Most protein-targeting drugs are prizes that drug developers have unearthed from G protein–coupled receptor (GPCR) interactomes. So, when GPCR-targeting drugs become harder to find, one might liken GPCR interactomes to depleted mines, and consider whether prospecting efforts should be directed elsewhere. Alternatively, one might continue digging into GPCR interactomes, albeit more deeply.
The latter option looks a little more promising with the introduction of a new excavation tool: a customized multiplexed suspension bead array (SBA) immunoassay. It was developed by Rockefeller University scientists, who described their work in Science Advances, in a paper titled, “Multiplexed mapping of the interactome of GPCRs with receptor activity–modifying proteins.”
To expand the field of GPCR-focused drug discovery, the scientists focused on the complexes that GPCRs form with accessory proteins called receptor activity–modifying proteins (RAMPs). RAMPs help transport GPCRs to the cell surface and can vastly alter how these receptors transmit signals by changing the receptor’s shape or influencing its location.
“RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail,” the article’s authors wrote. “To elucidate a comprehensive GPCR-RAMP interactome, we created a library of 215 dual epitope-tagged GPCRs representing all GPCR subfamilies and co-expressed each GPCR with each of the three RAMPs.
“Screening the GPCR-RAMP pairs with customized multiplexed SBA immunoassays, we identified 122 GPCRs that showed strong evidence for interaction with at least one RAMP. We screened for interactions in three cell lines and found 23 endogenously expressed GPCRs that formed complexes with RAMPs.”
“On the technical side, we can now study these receptors at unprecedented scale,” said first author Ilana Kotliar, a former graduate student in the Rockefeller University lab headed by Thomas P. Sakmar. “And on the biological side, we now know that the phenomenon of these protein-receptor interactions is much more widespread than originally thought, opening the door to future investigations.”
Sakmar, one of the corresponding authors and the Richard M. and Isabel P. Furlaud Professor, added, “You could have two cells in the body in which the same drug is targeting the same receptor—but the drug only works in one cell. The difference is that one of the cells has a RAMP that brings its GPCR to the surface, where the drug can interact with it. That’s why RAMPs are so important.”
Knowing this, Sakmar and colleagues were determined to develop a technique that would allow researchers to parse out each RAMP’s effect on every GPCR. Such a comprehensive map of GPCR-RAMP interactions would supercharge drug development, with the added benefit of possibly explaining why some promising GPCR drugs mysteriously haven’t panned out.
They hoped that such a map would also contribute to basic biology by revealing which natural ligands several so-called “orphan” GPCRs interact with. “We still don’t know what activates many GPCRs in the human body,” Kotliar admitted. “Screenings may have missed those matches in the past because they weren’t looking for a GPCR-RAMP complex.”
But wading through every GPCR-RAMP interaction was a daunting task. With three known RAMPs and almost 800 GPCRs, searching through every possible combination was impractical, if not impossible. In 2017 Emily Lorenzen, then a graduate student in Sakmar’s lab, began a collaboration with scientists at the Science for Life Laboratory in Sweden and Sweden’s Human Protein Atlas Project to create an assay capable of screening for GPCR-RAMP interactions.
The team started by coupling antibodies from the Human Protein Atlas to magnetic beads, each pre-colored with one of 500 different dyes. These beads were then incubated with a liquid mixture of engineered cells expressing various combinations of RAMPs and GPCRs. This setup allowed researchers to simultaneously screen hundreds of potential GPCR-RAMP interactions in a single experiment. As each bead passed through a detection instrument, color coding was used to identify which GPCRs were bound to which RAMPs, enabling high throughput tracking of 215 GPCRs and their interactions with the three known RAMPs.
“A lot of this technology already existed,” Sakmar noted. “Our contribution was an enabling technology built upon it. We developed a technique to test for hundreds of different complexes at once, which generates a huge amount of data, and answers many questions simultaneously.
“Most people don’t think in multiplex terms. But that’s what we did—500 experiments at once.”
While this work is the culmination of a team effort over a long period of time, Kotliar made herculean efforts to drag it across the finish line—shuttling samples and scarce reagents back and forth from Sweden in rare travel windows during COVID-19.
It paid off. The results provide a handful of long-awaited resources for GPCR researchers and drug developers: publicly available online libraries of anti-GPCR antibodies, engineered GPCR genes and, of course, the mapped interactions. “You can now type in your favorite receptor, find out what antibodies bind to it, whether those antibodies are commercially available, and whether that receptor binds to a RAMP,” Sakmar declared.
The findings increase the number of experimentally identified GPCR-RAMP interactions by an order of magnitude and lay the groundwork for techniques that could help detect combinations of GPCRs and identify harmful autoantibodies. “Ultimately, it’s a technology-oriented project,” Sakmar stressed. “That’s what our lab does. We work on technologies to advance drug discovery.”
