- Email: [email protected]
Treasures throughout the life-cycle of G-protein-coupled receptors
396
Research Update
TRENDS in Pharmacological Sciences Vol.22 No.8 August 2001
Meeting Report
Treasures throughout the life-cycle of G-protein-coupled receptors Thomas M. Wilkie The G-Protein-Coupled Receptors meeting was held on 30–31 March 2001 in Orlando, Florida, USA.
This meeting’s mantra was Earl Southerland’s proclamation that ‘pharmacology is biochemistry with a purpose’. Several speakers and almost half the attendees hailed from pharmaceutical and biotechnology companies. The mix was lively. Attention focused on how to identify ligands for orphan receptors (with all the practical questions that entailed) and the mechanisms that generate signaling specificity and duration. Topics covered in the meeting can be organized around the life-cycle of G-protein-coupled receptors (GPCRs): their synthesis and transport from the endoplasmic reticulum to the plasma membrane, dimerization, ligand binding, signal activation, desensitization and internalization (Fig. 1). Each step can be manipulated to help identify ligands for orphan receptors. This is good news because it is a huge undertaking to identify ligands for all GPCRs in the human genome (661, counted by Celera1).
diabetes insipidus2, could be stabilized by cell-permeant antagonists and routed to the plasma membrane. These so-called pharmacological chaperones could be promising tools for ligand screens if they helped boost cell-surface expression of orphan receptors and were subsequently displaced by agonists. Receptor activation
Tales of receptor dimerization (or oligomerization) created such a stir that some people confessed to wondering if monomeric GPCRs were functional in cells. Many GPCRs form homodimers without the presence of an agonist – for example, Sharon Chinault (Washington University, St Louis, MO, USA) presented convincing genetic and fluorescence resonance energy transfer (FRET) analysis of yeast alpha factor receptor oligomerization – but agonists can also enhance oligomerization. Graeme Milligan (University of Glasgow, UK) used intragenic complementation to show formation of functional oligomers of the α2-adrenoceptor. Sometimes only receptor heterodimers activate signaling. Julia White
(GlaxoSmithKline, Stevenage, UK) showed that GABAB(1) receptors were not expressed at the cell surface and were not functional, unless coexpressed with GABAB(2) receptors. Bouvier also demonstrated heterodimerization of these receptors by bioluminescence resonance energy transfer (BRET), which places the covalently tagged C-termini of these receptors within 100 Å of each other. Yogesh Patel (McGill University, Montreal, Canada) used photobleaching FRET (which measures signal transfer quench time) to show heterodimerization of somatostatin sst5 and dopamine D2 receptors. Amazingly, even C-terminal deletion mutants of the somatostatin receptor could heterodimerize and enhance D2 receptor signaling. These observations raise the intriguing possibility of expressing receptor combinations (with perhaps one inactive) to boost cell-surface expression of orphan receptors in ligand screens. William Clarke (University of Texas at San Antonio, TX, USA) presented examples of ligand-dependent differential signaling. Receptors that couple with two H Ligand binding
Ligand screens
Shelagh Wilson (GlaxoSmithKline, Essex, UK) opened by listing three key requirements of a ligand screen: (1) cellsurface expression of GPCRs; (2) cellular assays to detect ligand binding; and (3) libraries of putative natural and synthetic ligands. GPCRs are often not expressed on the surface of transfected cells – a major impediment to identifying ligands of orphan receptors and odorants that bind olfactory receptors. Jurgen Wess (NIDDK, Washington DC, USA) reported that cellsurface expression of mutant muscarinic acetylcholine M3 receptors is increased by addition of the antagonist atropine to the culture medium. Michel Bouvier (University of Montreal, Canada) showed that misfolded mutants of the vasopressin V2 receptor, known to cause nephrogenic http://tips.trends.com
α
β
γ α
E1
Dimerization (n>2)
RGS
Degradation Synthesis
γ
β
GDP GTP
Recycling
E2
GRK P
A
Signal
A ER
Internalization Lysosome
P
P P
Endosome
TRENDS in Pharmacological Sciences
Fig. 1. The life-cycle of a G-protein-coupled receptor (GPCR). A schematic representation of GPCR synthesis, dimerization (or oligomerization, represented by n≥2), signaling complex assembly, agonist activation, desensitization, internalization and resensitization or degradation. GPCR oligomerization might persist throughout the cycle. Abbreviations: A, arrestin; E, effector protein; ER, rough endoplasmic reticulum; GDP, guanine nucleotide diphosphate; GRK, G-protein-coupled receptor kinase; GTP, guanine nucleotide triphosphate; H, hormone; P, phosphate group; RGS, regulator of G-protein signaling3,8.
0165-6147/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(00)01736-3
Research Update
Key conference outcomes • G-protein-coupled receptor (GPCR) expression, dimerization and internalization can be manipulated for ligand identification screens. • Apparent ligand efficacy depends on competition between different GPCR conformations. • Regulators of G-protein signaling (RGS) proteins can both enhance and inhibit G-protein-coupled signaling. • Loco-domain proteins are Gα–GDIs (guanine nucleotide dissociation inhibitors).
TRENDS in Pharmacological Sciences Vol.22 No.8 August 2001
relocalization of arrestin marks activated GPCRs without needing to identify their downstream signaling pathways. The flip side of GPCR pharmaceutics is that some diseases are caused by constitutively active receptors. Frederick Leeb-Lundberg (University of Texas at San Antonio, TX, USA) used chimeric receptors to determine which regions of the bradykinin B2 receptor conferred high constitutive activity. Members of the signal transduction field eagerly await the day we can compare crystal structures of wild-type and constitutively active GPCRs. Regulators of G-protein signaling
or more classes of G proteins can differentially activate multiple signal transduction pathways, depending on the agonist. This could be missed during initial ligand screens but might confound later phases of drug testing if not recognized as signal redirection induced by different agonist analogs. A different twist on ligand efficacy was presented by Terry Kenakin (GlaxoSmithKline, Research Triangle Park, NC, USA). Empty receptors are distributed in various free-energy wells (or meta-stable conformations), described as a so-called ‘conformational cafeteria’. Different ligands can stabilize different conformations of the same receptor and thereby act as full, partial or inverse agonists. Apparent ligand efficacy might depend on which other ligands are present, which receptor conformations are available for binding, and what end-point is used to determine a response. This behavior could explain why a synthetic ligand might fully displace a natural ligand but only partially activate signaling, or alter the relative efficacy of activating two different signaling pathways. High-throughput screens depend on robust and adaptable assays. A potentially spectacular new approach exploits receptor internalization. Marc Caron (Duke University, Durham, NC, USA) showed that most, if not all, agoniststimulated GPCRs are eventually phosphorylated by GPCR kinases (GRKs) and then bind arrestin during desensitization and internalization. These interactions were monitored in cells by the recruitment of green fluorescent protein (GFP)- and yellow fluorescent protein (YFP)-tagged arrestin. Cellular http://tips.trends.com
Most attention in drug screens is on GPCR ligands but new horizons are appearing. Recent work by many investigators suggests that the regulators of G-protein signaling (RGS) proteins modulate Gi and Gq signaling by two combined activities. RGS proteins have scaffolding properties that help to initiate GPCR-catalyzed signaling rapidly; they are also GTPaseactivating proteins (GAPs) that can rapidly terminate signaling3. Tom Wilkie and Shmuel Muallem (University of Texas Southwestern, Dallas, TX, USA) presented evidence that RGS proteins use these combined activities to regulate Ca2+ oscillations initiated from GPCR complexes. Feedback regulation of RGS GAP activity by Ca2+-binding proteins and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] appear to act at an allosteric regulatory site for RGS GAP activity that is suitable for drug screens4. RGS proteins might be essential to diverse and physiologically important cellular responses such as cardiomyocyte hypertrophy and immune cell chemotaxis. Early in the 1990s, Henry Bourne made an audacious claim that not only GTP-bound G-protein α- and βγ-subunits could initiate signaling, but Gα–GDP might as well5! This theoretical possibility is finding empirical support. Stephen Lanier (Louisiana State University, New Orleans, LA, USA) presented the story of AGS3, one of several proteins with Loco domains that bind and stabilize Gα–GDP (Refs 6,7). What is the purpose of these guanine nucleotide dissociation inhibitors (GDIs)? Is it to prolong Gβγ signaling or to have direct effector functions? Loco domain proteins appear to be signal integrators in diverse processes such as asymmetric cell divisions, glial migration
397
and neurite outgrowth. With these stories and many others the ASPET Colloquium on G Protein Coupled Receptors accomplished its goals of stimulating new thinking about old concepts and rewarding creativity outside the box. References 1 Venter, J.C. et al. (2001) The sequence of the human genome. Science 291, 1304–1351 2 Birnbaumer, M. (1999) Vasopressin receptor mutations and nephrogenic diabetes insipidus. Arch. Med. Res. 30, 465–474 3 Ross, E.M. and Wilkie, T.M. (2000) GTPaseactivating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu. Rev. Biochem. 69, 795–827 4 Popov, S.G. et al. (2000) Ca2+/calmodulin reverses phosphatidylinositol 3,4,5trisphosphate-dependent inhibition of regulators of G protein-signaling GTPaseactivating protein activity. J. Biol. Chem. 275, 18962–18968 5 Bourne, H.R. et al. (1990) The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125–132 6 De Vries, L. et al. (2000) Activator of G protein signaling 3 is a guanine dissociation inhibitor for Gαi subunits. Proc. Natl. Acad. Sci. U. S. A. 97, 14364–14369 7 Natochin, M. et al. (2000) AGS3 inhibits GDP dissociation from Gα subunits of the Gi family and rhodopsin-dependent activation of transducin. J. Biol. Chem. 275, 40981–40985 8 Ferguson, S.S. et al. (1998) Molecular mechanisms of G protein-coupled receptor desensitization and resensitization. Life Sci. 62, 1561–1565
Thomas M. Wilkie Pharmacology Dept, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9041, USA. e-mail: [email protected]
Meeting Reports TiPS is pleased to publish highlights from international conferences. Conference participants who wish to cover a meeting should contact the Editor.
TiPS, Elsevier Science London, 84 Theobald’s Road, London, UK WC1X 8RR. ([email protected])
Research Update
TRENDS in Pharmacological Sciences Vol.22 No.8 August 2001
Meeting Report
Treasures throughout the life-cycle of G-protein-coupled receptors Thomas M. Wilkie The G-Protein-Coupled Receptors meeting was held on 30–31 March 2001 in Orlando, Florida, USA.
This meeting’s mantra was Earl Southerland’s proclamation that ‘pharmacology is biochemistry with a purpose’. Several speakers and almost half the attendees hailed from pharmaceutical and biotechnology companies. The mix was lively. Attention focused on how to identify ligands for orphan receptors (with all the practical questions that entailed) and the mechanisms that generate signaling specificity and duration. Topics covered in the meeting can be organized around the life-cycle of G-protein-coupled receptors (GPCRs): their synthesis and transport from the endoplasmic reticulum to the plasma membrane, dimerization, ligand binding, signal activation, desensitization and internalization (Fig. 1). Each step can be manipulated to help identify ligands for orphan receptors. This is good news because it is a huge undertaking to identify ligands for all GPCRs in the human genome (661, counted by Celera1).
diabetes insipidus2, could be stabilized by cell-permeant antagonists and routed to the plasma membrane. These so-called pharmacological chaperones could be promising tools for ligand screens if they helped boost cell-surface expression of orphan receptors and were subsequently displaced by agonists. Receptor activation
Tales of receptor dimerization (or oligomerization) created such a stir that some people confessed to wondering if monomeric GPCRs were functional in cells. Many GPCRs form homodimers without the presence of an agonist – for example, Sharon Chinault (Washington University, St Louis, MO, USA) presented convincing genetic and fluorescence resonance energy transfer (FRET) analysis of yeast alpha factor receptor oligomerization – but agonists can also enhance oligomerization. Graeme Milligan (University of Glasgow, UK) used intragenic complementation to show formation of functional oligomers of the α2-adrenoceptor. Sometimes only receptor heterodimers activate signaling. Julia White
(GlaxoSmithKline, Stevenage, UK) showed that GABAB(1) receptors were not expressed at the cell surface and were not functional, unless coexpressed with GABAB(2) receptors. Bouvier also demonstrated heterodimerization of these receptors by bioluminescence resonance energy transfer (BRET), which places the covalently tagged C-termini of these receptors within 100 Å of each other. Yogesh Patel (McGill University, Montreal, Canada) used photobleaching FRET (which measures signal transfer quench time) to show heterodimerization of somatostatin sst5 and dopamine D2 receptors. Amazingly, even C-terminal deletion mutants of the somatostatin receptor could heterodimerize and enhance D2 receptor signaling. These observations raise the intriguing possibility of expressing receptor combinations (with perhaps one inactive) to boost cell-surface expression of orphan receptors in ligand screens. William Clarke (University of Texas at San Antonio, TX, USA) presented examples of ligand-dependent differential signaling. Receptors that couple with two H Ligand binding
Ligand screens
Shelagh Wilson (GlaxoSmithKline, Essex, UK) opened by listing three key requirements of a ligand screen: (1) cellsurface expression of GPCRs; (2) cellular assays to detect ligand binding; and (3) libraries of putative natural and synthetic ligands. GPCRs are often not expressed on the surface of transfected cells – a major impediment to identifying ligands of orphan receptors and odorants that bind olfactory receptors. Jurgen Wess (NIDDK, Washington DC, USA) reported that cellsurface expression of mutant muscarinic acetylcholine M3 receptors is increased by addition of the antagonist atropine to the culture medium. Michel Bouvier (University of Montreal, Canada) showed that misfolded mutants of the vasopressin V2 receptor, known to cause nephrogenic http://tips.trends.com
α
β
γ α
E1
Dimerization (n>2)
RGS
Degradation Synthesis
γ
β
GDP GTP
Recycling
E2
GRK P
A
Signal
A ER
Internalization Lysosome
P
P P
Endosome
TRENDS in Pharmacological Sciences
Fig. 1. The life-cycle of a G-protein-coupled receptor (GPCR). A schematic representation of GPCR synthesis, dimerization (or oligomerization, represented by n≥2), signaling complex assembly, agonist activation, desensitization, internalization and resensitization or degradation. GPCR oligomerization might persist throughout the cycle. Abbreviations: A, arrestin; E, effector protein; ER, rough endoplasmic reticulum; GDP, guanine nucleotide diphosphate; GRK, G-protein-coupled receptor kinase; GTP, guanine nucleotide triphosphate; H, hormone; P, phosphate group; RGS, regulator of G-protein signaling3,8.
0165-6147/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(00)01736-3
Research Update
Key conference outcomes • G-protein-coupled receptor (GPCR) expression, dimerization and internalization can be manipulated for ligand identification screens. • Apparent ligand efficacy depends on competition between different GPCR conformations. • Regulators of G-protein signaling (RGS) proteins can both enhance and inhibit G-protein-coupled signaling. • Loco-domain proteins are Gα–GDIs (guanine nucleotide dissociation inhibitors).
TRENDS in Pharmacological Sciences Vol.22 No.8 August 2001
relocalization of arrestin marks activated GPCRs without needing to identify their downstream signaling pathways. The flip side of GPCR pharmaceutics is that some diseases are caused by constitutively active receptors. Frederick Leeb-Lundberg (University of Texas at San Antonio, TX, USA) used chimeric receptors to determine which regions of the bradykinin B2 receptor conferred high constitutive activity. Members of the signal transduction field eagerly await the day we can compare crystal structures of wild-type and constitutively active GPCRs. Regulators of G-protein signaling
or more classes of G proteins can differentially activate multiple signal transduction pathways, depending on the agonist. This could be missed during initial ligand screens but might confound later phases of drug testing if not recognized as signal redirection induced by different agonist analogs. A different twist on ligand efficacy was presented by Terry Kenakin (GlaxoSmithKline, Research Triangle Park, NC, USA). Empty receptors are distributed in various free-energy wells (or meta-stable conformations), described as a so-called ‘conformational cafeteria’. Different ligands can stabilize different conformations of the same receptor and thereby act as full, partial or inverse agonists. Apparent ligand efficacy might depend on which other ligands are present, which receptor conformations are available for binding, and what end-point is used to determine a response. This behavior could explain why a synthetic ligand might fully displace a natural ligand but only partially activate signaling, or alter the relative efficacy of activating two different signaling pathways. High-throughput screens depend on robust and adaptable assays. A potentially spectacular new approach exploits receptor internalization. Marc Caron (Duke University, Durham, NC, USA) showed that most, if not all, agoniststimulated GPCRs are eventually phosphorylated by GPCR kinases (GRKs) and then bind arrestin during desensitization and internalization. These interactions were monitored in cells by the recruitment of green fluorescent protein (GFP)- and yellow fluorescent protein (YFP)-tagged arrestin. Cellular http://tips.trends.com
Most attention in drug screens is on GPCR ligands but new horizons are appearing. Recent work by many investigators suggests that the regulators of G-protein signaling (RGS) proteins modulate Gi and Gq signaling by two combined activities. RGS proteins have scaffolding properties that help to initiate GPCR-catalyzed signaling rapidly; they are also GTPaseactivating proteins (GAPs) that can rapidly terminate signaling3. Tom Wilkie and Shmuel Muallem (University of Texas Southwestern, Dallas, TX, USA) presented evidence that RGS proteins use these combined activities to regulate Ca2+ oscillations initiated from GPCR complexes. Feedback regulation of RGS GAP activity by Ca2+-binding proteins and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] appear to act at an allosteric regulatory site for RGS GAP activity that is suitable for drug screens4. RGS proteins might be essential to diverse and physiologically important cellular responses such as cardiomyocyte hypertrophy and immune cell chemotaxis. Early in the 1990s, Henry Bourne made an audacious claim that not only GTP-bound G-protein α- and βγ-subunits could initiate signaling, but Gα–GDP might as well5! This theoretical possibility is finding empirical support. Stephen Lanier (Louisiana State University, New Orleans, LA, USA) presented the story of AGS3, one of several proteins with Loco domains that bind and stabilize Gα–GDP (Refs 6,7). What is the purpose of these guanine nucleotide dissociation inhibitors (GDIs)? Is it to prolong Gβγ signaling or to have direct effector functions? Loco domain proteins appear to be signal integrators in diverse processes such as asymmetric cell divisions, glial migration
397
and neurite outgrowth. With these stories and many others the ASPET Colloquium on G Protein Coupled Receptors accomplished its goals of stimulating new thinking about old concepts and rewarding creativity outside the box. References 1 Venter, J.C. et al. (2001) The sequence of the human genome. Science 291, 1304–1351 2 Birnbaumer, M. (1999) Vasopressin receptor mutations and nephrogenic diabetes insipidus. Arch. Med. Res. 30, 465–474 3 Ross, E.M. and Wilkie, T.M. (2000) GTPaseactivating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu. Rev. Biochem. 69, 795–827 4 Popov, S.G. et al. (2000) Ca2+/calmodulin reverses phosphatidylinositol 3,4,5trisphosphate-dependent inhibition of regulators of G protein-signaling GTPaseactivating protein activity. J. Biol. Chem. 275, 18962–18968 5 Bourne, H.R. et al. (1990) The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125–132 6 De Vries, L. et al. (2000) Activator of G protein signaling 3 is a guanine dissociation inhibitor for Gαi subunits. Proc. Natl. Acad. Sci. U. S. A. 97, 14364–14369 7 Natochin, M. et al. (2000) AGS3 inhibits GDP dissociation from Gα subunits of the Gi family and rhodopsin-dependent activation of transducin. J. Biol. Chem. 275, 40981–40985 8 Ferguson, S.S. et al. (1998) Molecular mechanisms of G protein-coupled receptor desensitization and resensitization. Life Sci. 62, 1561–1565
Thomas M. Wilkie Pharmacology Dept, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9041, USA. e-mail: [email protected]
Meeting Reports TiPS is pleased to publish highlights from international conferences. Conference participants who wish to cover a meeting should contact the Editor.
TiPS, Elsevier Science London, 84 Theobald’s Road, London, UK WC1X 8RR. ([email protected])