.E. of three independent experiments (PI, stimulation of PI hydrolysis; AC, stimulation of adenylyl cyclase). The symbols are defined as the percentage of maximum PI and cAMP responses induced by the wild type V1a and V2 receptor, respectively:

++++, 90-100%; +++, 80-90%; +, 10-30%; -, no significant response. J. Biol. Chem. 1996;271:8772

4.1 G-protein coupling domain…

Analysis of vasopressin receptor chimeras

AVP-induced cAMP accumulation mediated by wild type V1a, V2 and hybrid V1a/V2 vasopressin receptors. Transfected COS-7 cells transiently expressing the different receptors were incubated in 6-well plates for 1 h at 37 °C with the indicated AVP concentrations, and the resulting increases in intracellular cAMP levels were determined. The data are presented as fold increase in cAMP above basal levels in the absence of AVP. Each curve is representative of three independent experiments, each carried out in duplicate.

4.1 G-protein coupling domain…

Analysis of the potency of Vasopressin chimeras to stimulatecAMP accumulation (Gs response)

J. Biol. Chem. 1996;271:8772

Figure 3: AVP-induced stimulation of PI hydrolysis mediated by wild type V1a, V2 and hybrid V1a/V2 vasopressin receptors. Transfected COS-7 cells transiently expressing the various receptors were incubated in 6-well plates for 1 h at 37 °C with the indicated AVP concentrations, and the resulting increases in intracellular IP levels were determined. The data are presented as fold increase in IP above basal levels in the absence of AVP. Each curve is representative of three independent experiments, each carried out in duplicate.

4.1 G-protein coupling domain…

Analysis of the potency of Vasopressin chimeras to stimulateInositol-Phosphate (PI or lipid hydrolysis) accumulation (Gq response)

J. Biol. Chem. 1996;271:8772

Localization of FSHR Mutations and Functional Characterization of Mutant Receptors To characterize mutant FSHRs (A), COS-7 cells were transfected with the different constructs, and cAMP accumulation assays were performed as outlined in Materials and Methods. (B) Data obtained from three independent experiments, each performed in triplicate, are presented as -fold of basal cAMP levels of the FSHR(wt) (means ± SEM) .

4.1 G-protein coupling domain…

Molecular Endocrinology 1999, 13: 181-190

Fine structure mapping of G-protein interacting regionusing deletion analysis

Functional Analysis of Conserved Basic Amino Acids within the LHR i2 Loop by Alanine Scanning Mutagenesis. To identify a cationic contact site for D564, all conserved basic amino acids within the i2 loop were replaced by A using a site-directed mutagenesis approach (A). The various LHR mutants were expressed in COS-7 cells, and cAMP accumulation assays were performed (B). Basal (open bars) and agonist-induced cAMP levels are presented as means ± SEM of two independent experiments, each carried out in triplicate.

4.1 G-protein coupling domain…

Molecular Endocrinology 1999, 13: 181-190

Fine structure mapping of G-protein interacting regionusing alanine mutagenesis

Schematic models of ligand-receptor complexes for structurally diverse ligands interacting with GPCRs.

4. Functional regions within GPCRs4.2 Ligand binding domain 3-D models

Mol Pharmacol. 2004 Jun;65(6):1323-32.

4. Functional regions within GPCRs

4.2 Ligand binding domain

Two different receptors onebinds FSH and the other bindsLH

Using GPCR chimeras to identify Functional domains within receptors

A wide repertoire of physiological effects of CCK and/or gastrin which mediated CCK1 and/or CCK2 receptors has been identified. Among actions of CCK which are mediated by the CCK1R, control of satiety, gallbladder contraction, pancreatic exocrine secretion, gastric pepsinogen and leptin secretions, gastric emptying and gut motility are the best known. Actions of CCK which occur through the CCK2R include modulation of anxiety and pain perception; these actions involve CCK2 receptors of the central nervous system. The wide spectrum of biological functions regulated by the CCK1R and CCK2R makes them candidate targets for a therapeutic approach in a number of diseases. This led a number of academic and pharmaceutical research groups to design specific and highly potent agonists and antagonists for those receptors.As for other G-protein-coupled receptors, the cloning of CCK receptor cDNAs and genes have stimulated generation of new biological models (transgenic animals, genetically modified cells   ) and opened new avenues for research in the physiology, pathophysiology, molecular pharmacology and structure-function relationships of the receptors. Among these themes, delineation of CCK receptor binding sites represents a prerequisite for the understanding of the molecular basis for ligand recognition, partial agonism, ligand-induced traffiking of receptor signalling etc.

Structure of Cholecystokinin R eceptor Binding Sites and Mechanism of Activation/Inactivation by Agonists/Antagonists

Pharmacology & Toxicology 2002 91;313

4.2 Ligand binding domain…

Pharmacology & Toxicology 91:313, 2002

Criteria used to identify residues of CCKR binding site in an example of analysis of a putative ionic interaction between a positively charged aminoacid Y+ of the receptor binding site, and its negatively charged partner residues X in the ligand.

4.2 Ligand binding domain…

Schematic representations of the CCK1R with residues of the CCK binding site.

Pharmacology & Toxicology 91:313, 2002

Localizations of residues in biogenic amine receptors that are important for ligand binding (gray color) within a membrane topology model of the rat 5HT2A receptor (N terminus and most

of C terminus are not shown).

Pharmacol Ther. 2004 Jul;103(1):21-80

4.2 Ligand binding domain…Detailed understanding of residues involved in ligandbinding allows detailed ligand/Receptor modeling and permits the application of rational drug design to develop new agonistsand antagonist that can be usedas receptor specific drugs.

Generic diagram of sequence regions involved in post-translational modification and modulation of functions. Summary of findings based on receptor mutagenesis and construction of receptor chimeras for a variety of GPCR.

Molecular Interventions 4:326-336, (2004)

4. Functional regions within GPCRs

Summary

(a) Concentration-dependent increase in cAMP in response to AM (EC50 3.2±0.7 nM, filled squares) and CGRP at 1 µM

(no effect, filled triangle) in Rat-2 fibroblasts. (b) Concentration-dependent attenuation of AM cAMP

responses following preexposure to AM. Rat-2 cells were incubated with SFM or various concentrations (as indicated) of AM for 1 h followed by a washout period and a restimulation of 15 min with 10 nM AM Data are expressed as a percentage of the response in cells not preincubated with AM, that is, nondesensitised controls. Cells incubated with AM for 1 h were less able to elevate cAMP upon restimulation with AM when compared with cells incubated with SFM for the same time.

(c) The time course for AM receptor desensitisation in Rat-2 cells was determined by varying the preincubation time period (as indicated) of exposure to 100 nM AM. Cells were then washed and restimulated, and the cAMP response was measured. The cAMP response elicited by 10 nM AM was attenuated in a time-dependent manner. Cells preexposed to 100 nM AM for 1 min were capable of only 59.1±7% of the response of cells not preincubated with AM. After 2 h preincubation with 100 nM AM, the level of cAMP response was 19.3±2.2% of control stimulation.

Regul Pept. 2003;112:139

5. Inactivation of GPCR signalling-Tachyphylaxis of the adrenomedulin (AM) receptor

5. Inactivation of GPCR signalling…

GPCR Kinase and -arrestin-dependent desensitization and internalization of GPCRs

Fergusson, 2001

Translocation of ß-arrestin 2-GFP to the ß2-adrenergic receptor (ß2AR). HEK 293

cells stably overexpressing the ß2AR were transiently transfected with ß-arrestin 2-

GFP. The distribution of ß-arrestin 2-GFP fluorescence was visualized by confocal microscopy before (−Iso) and after a 5 min treatment with isoproterenol (+Iso; is a ßAR agonist, 10−8, 10−6 M) at 37 °C. Before agonist-stimulation, ß-arrestin 2-GFP is uniformly distributed throughout the cytosol. Upon agonist addition, ß-arrestin 2-GFP translocates from the cytosol to the plasma membrane where it is found colocalizing with the receptor in punctuated areas of the plasma membrane.

Prog Neurobiol. 2002 Feb;66(2):61-79

5. Inactivation of GPCR signaling…

Visulialization of GPCR internalization following receptor stimulation

5. Inactivation of GPCR signaling…Model depicting the regulation of β2AR internalization, trafficking, dephosphorylation, and recycling

Fergusson, 2002

Fig. 8. Pathways involved in desensitization and resensitization of GPCR signaling. Typically activation of a GPCR leads to (1) activation and inhibition of specific signaling pathways in the cell, (2) short-term desensitization mediated by phosphorylation of GPCRs by GRKs followed by β-arrestin binding to GPCRs that uncouple the receptor at the plasma membrane from the G-protein, (3) endocytosis of the receptor, followed by postendocytic sorting of the receptor either (4) back to the plasma membrane or (5) to lysosomes for degradation.

Molecular Interventions 4:326-336, (2004)

5. Inactivation of GPCR signaling…Model depicting the regulation of GPCR internalization,trafficking, dephosphorylation, and recycling.

Schematic representation of the domain architecture for GRK1-GRK7. The amino-terminal GPCR-binding domain of GRK1-GRK7 contains a conserved RGS domain. The plasma membrane targeting of each of the GRKs is mediated by distinct mechanisms that involves their carboxyl-terminal domains. GRK1 and GRK7 are farnesylated at CAAX motifs in their carboxyl termini. The carboxyl-terminal domains of GRK2 and GRK3 contain a βγ -subunit binding domain that exhibits sequence homology to a pleckstrin homology domain. The GRK5 carboxyl-terminal domain contains a stretch of 46 basic amino acids that mediate plasma membrane phospholipid interactions

Pharmacol Rev. 2001;53:1-24.

5. Inactivation of GPCR signaling…Structure of GRKs

Nature Reviews Molecular Cell Biology 3; 639-650 (2002)

5. Inactivation of GPCR signaling…

Extracellular Environment

GDP GTP

N

C

+Pi

RGS

Downstream effectors

5. Inactivation of GPCR signaling…

Regulators of G-protein Signalling (RGS) negatively regulate GPCR signalling

Wieland and Mittmann 2003

5. Inactivation of GPCR signaling…

Prototypical RGS members

Wieland and Mittmann 2003

5. Inactivation of GPCR signaling…Up-regulation of RGS4 desensitises endothelin-1 signalling in failing human myocardium

Altered RGS expression leads to changes in GPCR signalling.

In Congestive Hearts or Sepsis

Also see increases in RGS1, 3, 16

Schematic representation of the wild-type human M2 and rat M3, the fragments M2-

trunc and M3-tail, and the mutants M3-short and M2 (Asn404 to Ser) muscarinic

receptors. The truncated fragment, M2-trunc, contains the amino-terminal domain,

the first five hydrophobic transmembrane regions and the initial portion (56 amino acids) of the i3 loop of the wild-type muscarinic M2 receptor. The M3-tail fragment

contains the final portion of the i3 loop (105 amino acids), the last two hydrophobic transmembrane regions, and the carboxy-terminal segment of the wild-type M3

muscarinic receptor. The short construct (M3-short) represents a receptor in which

196 amino acids of the i3 loop have been deleted; the remaining i3 loop is 43 amino acids long. The point mutant M2 (Asn404 to Ser) has the asparagine 404 replaced

with serine.

6. GPCR dimerization:The Split Receptor

Figure 1 | Role of homo- and heterodimerization in the transport of G-protein-coupled receptors.   When expressed alone, the GABABR1 (GBR1) receptor is retained as an immature protein in the endoplasmic reticulum (ER) of cells and never reaches the cell surface. By contrast, the GBR2 isoform is transported normally to the plasma membrane but is unable to bind GABA and thus to signal. When coexpressed, the two receptors are properly processed and transported to the cell surface as a stable dimer, where they act as a functional metabotropic GABAB receptor.

Nat Rev Neurosci. 2001 Apr;2(4):274-86

6. GPCR dimerization…

Molecular determinants of G-protein-coupled-receptor dimerization.   Distinct intermolecular interactions were found to be involved for various G-protein-coupled receptors. Covalent disulphide bonds were found to be important for the dimerization of the calcium-sensing and metabotropic glutamate receptors. A coiled-coil interaction involving the carboxyl tail of the GBR1 and GBR2 receptors is involved in the formation of their heterodimer. Finally, for monoamine receptors such as the β2-adrenergic and dopamine receptors, interactions between transmembrane helices were proposed to be involved.

6. GPCR dimerization…

Alternative three-dimensional models showing dimers of G-protein-coupled receptors.   Two models have been proposed for the general three-dimensional organization of G-protein-coupled-receptor dimers. a | First is the domain-swapping model in which each functional unit within the dimer is composed of the first five transmembrane domains of one polypeptide chain and the last two of the other. Such a model is useful to rationalize the functional complementation observed when mutant or chimeric receptors are coexpressed. b | Second is the contact model in which each polypeptide forms a receptor unit that touches the other through interactions involving transmembrane domains five and six.

6. GPCR dimerization…

Heterodimerization of CRLR and RAMP. HOMOTROPIC interactions between G-protein-coupled receptors (GPCRs) are not the only type of protein–protein interaction shown to influence their functional expression. Recently, a new class of membrane proteins that can interact with GPCRs and affect their activity profile has been identified. These new proteins were discovered while studying the expression of a complementary DNA that encoded a putative GPCR, which did not lead to the expression of a functional receptor. Specifically, a cDNA named calcitonin-receptor-like receptor (CRLR), which showed 55% overall identity to the calcitonin-receptor gene, was proposed to encode the receptor for the calcitonin-gene-related peptide (CGRP). However, various attempts to show that it was indeed the CGRP receptor failed because it was impossible to demonstrate any type of functional expression.

6. GPCR dimerization…

EMBO Rep. 2004; 5:30-4.

Potential roles of G-protein-coupled receptor (GPCR) dimerization during the GPCR life cycle.(1) In some cases, dimerization has been shown to have a primary role in receptor maturation and allows the correct transport of GPCRs from the endoplasmic reticulum (ER) to the cell surface. (2) Once at the plasma membrane, dimers might become the target for dynamic regulation by ligand binding. (3) It has been proposed that GPCR heterodimerization leads to both positive (+) and negative (-) ligand binding cooperativity, as well as (4) potentiating (+)/attenuating (-) signalling or changing G-protein selectivity. (5) Heterodimerization can promote the co-internalization of two receptors after the stimulation of only one protomer. Alternatively, the presence of a protomer that is resistant to agonist-promoted endocytosis, within a heterodimer, can inhibit the internalization of the complex. G, G protein; L, ligand.

6. GPCR dimerization…

Summary of studies on hetero-dimerization in different GPCR classes and the possible functional roles of GPCR dimerization

6. GPCR dimerization…

6. GPCR dimerization…

Summary of studies on hetero-dimerization in different GPCR classes and the possible functional roles of GPCR dimerization (cont’d)

The coupling of GPCRs with multiple G-proteins is selectively regulated at different levels. First, the nature of the response is dependent on the agonist used, which may selectively favour the coupling with a subset of G-proteins. In addition, when multiple couplings occur, several studies have demonstrated that the agonist elicited the responses with different potencies. Therefore, the involvement of multiple G-proteins is influenced by the concentration of agonist. Secondly, alterations in the expression or in the structure of the receptor have been shown to affect the coupling profile with multiple G-proteins. Thus, distinct coupling properties have been observed for related splice variants of a same receptor. Post-translational modifications, such as palmitoylation and phosphorylation, are also involved in the dynamic regulation of the G-protein coupling specificity. Many recent studies have shed light on the critical role played by GPCR-interacting proteins in determining the efficiency of coupling with distinct G-proteins. Finally, the availability of distinct G-proteins and the selective modulation of their expression, localisation, and activity contribute to determining the specificity of the intracellular signalling triggered after the receptor activation.

Pharmacol Ther. 2003 99:25-44

Summary of factors effectingGPCR signalling

Figure 1. HIV-1 fusion according to current models Env is composed of a surface subunit gp120 and transmembrane subunit gp41, which are non-covalently associated and then assembled as a trimer. The CD4 binding region of gp120 is exposed, but variable regions of gp120 screen important conserved structures involved in chemokine receptor interactions. Each gp41 molecule contains two alpha-helices that form a hairpin configuration, and the N-terminal hydrophobic fusion peptide is buried within the complex. gp120 binds to CD4 (A) and undergoes conformational changes that create or unmask the co-receptor binding site (B) and enable binding to the chemokine receptor (C). Structural changes are then induced in gp41 that extend the helical domains to form a ‘pre-hairpin intermediate’ (D). The hydrophobic fusion peptide inserts into the target cell membrane, causing gp41 to span between the virus and cell membranes. The gp41 helices then fold into a six-helix bundle, bringing together the N-terminal and C-terminal domains and thus the viral and cellular membranes (E). Contact between the membranes allows mixing of the outer leaflets followed by the development of a fusion pore (G). gp120 is omitted from panels F and G for the sake of clarity. Modified after Starr-Spires and Collman [6]. From:   Shaheen: Curr Opin Infect Dis, Volume 17(1).February 2004.7-16

7. Alternative functions of GPCRs

                       

                                                         

                           

                                                                        

                                       

The Nobel Prize in Physiology or Medicine 2004

"for their discoveries of odorant receptors and the organization of the olfactory system"

 

                    

      

                    

      

Richard Axel Linda B. Buck

    1/2 of the prize     1/2 of the prize

USA USA

Columbia University New York, NY, USA; Howard Hughes Medical Institute

Fred Hutchinson Cancer Research Center Seattle, WA, USA; Howard Hughes Medical Institute

b. 1946 b. 1947

B1. Odorant receptors

The sense of smell: genomics of vertebrate odorant receptors.

Olfactory receptor (OR) proteins interact with odorant molecules in the nose, initiating a neuronal response that triggers the perception of a smell. The OR family is one of the largest known mammalian gene families, with around 900 genes in human and 1500 in mouse. After discounting pseudogenes, the functional repertoire in mouse is more than three times larger than that of human. OR genes encode G-protein-coupled receptors containing seven transmembrane domains. ORs are arranged in clusters of up to 100 genes dispersed in 40-100 genomic locations. Each neuron in the olfactory epithelium expresses only one allele of one OR gene. The mechanism of gene choice is still unknown, but must involve locus, gene, and allele selection. The gene family has expanded mainly by tandem duplications, many of which have occurred since the divergence of the rodent and primate lineages. Interchromosomal segmental duplications including OR genes have also occurred, but more commonly in the human than the mouse family. As a result, many human OR genes have several possible mouse orthologs, and vice versa. Sequence and copy number polymorphisms in OR genes have been described, which may account for interindividual differences in odorant detection thresholds.

Hum Mol Genet. 2002,11:1153

B1. Odorant receptors…

Figure 32.2. The Main Nasal Epithelium. This region of the nose, which lies at the top of the nasal cavity, contains approximately 1 million sensory neurons. Nerve impulses generated by odorant molecules binding to receptors on the cilia travel from the sensory neurons to the olfactory bulb.

Biochemistry.

Berg, Jeremy M.; Tymoczko, John L.; and Stryer, Lubert

B1. Odorant receptors…

The olfactory systemThe olfactory epithelium contains millions of olfactory neurons, which send messages directly to the olfactory bulb of the brain.The olfactory receptor cells are the only neurons in the nervous system exposed directly to the external environment.

 Species differencesThe area of the olfactory epithelium (red) in dogs is some forty times larger than in humans. Mice – the species Axel and Buck studied – have about one thousand different odorant receptor types.Humans have a smaller number than mice; some of the genes have been lost during evolution. There are several millions of olfactory receptor cells in our olfactory epithelium.

B1. Odorant receptors…

                                                         

   

A large family of odorant receptorsRichard Axel and Linda Buck published their fundamental paper in 1991, in which they described the genes coding for a large family of odorant receptors.The odorant receptors are located on the olfactory receptor cells in the nasal cavity. Each olfactory receptor cell expresses only one type of odorant receptor, and each receptor can detect a limited number of odorant substances. 

 

The olfactory receptorEach receptor consists of a protein chain that traverses the cell membrane seven times.When an odorant substance attaches to an olfactory receptor, the shape of the receptor protein is altered, leading to a G protein activation.An electric signal is triggered in the olfactory receptor neuron and sent to the brain via nerve processes.

 

Small variationsAll odorant receptors are related proteins and differ only in some amino acid residues (indicated in green, blue and red).The subtle differences in the protein chains explain why the receptors are triggered by different odorant molecules.

(A) The combinatorial code of olfaction. Neurons expressing a given receptor can respond to more than one type of odorant (e.g. green receptor). Each odorant can elicit responses from several receptors, perhaps with different response amplitudes (here, the red receptor reacts strongly and the green receptor less strongly). Thousands of neurons expressing a given olfactory receptor are spread throughout one zone of the olfactory epithelium, but their axons converge on one or two glomeruli in the olfactory bulb. (B) Sources of phenotypic variation in olfaction. Individuals with different genotypes may (1) be homozygous for a given olfactory receptor, (2) express sequence variants with slightly different odorant-binding capabilities, (3) possess non-functional variants (hatched receptor) and/or (4) have duplicate gene copies, perhaps changing relative numbers of responsive neurons in the olfactory epithelium.

Human Molecular Genetics, 2002, 11, 1153

B1. Odorant receptors…

Genomic organization of olfactory receptor genes. Top: OR genes have a single main coding exon (black) and typically have several 5' untranslated exons. Alternate splicing is seen in many genes. Middle: OR genes are clustered in the genome in groups of 1 to over 100 genes (green arrows) and pseudogenes (red arrows) in both transcriptional orientations. Bottom: OR clusters are dispersed around the genome in more than 40 (mouse) or over 100 (human) locations.

B1. Odorant receptors…

Models of odorant receptor (OR) transcriptional regulation. (a) The short promoter model. The sequences immediately upstream of OR genes contain transcription factor binding sites sufficient to regulate receptor transcription. Which OR gene is expressed depends on the transcription factor(s) expressed in the cell. (b) The locus control region (LCR) model. Proximal promoters contain transcription factor binding sites necessary to drive specific receptor transcription, but are unavailable until factors binding to a distal LCR makes the region transcriptionally accessible. (c) The recombination model. OR genes are translocated by recombination or copied by gene conversion into a single active locus for expression. Transcription promoting factors are depicted as ovals.

Trends Genet. 2002 Jan;18(1):29-34

Figure 1. Gene-translocation mechanisms that activate one member of the multigene family. (a) DNA recombination. As seen in the mouse immunoglobulin (Ig) κ light-chain genes, DNA deletion brings a promoter carried by each variable (V) gene segment and the enhancer region between the joining (J) and constant (C) gene segments into proximity, thus activating the translocated gene. (b) Gene conversion. This is another gene-translocation mechanism that activates one particular member of the multigene family. A copy of the gene to be activated is transferred into the expression cassette located remotely from the gene cluster. This activation mechanism can be found in the yeast mating-type choice and antigenic variation in African trypanosomes. Abbreviations: P, promoter; E, enhancer.

Trends Genet. 2004 Dec;20(12):648-53

B1. Odorant receptors…

Figure 32.5. The Olfactory Signal-Transduction Cascade. The binding of odorant to the olfactory receptor activates a signaling pathway similar to those initiated in response to the binding of some hormones to their receptors The final result is the opening of cAMP-gated ion channels and the initiation of an action potential.

Sensory transduction. Within the compact cilia of the OSNs a cascade of enzymatic activity transduces the binding of an odorant molecule to a receptor into an electrical signal that can be transmitted to the brain. This is a classic cyclic nucleotide transduction pathway in which all of the proteins involved have been identified, cloned, expressed and characterized. Additionally, many of them have been genetically deleted from strains of mice, making this one of the most investigated and best understood second-messenger pathways in the brain. AC, adenylyl cyclase; CNG channel, cyclic nucleotide-gated channel; PDE, phosphodiesterase; PKA, protein kinase A; ORK, olfactory receptor kinase; RGS, regulator of G proteins (but here acts on the AC); CaBP, calmodulin-binding protein. Green arrows indicate stimulatory pathways; red indicates inhibitory (feedback).

 Combinatorial receptor codesThe odorant receptor family is used in a combinatorial manner to detect odorants and encode their unique identities. Different odorants are detected by different combinations of receptors and thus have different receptor codes. These codes are translated by the brain into diverse odour perceptions.The immense number of potential receptor combinations is the basis for our ability to distinguish and form memories of more than 10,000 different odorants.

B1. Odorant receptors…

Smell Molecule NameChemical Formula

Shape

Fruity ethyl octanoate C10H20O2                   

Minty beta-cyclocitral C10H13O                   

Minty p-anisaldehyde C8H8O2                   

Nutty,Medicinal2,6-dimethyl pyrazine

C6H8N2                   

Nutty,Medicinal 4-heptanolide C7H12O2                   

Nutty,Medicinal p-cresol C7H8O                   

B1. Odorant receptors…

Testes and the nose both express members of the odorant receptor super-family of G protein-coupled receptors.The testes odorant receptor hOR 17-4, previously shown to interact with the floral odor bourgeonal, is now shown to be expressed in the nose as well. This suggests that hOR 17-4 has evolved a dual role in chemoreception: perhaps guiding sperm to the egg and providing a conscious perception of odors through the nose. A gradient of bourgeonal is shown to attract sperm (left) and to be smelled by the nose (right).

Curr Biol. 2004 Nov 9;14(21):R918-20

B1. Odorant receptors…

Schematic representation of the number and classification of liganded and orphan GPCRs.

Annu Rev Pharmacol Toxicol. 2004;44:43-66.

B2-GPCRs as drug targets: orphan and known GPCRs

Box 1 | Methods for 'de-orphanizing' seven-transmembrane (7TM) receptors  Three main approaches have been taken to identify the endogenous ligands for orphan receptors. First, peptides or other putative ligands that are known to have bioactivity in distant species have been found to exist in mammals, and subsequently matched as agonists for orphan receptors. A second related approach has been to identify potential peptides, either biochemically from tissue extracts or by predictive means using the sequences of apparent neuropeptide precursors. These peptides are then synthesized and tested for bioactivity at orphan and known receptors. A distinct third approach has been to use the orphan receptors themselves in functional screens of fractionated tissues, and those that gave rise to an 'active peak' (that is, they include a component that binds to the receptor) are then sequenced or subjected to mass spectroscopy to identify the active principle. This approach is shown above.

Nature Reviews Molecular Cell Biology 3; 639-650 (2002);

B2- GPCRs as drug targets: orphan and known GPCRs…

Strategy for the identification of ligands at orphan GPCRs. Orphan GPCRs are expressed in a recombinant expression system, such as mammalian cells, yeast, or Xenopus melanophores. Following expression, it is usual to generate an assay amenable to the screening of candidate ligands in 96 well or 384 well microtitre plate formats. Candidate ligands, including small molecules, peptides, proteins, lipids, or tissue extracts, are screened in the assay. The identification of an activating ligand is detected according to the activation of an intracellular signaling cascade (see text for details). An activating ligand, often termed a hit molecule, will be identified according to its ability to cause a concentration-dependent increase in the activity of a signaling cascade. Once identified, the ligand may be further characterized against other GPCRs to determine its activity and selecticity profile prior to being used in cell-based, tissue, and in some cases whole-animal experiments in order to study the physiological role of the newly liganded receptor.

Annu Rev Pharmacol Toxicol. 2004;44:43-66.

B2- GPCRs as drug targets: orphan and known GPCRs…

Characterization of HM74 as a G-protein coupled receptor that responds to nicotinic acid. (A) Application of 300  µM (hashed columns) and 1 mM (filled columns) nicotinic acid to membranes from HEK293/T cells expressing a variety of orphan G protein coupled receptors was found to stimulate [35S]-GTPγS binding in membranes from cells expressing HM74 (open columns, basal conditions). (B) Nicotinic acid stimulated a dose-dependent increase

in [35S]-GTPγS binding in cells expressing HM74.

Annu Rev Pharmacol Toxicol. 2004;44:43-66.

B2- GPCRs as drug targets: orphan and known GPCRs…

Trends Pharmacol Sci. 2003 Jan;24(1):30-5

Recent peptide/GPCR pairings

B2- GPCRs as drug targets: orphan and known GPCRs…

Natural ligand Sites of action G protein coupling profile

Kyotorphin CNS Gi/oNocistatin Brain,spinal cord Gi/oProsaposin Brain Gi/o Chromostatin Adrenal gland Gi/oPancreastatin Heart, liver, adipose Gq/11>Gi/o

Examples of naturally occurring ligands that are likely to mediate their biological effects via GPCRs

Annu Rev Pharmacol Toxicol. 2004;44:43-66.

B2- GPCRs as drug targets: orphan and known GPCRs…

Annu Rev Pharmacol Toxicol. 2004;44:43-66.The identification of ligands at orphan G-protein coupled receptors.Wise A, Jupe SC, Rees S.7TMR Systems Research Europe, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, United Kingdom

FEBS Lett. 2005 Jan 3;579(1):259-64.        Identification and characterisation of a novel splice variant of the human CB1 receptor.Ryberg E, Vu HK, Larsson N, Groblewski T, Hjorth S, Elebring T, Sjogren S, Greasley PJ.Department of Molecular Pharmacology, AstraZeneca R&D, Molndal, Sweden

J Mol Graph Model. 2004 Sep;23(1):15-21. Design of a gene family screening library targeting G-protein coupled receptors.Lamb ML, Bradley EK, Beaton G, Bondy SS, Castellino AJ, Gibbons PA, Suto MJ, Grootenhuis PD.Deltagen Research Laboratories, 740 Bay Rd., Redwood City, CA 94063, USA

J Med Chem. 2004 Sep 9;47(19):4677-83.Indolebutylamines as selective 5-HT(1A) agonists.Heinrich T, Bottcher H, Bartoszyk GD, Greiner HE, Seyfried CA, Van Amsterdam C.Preclinical Pharmaceutical Research, Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany.

Drug Discov Today. 2005 Jan 1;10(1):69-73 Predicting ligands for orphan GPCRs.Huang ES.Pfizer Research Technology Center, 620 Memorial Drive, Cambridge, MA 02139 USA

Examples of Pharmaceutical GPCR research

Recruitment, localisation, and tissue exit of circulating leucocytes. Tissue localisation of leucocytes involves two distinct and sequential processes, termed extravasation and chemotaxis. During extravasation, blood leucocytes interact with adhesion molecules on the luminal side of blood vessels and, upon chemokine receptor triggering, become firmly attached and transmigrate through the epithelial barrier. Subsequent chemotaxis guides the perivascular leucocytes to the cellular source(s) of chemokines, which enables cellular colocalisation and subsequent execution of leucocyte function. Eventually, leucocytes exit the tissue via afferent lymphatic vessels to reach draining lymph nodes (LNs) and peripheral blood.

Ann Rheum Dis. 2004 Nov;63 Suppl 2:ii84-ii89

Chemokines. Chemokines are bioactive peptides that regulate leukocyte activation and migration. They are essential mediators of inflammation and are crucial for the control of viral infections. With around 50 members, the chemokine superfamily can be divided — on the basis of the arrangement of the first highly conserved N-terminal cysteine residue (or residues) — into four distinct classes: CXC (  ), CC (   ), CX3C (  ), and C (   ) (where C is cysteine and X is any amino acid; see figure). All chemokines are structurally similar, having at least three   -pleated sheets and a C-terminal  -helix, with disulphide bonds connecting these cysteine residues. There is a comparably large number of chemokine receptors (19 in humans), and each chemokine class has a preference for a subfamily of these G-protein-coupled receptors (GPCRs), which are often expressed in different subsets of leukocytes as well as in other cells — for example, in endothelial cells and neurons. CXC-chemokines bind preferentially to CXC-chemokine receptors (CXCRs) and attract neutrophils, whereas CC-chemokines generally bind to CC-chemokine receptors (CCRs) and tend to attract monocytes, lymphocytes, eosinophils and BASOPHILS. The C-chemokine, lymphotactin (XCL1), binds to XCR1 and induces neutrophil and T- and B-cell migration. Finally, the CX3C-chemokine, fractalkine (CX3CL), is capable of functioning as a membrane-anchored ligand, or as a shed ligand, and binds CX3CR1 on blood-derived neutrophils, monocytes, natural killer cells and T lymphocytes.

Leukocyte expression and ligand specificity of chemokine receptors at a glance. Receptors, selected cognate ligands and predominant receptor repertoires in different leukocyte populations are listed. The selected ligands are identified with one old acronym and with the new nomenclature, in which the first part of the name identifies the family and L stands for ‘ligand’ followed by a progressive number. Red identifies predominantly ‘inflammatory’ or ‘inducible’ chemokines, green ‘homeostatic’ agonists, yellow molecules belonging to both realms. Chemokine acronyms shown are as follows: BCA, B cell activating chemokine; BRAK, breast and kidney chemokine; CTACK, cutaneous T-cell attracting chemokine; ELC, Epstein–Barr virus-induced receptor ligand chemokine; ENA-78, epithelial cell-derived neutrophil-activating factor (78 amino acids); GCP, granulocyte chemoattractant protein; GRO, growth-related oncogene; HCC, hemofiltrate CC chemokine; IP, IFN-inducible protein; I-TAC, IFN-inducible T-cell α chemoattractant; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine; Mig, monokine induced by gamma interferon; MIP, macrophage inflammatory protein; MPIF, myeloid progenitor inhibitory factor; NAP, neutrophil-activating protein; PARC, pulmonary and activation-regulated chemokine; RANTES, regulated upon activation normal T cell-expressed and secreted; SCM, single C motif; SDF, stromal cell-derived factor; SLC, secondary lymphoid tissue chemokine; TARC, thymus and activation-related chemokine; TECK, thymus expressed chemokine. Abbreviations: PMN, neutrophils; Eo, eosinophils; Ba, basophils; MC, mast cells; Mo, monocytes; Mø, macrophages; iDCs, immature dendritic cells; mDCs, mature DCs; T naïve, naïve T cells; T act, activated T cells; T skin, skin-homing T cells; T muc, mucosal-homing T cells; Treg, regulatory T cells

Model of Chemokine Regulation of Breast-Cancer Metastasis. Metastasis is an orderly, multistep process involving the movement of cancer cells from the primary tumor to specific organs under the guidance of specific chemokines. First, cancerous mammary epithelial cells undergo clonal proliferation, invade local tissue, induce angiogenesis, and express CXC chemokine receptor 4 (CXCR4) on their surface. Then, cancer cells detach from the primary tumor, migrate across lymphatic and vascular walls in the tumor, and enter the systemic circulation. Cancer cells are arrested in vascular beds in organs that produce high levels of the CXCR4 ligand (CXCL12), which is expressed on the surface of vascular endothelial cells. Binding of CXCL12 to CXCR4 induces the migration of cancer cells into normal tissue, where the cells proliferate, induce angiogenesis, and form metastatic tumors. Breast-cancer cells do not usually metastasize to organs that produce low levels of CXCL12, such as the kidney.

N Engl J Med. 2001 Sep 13;345(11):833

Figure 1 | Viral pathogen hijacking of intracellular signalling networks is regulated by GPCRs.   a | On binding by chemokines (among many other agonists), cellular G-protein-coupled receptors (GPCRs) signal through a complex system of intracellular pathways. b | Viruses (such as HIV) might use cellular GPCRs as co-receptors for viral entry; viral envelope glycoproteins (gps) might function as agonists or antagonists of cellular GPCRs, modulating their downstream signalling pathways. c | Viruses might encode their own GPCRs, which often constitutively signal to a network of intracellular cascades; cellular chemokines could function as agonists or antagonists of viral GPCRs, thereby modulating these signalling networks. d | Virally encoded chemokines (virokines) might also function as agonists or antagonists of cellular (or viral) GPCRs. e | Virally encoded chemokine-binding proteins (CKBPs) bind to and sequester cellular chemokines, thereby preventing the activation of cellular GPCRs by endogenous chemokines.

Nat Rev Mol Cell Biol. 2004;5:998-1012

Comparison of Kaposi's sarcoma-like lesion at the nose of a mouse and a man. Kaposi's sarcoma-like lesion at the nose of a mouse transgenic expressing ORF74 from HHV8 under control of the CD2 promoter (left side) compared to a Kaposi's sarcoma lesion at a human nose

Oncogene. 2001; 20:1582-93.

Figure 2 | Signalling downstream of HIV gp120–CCR5 interactions.   Stimulation of CC-chemokine receptor-5 (CCR5) by R5 glycoprotein (gp)120 in macrophages activates proliferative signalling pathways such as the mitogenic signalling cascade from Raf to MEK (mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase) to ERK. This thereby promotes the proliferation of virally infected cells. Concurrently, stimulation of stress-activated kinases (such as p38 and Jun N-terminal kinase (JNK), which are downstream of MAPK kinase 3/6 (MKK3/6) and MEK kinase-1 (MEKK1), respectively; see Box 2) might enhance the transcriptional activity of key transcription factors (such as nuclear factor (NF)-  B and activator protein-1 (AP-1)), and lead to the secretion of cellular cytokines that could promote the recruitment of target cells or further enhance infected-cell proliferation through paracrine mechanisms. Collectively, these pathways could facilitate HIV replication and promote viral transmission.

Figure 3 | Interaction of CXCR4 with HIV gp120 promotes lymphocyte migration and apoptosis.   Binding of CXC-chemokine receptor-4 (CXCR4) by X4 glycoprotein (gp)120 in T cells leads to the stimulation of focal adhesion kinase (FAK) or protein tyrosine kinase-2 (PYK2), and SRC (left-hand pathway). When these tyrosine kinases are stimulated, adaptor molecules such as CRK-associated substrate (CAS), paxillin and CRK promote the recruitment/activation of the engulfment and cell motility protein (ELMO)–Dock180 complex, which is a Rac guanine nucleotide-exchange factor. ELMO–Dock180 activation triggers the exchange of GDP for GTP by Rac. GTP-bound (active) Rac stimulates actin polymerization and cell adhesion and migration through p21-activated kinase-1 (PAK1). Activation of FAK/PYK2 by gp120 binding to CXCR4 might also stimulate signalling through phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent protein kinase (PDK) and AKT/protein kinase B (AKT/PKB; right-hand pathway), thereby promoting host-cell survival. In certain cases, however, CXCR4 activation might lead to T-cell apoptosis — as depicted in Fig. 4 for neuronal cells. Collectively, these pathways could help to ensure successful viral infectivity and propagation, and promote disease progression. Similar pathways are also activated by R5 gp120 stimulation of CCR5 (not shown).

Figure 5 | Role of KSHV-GPCR signalling pathways in Kaposi's sarcomagenesis.   a | The expression of Kaposi's-sarcoma-associated herpesvirus (KSHV) G-protein-coupled receptor (GPCR) in KSHV-infected endothelial cells might be essential for triggering Kaposi's sarcomagenesis. KSHV-GPCR-expressing cells release angiogenic growth factors that recruit (and transform) adjacent endothelial cells through paracrine mechanisms. These endothelial cells might be latently infected with KSHV, which would further promote cell proliferation and survival. b | Signalling pathways that are upregulated by KSHV GPCR in endothelial cells might induce endothelial-cell transformation through direct and indirect (paracrine) mechanisms. KSHV GPCR, by releasing       subunits (Box 2), promotes signalling through phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent kinase (PDK) and AKT/protein kinase B (AKT/PKB). Concurrently, KSHV-GPCR-mediated activation of mitogen-activated-protein-kinase cascades (such as Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38; Box 2) through the small GTPases Ras and Rac1 stimulates the activity of key cellular transcription factors (such as activating protein-1 (AP-1), nuclear factor (NF)-  B, and hypoxia-inducible factor-1 (HIF1)). These transcription factors upregulate the transcription and secretion of pro-angiogenic growth factors (such as interleukin-8 (IL-8)/CXC-chemokine ligand-8 (CXCL8), Gro  /CXCL1, and vascular endothelial growth factor (VEGF)). Secreted growth factors might then bind to and activate endogenous cellular receptors, like kinase insert deaminase receptor (KDR), which leads to the indirect (paracrine) activation of AKT/PKB. Several of these secreted cytokines could also function as agonists for KSHV GPCR, enhancing its signalling through an autocrine mechanism.