Receptors as Kissing Cousins

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Science  07 Apr 2000:
Vol. 288, Issue 5463, pp. 65-67
DOI: 10.1126/science.288.5463.65

Cellular processes as different as growth factor signaling and transcription depend on interactions between proteins [HN1]. Given this, it may not be surprising that certain receptors [HN2] bind to related but different receptors as well as to each other. However, the dogma has been that members of the seven-transmembrane helix G protein-coupled receptor (GPCR) [HN3] family pair up with their own kind but do not bind to other family members (1). Several recent studies, including a report by Rocheville et al.[HN4] (2) on page 154 of this issue, provide evidence that GPCRs can pair up with even rather distantly related relatives to form heterodimeric receptors with distinct properties. Rocheville and co-workers show that the dopamine D2 receptor [HN5] and the somatostatin SST5 receptor [HN6] form heterodimers [HN7]. Although pharmacologically distinct, these two GPCRs are coexpressed in striatal and pyramidal neurons of the cortex. If the reported interaction between the d and k opioid receptors [HN8] (3) (closely related GPCR family members) can be considered as a pairing of brother and sister, then the union of the D2 and SST5 receptors is more akin to a marriage between kissing cousins.

These findings have caused something of a shock. This is despite earlier experiments in which the coexpression of two mutant (nonfunctional) angiotensin II receptors [HN9] resulted in formation of a homodimer that once more could activate signal transduction pathways after binding ligand (4). Clear evidence emerged last year that formation of heterodimers between GABA (γ-aminobutyric acid) R1 and R2 receptors was necessary for a fully functional GABAB receptor [HN10] (5).

The usual strategy for studying receptor heterodimerization is to coexpress differentially tagged forms of the receptors and then to immunoprecipitate [HN11] them (3). Although standard for elucidating the interactions between cytoplasmic proteins, the highly hydrophobic nature of GPCRs mandates their extraction from the membrane before immunoprecipitation. This necessity renders the approach less than ideal because artifacts may arise from aggregation of GPCRs. Rocheville et al. decided to apply photobleaching fluorescence resonance energy transfer (FRET) [HN12] to see whether GPCR relatives were cohabiting. They produced cell lines that stably coexpressed modest levels of the D2 and SST5 receptors (which were each labeled with a specific dye that could be visualized by FRET). Because energy transfer only occurs when donor and acceptor molecules are in close proximity, FRET offers an ideal way to follow the interactions of receptors in single living cells.

Surprisingly, the investigators found little indication of heterodimerization between D2 and SST5 receptors unless a neurotransmitter agonist [HN13] for either receptor was present. The neurotransmitter for either receptor promoted heterodimerization, but the presence of both ligands did not produce an additive or synergistic interaction. Previous studies often demonstrated high levels of GPCR heterodimerization in the absence of agonist ligands and variations in the ability of ligands to alter this status (5, 6). These earlier findings may reflect both the high-level expression of receptors and their capacity to form nonspecific aggregates in the absence of a membrane environment. Because the D2 and SST5 receptors are known to form homodimers, it would be predicted that each individual agonist would encourage homodimerization as well as (or, perhaps, rather than) heterodimerization. Although not studied by Rocheville et al. (2), it would be interesting to compare the formation of homo- and heterodimers between the D2 and SST5 receptors in the presence of either ligand.

The GPCR family is probably the largest in the human genome. If ligand-induced heterodimerization turns out to be common, then the array of GPCR combinations will be truly bewildering. Both biochemical and physiological data had hinted at the interaction of D2 and SST5. For example, coexpression of the two GPCRs resulted in reciprocal shifts in ligand binding affinity. Furthermore, although the somatostatin ligand was unable to block adenylyl cyclase [HN14] activity in a mutant SST5 receptor (the GPCR signal transduction pathway is activated through inhibition of adenylyl cyclase by Gi proteins), coexpression of the D2 receptor resulted in rescue of somatostatin function (2). This suggested that binding of somatostatin to the SST5-D2 heterodimer induced direct activation of the D2 receptor. All of the somatostatin receptors and three of the dopamine receptors (D2, D3, and D4) activate members of the same family of G proteins, which inhibit adenylyl cyclase and regulate ion flow through a group of Ca2+ and K+ channels [HN15]. No doubt heterodimers between other somatostatin and dopamine receptors will be identified soon.

Two separate gene families encode GPCRs and ion channel proteins, and, until recently, there was little evidence for interactions between such strangers. But, the repertoire of proteins that interact with GPCRs is expanding dramatically (7). The purpose of some interactions is clear—the interaction of Homer/Vesl proteins with metabotropic glutamate receptors [HN16] (8) results in release of these GPCRs from the endoplasmic reticulum and their expression in the plasma membrane [HN17]; the intracellular trafficking of the b2-adrenergic receptor is directed by its interaction with the phosphoprotein EBP50 [HN18] (9). But there are other cases where the consequences of interactions between GPCRs and unrelated proteins remain obscure (10, 11).

Recently, Liu et al. (12) provided strong biochemical and functional evidence for a direct interaction between the D5 dopamine GPCR and a GABAA receptor with a γ2 subunit (see the figure) [HN19]. GABAA receptors (activated by the inhibitory neurotransmitter GABA) are Cl ion channels composed of subunits from five related protein families. D1 and D5 dopamine receptors stimulate adenylyl cyclase activity (unlike D2 which inhibits it). Their transmembrane regions are highly conserved but their carboxyl terminal tails are not. The investigators studied interactions between combinations of fusion proteins composed of the carboxyl terminal tail of the D1 or D5 receptor and the second intracellular loop of the five GABAA receptor subunits. They found a high-affinity interaction between the D5 carboxyl terminus and the second intracellular loop of the GABAA receptor γ2 subunit (see the figure). Remarkably, this interaction was only observed after exposure to agonists for both receptors. Stimulation of adenylyl cyclase by D5 (but not D1) was inhibited in a concentration-dependent manner by the γ2 subunit of GABAA in the presence of GABA. This effect could be transferred by exchanging the carboxyl terminus of D1 for that from D5. As a corollary, in cells coexpressing GABAA (containing a γ2 subunit) and D5, GABA-induced ion currents decreased when dopamine was added. As in the Rocheville study (2), direct evidence from earlier experiments for the coexpression of the GABAA and D5 receptors in hippocampal neurons provided the impetus to investigate possible interactions between these two classes of GPCRs.

Family and friends.

Interactions between GPCRs and ion channels. Each GABAA receptor ion channel consists of five polypeptides. The predominant type of GABAA is composed of two α, two β, and one γ subunits. As γ2 is the predominant γ isoform, there is the potential for interactions (with a likely 1:1 stoichiometry) between GABAA and D5 dopamine receptors in most neurons that coexpress both. The site of contact between the carboxyl-terminal tail of the D5 receptor and the second intracellular loop of the γ2 subunit have not been explored but agonists for both receptors (dopamine and GABA, respectively) are required for the interaction.


Large-scale protein-protein interaction screens in model systems (13) together with global gene expression profiling after receptor activation (14) promise the rapid identification of many more unexpected interactions between GPCRs and other proteins (15). The next step will be to work out the functional importance of all these kissing cousins.

HyperNotes Related Resources on the World Wide Web

General Hypernotes

The On-line Medical Dictionary is available from CancerWEB.

D. Glick's Glossary of Biochemistry and Molecular Biology is provided on the Web by Portland Press.

A neuroscience glossary is provided for a course on computational neuroscience at the University of Wisconsin.

The Bio Netbook is a searchable database of Internet resources provided by the Institut Pasteur, Paris.

P. Gannon's Cell & Molecular Biology Online is a collection of annotated links to Internet resources.

Neurosciences on the Internet is a searchable and browsable index of neuroscience resources available on the Internet.

The Virtual Library of Neuroscience is maintained by the Department of Neurology and Neuroscience at Cornell University Medical College.

The library of the Karolinska Institutet, Stockholm, Sweden, provides a collection of links to biomedical information on the Web. Sections on cell biology and neuroscience are included.

The CMS Molecular Biology Resource is a compendium of electronic and Internet-accessible tools and resources for molecular biology, biotechnology, molecular evolution, biochemistry, and biomolecular modeling.

J. Kimball presents Kimball's Biology Pages, an online biology textbook and glossary.

The MIT Biology Hypertextbook provides background information on the biology of cells. Receptors describes the action of ligands and receptors and Central Dogma describes the process of translation and the biosynthesis of peptides.

P. Katz, Department of Biology, Georgia State University, Atlanta, provides lecture notes for course in neurobiology.

R. M. Robertson, Department of Biology, Queen's University, Kingston, Canada, provides lecture notes for a course on integrative neurobiology and neuroethology.

D. Bourque, Department of Biochemistry, University of Arizona, offers lecture notes on protein synthesis and protein targeting for a course in nucleic acid biochemistry.

M. Blaber, Department of Chemistry, Florida State University, provides lecture notes for courses on molecular biology and biotechnology and protein structure and stability.

The Glaxo Wellcome R&D Web site provides a pharmacology guide that offers presentations on agonist and antagonist action and receptor binding, as well as a glossary of terms.

GeneCards: Human genes, proteins and diseases is a database maintained by the Bioinformatics Unit of the Genome Center, Weizmann Institute of Science, Israel. There is a U.S. mirror of the site.

The ExPASy Molecular Biology Server is provided by the Swiss Institute of Bioinformatics. SWISS-PROT (annotated protein sequences) and PROSITE (protein families and domains) are two of the databases available. Amos' WWW Links is the ExPASy list of Internet biomolecular resources.

GPCRDB (G Protein-Coupled Receptor Data Base) is an information system for G protein-coupled receptors (GPCRs) developed by a partnership with funding by the European Community's biotechnology program. GPCR literature and a collection of GPCR-related Internet resources are provided.

GCRDb (G-Protein-Coupled Receptor Database) is maintained by L. Kolakowski, Department of Pharmacology, University of Texas Health Science Center at San Antonio. An introduction to GPCR families is provided.

Drugs, Brains and Behavior, an online text by C. R. Timmons and L. Hamilton, Department of Psychology, Rutgers University, includes an introduction to the chemistry of the brain.

The 1994 Nobel Prize in Physiology or Medicine was awarded to Alfred G. Gilman and Martin Rodbell “for their discovery of G-proteins and the role of these proteins in signal transduction in cells.” An illustrated presentation on G proteins and signal transduction is provided.

Numbered Hypernotes

1. Protein is defined and illustrated in the Glossary of Genetic Terms provided by the National Human Genome Research Institute. provides an Encyclopædia Britannica article on protein. D. Jaffe, Department of Mathematics and Statistics, University of Nebraska, provides a presentation on proteins for a course on algorithms in biological sequence analysis. The THCME Medical Biochemistry Page, maintained by M. King, Terre Haute Center for Medical Education, IN, provides an introduction to protein structure. Kimball's Biology Pages provides an introduction to proteins and transcription. D. Clark, Department of Microbiology, Southern Illinois University, Carbondale, offers lecture notes on transcription for a molecular biology course. T. Terry, Department of Molecular and Cellular Biology, University of Connecticut, provides lecture notes on cell signaling for a biology course. For a course on molecular and cell biology, the Molecular Biology Program, New Mexico State University, provides lecture notes on cell signaling.

2. Receptor is defined in the neuroscience glossary from the University of Wisconsin. J. Brown, Department of Molecular Biosciences, University of Kansas, provides an introduction to receptors and what they do. The Graphics Gallery of Access Excellence illustrates the binding site of a protein.

3. G protein is defined in the Glossary of Biochemistry and Molecular Biology. The Protein NMR Structure Gallery presented by the Protein NMR Spectroscopy Laboratory, Center for Advanced Biotechnology and Medicine, Rutgers University, provides an introduction to G proteins. The Biochemistry Web site of the School of Biomedical Sciences, University of Nottingham, UK, offers a presentation on G proteins in the course notes for a biochemistry course. The KUMC Medical Biochemistry Web site from the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, includes a section on signal transduction that includes a presentation on G proteins; a study guide to signal transduction is also available. ExPASy's PROSITE defines and lists known G protein-coupled receptors (GPCRs). K. Rice, SmithKline Beecham, Upper Merion, PA, provides an introduction to GPCRs. The MIT Biology Hypertextbook includes a presentation on the G protein receptor in the cell biology chapter. The THCME Medical Biochemistry Page has an introduction to G protein receptors in the section about signal transduction. The Department of Biology, University of Virginia, makes available lecture notes with a section on signal transduction by GPCRs for a biology course. The KEGG (Kyoto Encyclopedia of Genes and Genomes) database has an entry for GPCRs with links to Internet resources.

4. M. Rocheville, U. Kumar, and Y. Patel are at the Fraser Laboratories, Royal Victoria Hospital, and Faculty of Medicine, McGill University, Montreal. D. Lange and R. Patel are in the Department of Chemistry, Clarkson University, Potsdam, NY.

5. Dopamine is defined in the On-line Medical Dictionary. A reference guide to neurotransmitters by C. Burdess summarizes information about dopamine. Tocris Cookson, a company that produces chemicals for pharmacology and neurochemistry, offers a dopamine overview by P. Strange. T. Faulkner, College of Pharmacology, Ohio Northern University, provides a lecture supplement about dopamine. In an undergraduate thesis submitted to the School of Community Medicine, University of New South Wales, Australia, K. Johnson included information on dopamine and dopamine receptors. GeneCards has an entry for dopamine receptor D2. SWISS-PROT has an entry for D(2) dopamine receptor. Online Mendelian Inheritance in Man (OMIM) from the National Center for Biotechnology Information has an article about the dopamine receptor D2.

6. Somatostatin is defined in the On-line Medical Dictionary. offers an Encyclopædia Britannica article about somatostatin. Kimball's Biology Pages defines dopamine and somatostatin and includes links to further information. GeneCards has an entry for somatostatin receptor 5 with links to other databases. SWISS-PROT has an entry for somatostatin receptor type 5. OMIM has an article on somatostatin receptor 5.

7. Heterodimer is defined in the On-line Medical Dictionary.

8. GeneCards has entries for opioid receptor, delta 1 and opioid receptor, kappa 1. Tocris Cookson makes available a review of opioid receptors by A. Corbett et al . The Society for Neuroscience provides a briefing on opiate receptors.

9. The On-line Medical Dictionary defines angiotensin. GeneCards has an entry for angiotensin receptor 2.

10. The THCME Medical Biochemistry Page provides an introduction to GABA in the presentation on neurotransmitters. The Webvision Web site includes an introduction to the properties of GABA receptors in a presentation by H. Qian. GeneCards has an entry for GABA-B receptor. OMIM has an entry for GABA-B receptor 1.

11. Immunoprecipitation is described in the Signal Transduction Laboratory Manual provided by the Research Division, Peter MacCallum Cancer Institute, East Melbourne, Australia. P. J. Hansen, Department of Dairy and Poultry Sciences, University of Florida, provides information about the immunoprecipitation protocol used in his laboratory.

12. The Handbook of Fluorescent Probes and Research Chemicals, made available by Molecular Probes, Inc., Eugene, OR, includes information about fluorescence resonance energy transfer (FRET). Sections on photobleaching and FRET are included in the presentation titled “Fluorescence excitation and emission” provided by Integrated DNA Technologies, Coralville, IA. A discussion of FRET is included in the Techsight article by A. Mendelsohn and R. Brent titled “Protein interaction methods - Toward an endgame” in the 18 June 1999 issue of Science .

13. An introduction to receptor agonists is provided in a presentation on the chemistry of the nervous system by R. Sikes, Program in Physical Therapy, Northeastern University. E. Chudler, Department of Anesthesiology, University of Washington, presents an introduction to neurotransmitters and neuroactive peptides in his educational Exploring Neuroscience Web site. The Addiction Science Research and Education Center, University of Texas College of Pharmacy, offers a presentation on neurotransmitters; a section on dopamine is included. The NeuroPage presented by M. Jamali, College of Nursing and Health Professions, Arkansas State University, provides lecture notes on neurotransmitters. P. Patton, Neuronal Pattern Analysis Group, Beckman Institute, University of Illinois, provides lecture notes on neurotransmitters and their release for a course on neurobiology.

14. Information about adenylyl cyclase is provided in the glossary for the Biomedical Hypertexts offered by the College of Veterinary Medicine and Biomedical Sciences, Colorado State University. The ExPASy Enzyme database has an entry for adenylate cyclase (adenylyl cyclase). S. Sprang's Protein Structure Laboratory, Howard Hughes Medical Institute at the University of Texas Southwestern Medical Center, Dallas, offers a presentation on adenylyl cyclase.

15. A presentation on the brain, provided by the Instituto de Fisiologia Celular, Universidad Autónoma de México, includes an introduction to ion channels. The Channelwork Laboratory of CeNeS Pharmaceuticals, Cambridge, UK, offers an introduction to ion channels. The Chapman Laboratory, Harvard Medical School, provides background information on ion channels in a presentation titled “G protein-gated potassium-selective ion channels.” The Molecular & Cellular Classifications Web site from the Neuromuscular Disease Center, Washington University School of Medicine, provides an information page about ion channels, transmitters, receptors, and disease. The Albert and Mary Lasker Foundation provides an introduction by B. Culliton to the 1999 Lasker Awards for ion channel proteins research with links to further information on the recipients and their research.

16. Glutamate receptor is defined by the On-line Medical Dictionary. The Webvison Web site makes available information on glutamate receptors and metabotropic glutamate receptors in a presentation by V. Connaughton titled “Glutamate and glutamate receptors in the vertebrate retina.” D. Gellerman, Department of Psychiatry, University of California, Davis, offers a presentation titled “Structure-activity relationships of glutamate receptor ligands.”

17. M. Dalton offers a presentation on the endoplasmic reticulum for a Web cell biology course. NetBiochem provides a presentation on the cell membrane. Kimball's Biology Pages offers an introduction to cell membranes.

18. GeneCards has entries for beta-2 adrenergic receptor and SLC9A3R1 (EBP50). SWISS-PROT has an entry for beta-2 adrenergic receptor.

19. F. Liu is at the Department of Psychiatry, University of Toronto, Ontario, Canada. The OMIM entry for GABA-A receptor gives a brief summary of Liu's research. GeneCards has an entry for GABA-A receptor, gamma 2.

20. G. Milligan is in the Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland.


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