GPCR-RAMP interactome maps could help drug developers find buried treasure

Most drugs targeting proteins are treasures that drug developers have dug out of the G protein-coupled receptor (GPCR) interactomes. So, as drugs targeting GPCRs become increasingly difficult to find, one might compare GPCR interactomes to exhausted mines and consider whether prospecting efforts should not be directed elsewhere. Alternatively, one could continue to look at GPCR interactomes, albeit more deeply.

The latter option looks a little more promising with the introduction of a new excavation tool: a custom-designed multiplex suspension bead array immunoassay (SBA). It was developed by scientists at Rockefeller University who conducted their work in Scientific advancesin an article titled “Multiplex mapping of the interactome of GPCRs with receptor activity-modifying proteins.”

To expand the field of GPCR-focused drug discovery, 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 significantly alter the way these receptors transmit signals by changing the shape of the receptor or affecting its location.

“RAMP interactions have been identified for about 50 GPCRs, but few GPCR-RAMP complexes have been studied in detail,” the authors of the article wrote. “To determine a comprehensive GPCR-RAMP interactome, we constructed a library of 215 GPCRs with dual epitope tags representing all GPCR subfamilies and co-expressed each GPCR with each of the three RAMPs.

“By examining GPCR-RAMP pairs using customized multiplex SBA immunoassays, we identified 122 GPCRs that showed strong evidence of interaction with at least one RAMP. We screened three cell lines for interactions and found 23 endogenously expressed GPCRs that formed complexes with RAMPs.”

Based on this work, the scientists concluded that mapping the GPCR-RAMP interactome could contribute to the development of selective therapeutics for GPCR-RAMP complexes.

“Technically, we can now study these receptors on a scale never before possible,” said lead author Ilana Kotliar, a former graduate student in the Rockefeller University lab led by Thomas P. Sakmar. “And biologically, we now know that the phenomenon of these protein-receptor interactions is much more widespread than originally thought, which opens the door for future investigations.”

Sakmar, one of the corresponding authors and the Richard M. and Isabel P. Furlaud Professor, added: “There could be two cells in the body where the same drug targets 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.”

With this in mind, Sakmar and his colleagues wanted to develop a technique that would allow researchers to analyze the effect of each RAMP on each GPCR. Such a comprehensive map of GPCR-RAMP interactions would speed up drug development and potentially explain why some promising GPCR drugs mysteriously failed to achieve the desired success.

They hoped that such a map would also contribute to basic research in biology by providing information about 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. “Previous screens may have missed these matches 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 nearly 800 GPCRs, searching for every possible combination was impractical, if not impossible. In 2017, Emily Lorenzen, then a PhD student in Sakmar's lab, began collaborating with scientists at the Science for Life Laboratory in Sweden and the Swedish Human Protein Atlas Project to develop an assay to study GPCR-RAMP interactions.

The team began by coupling antibodies from the Human Protein Atlas to magnetic beads, each pre-stained with one of 500 different dyes. These beads were then incubated with a liquid mixture of genetically engineered cells expressing different combinations of RAMPs and GPCRs. This setup allowed the researchers to study hundreds of potential GPCR-RAMP interactions simultaneously 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. This enabled 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 a technology built on top of it. We developed a technique that can test hundreds of different complexes simultaneously. This generates huge amounts of data and answers many questions at once.”

“Most people don't think in multiplex terms. But that's exactly what we did – 500 experiments at once.”

Although this work represents the culmination of many years of teamwork, Kotliar made herculean efforts to get it across the finish line – transporting samples and rare reagents back and forth between Sweden and Turkey during rare travel windows during the COVID pandemic.

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 and find out which antibodies bind to it, whether those antibodies are commercially available, and whether that receptor binds to a RAMP,” Sakmar explained.

The results increase the number of experimentally identified GPCR-RAMP interactions by an order of magnitude and lay the foundation for techniques that could help detect combinations of GPCRs and identify harmful autoantibodies. “Ultimately, it's a technology-driven project,” Sakmar emphasized. “That's the job of our lab. We're working on technologies to advance drug discovery.”