Novel Structural Demonstration of GPCR Binding and Activation Mechanism Has Implications for Understanding Disease, Drug Discovery
NEW YORK (November 29, 2005) — A family of proteins called G protein-coupled receptors (GPCRs) lies on the surface membranes of cells and is the target for about half of all currently used drugs.
But until now, experts haven't clearly understood — or had the tools to better understand — just how pharmaceuticals switch these receptors on or off.
That may have all changed with the publication of a joint paper from researchers at the Weill Medical College of Cornell University and Columbia University College of Physicians and Surgeons, both in New York City.
Their findings appear in today's Proceedings of the National Academy of Sciences.
The research team has streamlined a method that, for the first time, provides a structural demonstration of how a pairing up of these GPCR protein molecules — "dimerization" — occurs at the cell surface. In the process, the investigators have also shed new light on a higher organization phenomenon, called "oligomerization," that organizes the dimers and may also play a key role in this drug-receptor interface.
"We're starting to identify, for the first time, which parts of the interface between GPCR pairs in the cell membrane are 'talking' to each other and how that information moves from one player to the other," explained senior researcher Dr. Jonathan A. Javitch, Associate Professor of Psychiatry and Pharmacology at Columbia University Medical Center.
Besides expanding our knowledge of how GPCR receptors work and how medicines affect these receptors, the findings could "improve our ability to find targets for drug design that do not interfere with the binding site, thereby reducing or removing side effects," added co-senior researcher Dr. Harel Weinstein, the Maxwell M. Upson Professor of Physiology and Biophysics, Chairman of the Department of Physiology and Biophysics at Weill Cornell Medical College, and Director of the College's Institute for Computational Biomedicine.
The Weill Cornell-Columbia team focused their research on one type of GPCR, called dopamine receptors. "These receptors are of major importance in the treatment of psychiatric and neurological disease — including schizophrenia and Parkinson's disease — where medications act as dopamine receptor antagonists (switching the receptor off) or agonists (turning the receptor on)," Dr. Javitch explained.
But just how does this biochemical switch get flipped?
Experts had long assumed that receptors function as monomers in the membrane, but, over the past several years, increasing experimental and theoretical evidence began to highlight the functional importance of receptors organizing in pairs — dimerizing — as they bind and react to other molecules (ligands).
What was needed was a structural interpretation of this theory, however.
To achieve that, Dr. Weinstein's lab at Weill Cornell designed and used a complex computer model to predict which parts of GPCRs might link up with which part of a particular pharmaceutical molecule to turn the receptor on or off.
Next came the even tougher trick of identifying the interfaces between two interacting GPCRs, and how this interaction is affected by the binding of the pharmaceutical agent. At this point, the predictions and insights from the computational work were evaluated by concrete experiments on the cell systems. Dr. Javitch's group spent nearly two years honing a method that mimicked the activity of either GPCR agonist or antagonist drugs on the GPCRs and their dimers, using an approach called cysteine cross-linking.
"Although it involved elements that had been established before, putting it together to help map membrane protein function was a time-consuming and complex feat," he said.
Cysteine cross-linking "chemically traps two positions together, forcing them to come together in space — even in the absence of the drug ligand," Dr. Javitch explained. Using this technique, they were able to switch the dopamine receptor "on" when cross-linking mimicked the activity of an agonist drug, or switch it "off" when cross-linking replicated binding with an antagonist compound.
In doing so, the study provided the first structure-based demonstration of how drugs work on the receptor and affect the GPCR interface — confirming and elaborating on the role dimerization plays in this process.
"The structural demonstration validated the elements of our computational model, but taught us many new things," Dr. Weinstein said. "In fact, the methodology itself is a breakthrough — we believe it can be used to glean insights into a wide range of membrane proteins."
Researchers have also found that dimerization can occur between two different types of receptors.
"Again, this has implications for drug discovery, because disparate receptors seem to form something entirely new — a new entity with novel pharmacological properties," Dr. Weinstein said. The Javitch and Weinstein laboratories are also collaborating on mapping the interfaces formed between such disparate receptors and comparing this with the interface identified in this study between like receptors.
The research also led to one more key finding.
"Work on the light receptor rhodopsin has suggested that these receptors are not only arranged in pairs but also that these pairs are arranged in rows — something we call 'oligomerization,'" Dr. Javitch said.
"Based on our experiments, we now propose — although we haven't proven this — that the activation or inactivation of this entire family of receptor involves a rearrangement of the interface, not only between pairs, but through a shift in these rows of pairs, called oligomers," he said.
If proven true by future research, this mechanism could point to a whole new mechanism for driving and refining the activity of GPCR-linked drugs.
"It's probably not an alternative mechanism, but rather a partner, working together with the GPCR-ligand interaction we've so far assumed is the only mechanism of importance," Dr. Weinstein said.
The existence of a second, oligomerization-based mechanism might help researchers design drugs that don't interfere with the primary GPCR binding site. That could potentially reduce drug side effects for patients, the Weill Cornell researcher explained.
Much of this new proposal remains speculative, the researchers stressed. However, the validated findings about the movements at the receptor interface strongly suggest that the way pharmaceuticals act on GPCRs is much more complex — and promising, in terms of drug discovery — than ever imagined before.
"We used to think that these medications worked through a single receptor to initiate the signaling changes that helped fight disease," Dr. Javitch said.
"We're beginning to understand that it's not that simple. We hope that this understanding will lead to a deeper knowledge of the role of these receptors in a wide range of illnesses, and how better to fight them."
The study was funded by grants from the National Institutes of Health to the two laboratories of the senior investigators, the National Alliance for Research on Schizophrenia and Depression Vicente Investigator Award, and the Lieber Center for Schizophrenia Research at Columbia University Medical Center.
Co-authors include lead author Dr. Wen Guo of Columbia University College of Physicians and Surgeons, and Drs. Lei Shi and Marta Filizola of Weill Cornell Medical College.
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