GPCRs: Researchers Disprove the Single Polypeptide Theory

For this article, Jim Kling interviewed Bryen A. Jordan, postdoctoral scientist in the lab of Edward Ziff at the New York University School of Medicine's department of biochemistry; Theresa Branchek, vice president of research at Synaptic Pharmaceutical Corp. (Paramus, NJ); and Christophe Gerald, vice president of target discovery and assessment at Synaptic Pharmaceutical. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. K.A. Jones, B. Borowsky, J.A. Tamm, D.A. Craig, M.M. Durkin, M. Dai, W.J. Yao, M. Johnson, C. Gunwaldsen, L.Y. Huang, C. Tang, Q.R. Shen, J.A. Salon, K. Morse, T. Laz, K.E. Smith, D. Nagarathnam, S.A. Noble, T.A. Branchek, C. Gerald, "GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2," Nature, 6712: 674-9, Dec. 17, 1998. (Cited in 176 papers) B.A. Jordan, L.A. Devi, "G-protein-coupled receptor heterodimerization modulates receptor function," Nature, 6737: 697-700, June 17, 1999. (Cited in 133 papers) Courtesy: Nature Co-localization of receptors in HEK293 cells by dual-wavelength scanning confocal microscopy. ABABR1 (green) and ABABR2 (red) strongly colocalize (a-c) whereas HA-GABABR2 (red) and Flag-Neuropeptide-Y Y5 (green) do not (d). Working out the interactions between G-protein-coupled receptors (GPCRs) and their ligands is a tricky business. Many GPCR ligands are unknown. In the case of the three known types of opioid receptors-m,d, and K-there are nine known opioid ligands, most of which have not been matched to a corresponding receptor. Considering that roughly 30 percent of drugs in current development target GPCRs, the issue takes on more scientific, clinical, and economic significance says Bryen Jordan, a postdoctoral scientist in the lab of Edward Ziff, at New York University School of Medicine's department of biochemistry. "They make up 2 to 3 percent of the entire mammalian genome, so it is an extremely important class [of receptors] to work with," he says. This well-known family of seven-transmembrane-domain cell surface receptors respond to many signals, including photons, biogenic amines, lipids, peptides, and proteases. The receptors have many functions, most of them related to homeostatic control of processes such as blood pressure, respiration, and appetite.1 Studied for nearly 30 years, researchers thought they had a decent idea of what GPCRs looked like. "GPCRs have historically been thought to be single polypeptides," says Theresa Branchek, vice president of research at Synaptic Pharmaceutical Corp. Paramus, NJ. The End of the Single-Polypeptide Myth Two papers, one co-authored by Jordan during his graduate school days with Lakshmi A. Devi's lab at NYU's department of pharmacology, the other by Branchek and her colleagues, are changing the way researchers look at GPCRs. Jordan's team studied the K and d opioid receptors, while Branchek's focused on receptors for the primary inhibitory neurotransmitter y-aminobutyric acid (GABA). Both came up with similar results: Two distinct GPCRs formed a heterodimer with novel binding and pharmacological properties. The work at Synaptic Pharmaceuticals grew out of the company's focus on GPCRs in general. The GABA receptors include three types, A, B, and C, and the company focused on GABAB, in part because it is the only one of the three that is a GPCR, and also because Synaptic says it believes that the GABAB receptor will allow them to indirectly modulate pathways, perhaps producing a more subtle and selective effect. "Biochemically, you're affecting a second messenger pathway and modulating the activity of ion channels. In the case of the ionotrophic GABA receptors [GABAA and GABAC], ... you're directly affecting current flow through ion channels, rather than modulating them," says Branchek. The researchers found that cells expressing cloned GABAB receptors could bind GABA but not be activated by it, so they looked for related receptors that might have functional activity. Using the GABAB sequence (now known as GABABR1) to conduct a search on expressed sequence tags in GenBank,2 researchers identified a novel receptor known as GABABR2, which turned out to be 35 to 41 percent homologous to GABABR1. Despite the discovery of the second receptor, the team could not coax any response out of either one. "Finally, Ken Jones [then a senior scientist in the department of molecular biology] tried to co-express these receptors, and we heard a huge shout," recalls Christophe Gerald, Synaptic's vice president of target discovery and assessment. "It was him looking at a functional readout of the receptors, which had never happened before." Says Branchek, "Jones had the notion that since these receptors appear to co-localize to a large extent, they may need to be expressed together in order to function. That stimulated him to do an experiment. When co-expressed, now all of a sudden, where you couldn't get signals before, now you were getting whopping signals in response to GABA. I believe we were the first to understand that none of these species had any functionality when expressed in vitro one at a time .... That was the first elucidation of the heterodimerization of a GPCR on a functional basis." Like Branchek's team, Jordan found that the theory of single polypeptide GPCRs couldn't explain what he saw. His team had already seen evidence that K and d receptors associated. So, using cells that expressed both, Jordan performed competitive binding assays between selective opioid ligands and a general opioid receptor antagonist, but saw no competition at all. This was a major surprise, because those ligands would have bound to their respective receptors. It was the sort of experimental artifact that leads one to wonder about the labels on the bottles, but "I knew I wasn't making any mistakes," Jordan recalls. "At one point I decided to put both ligands [U-69553 and DPDPE] in together. I'm not sure why I did it; maybe I just had extra ligand in a tube. All of a sudden, I got a signal." That experiment finally provided functional support to the biochemical evidence already collected. The team had seen signs of the interaction, but could not figure out its import. "This was when we knew we had something extremely important, because a receptor is defined by its pharmacology. Purely pharmacologically, our receptor was different," Jordan says. They looked closer and found that the new, combined receptor resembled the pharmacological profile of a receptor that other groups had observed but never cloned, a conundrum easily explained by the receptor's dual nature. A Molecular Explanation The important question, all agree, is how widespread heterodimerization is in the world of GPCRs. "If you're modeling your receptor as a monomer, it would perhaps be more profitable if you model it as a dimer... most GPCRs have now been shown to be dimers," says Jordan. The work could also explain synergy that is observed when animals are treated with ligands for two different opioid receptors. "We think we have a molecular explanation." Jordan's former colleagues continue to investigate interactions between opioid receptors and have documented similar heterodimerizations between d and m opioid receptors, and between b-adrenergic receptors and opioid receptors.3,4 Synaptic Pharmaceuticals continues its focus on GPCR-based drug discovery. They expect to start their first clinical trials this spring. In the end, both papers led to, but didn't answer, the same question: What other heterodimerization processes occur in GPCRs? Says Branchek, "That's an open question that will go on in many labs for many years to come, and that's the broader significance of this discovery." Jim Kling (jkling@nasw.org) is a freelance writer in Washington, D.C. References 1. J. Kling, "A role for reverse pharmacology," The Scientist, 14[6]:18, March 20, 2000. 2. GenBank can be accessed at www.ncbi.nlm.nih.gov. 3. B.A. Jordan et al., "Oligomerization of opioid receptors with beta 2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation," Proceedings of the National Academy of Sciences, 98[1]:343-8, Jan. 2, 2001. 4. I. Gomes et al., "Heterodimerization of mu and delta opioid receptors: A role in opiate synergy," Journal of Neuroscience, 20[22]:U22-6, Nov. 15, 2000.

GPCRs: Researchers Disprove the Single Polypeptide Theory

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