One in three FDA-approved drugs targets a single superfamily of receptors spread across the surfaces of human cells. From beta blockers to antihistamines, these essential, life-saving medications trigger winding biochemical pathways through these receptors to ultimately prevent a heart attack or stop an allergic reaction.
But scientists have discovered that their story is much more complicated than first thought: Some of these drugs actually target a complex consisting of one receptor and one associated protein. Now, a new study in Scientific progress introduces a new approach for mapping the interactions between such receptors and the three proteins with which they form complexes. The findings dramatically increase the understanding of these interactions and their therapeutic potential.
“Technically, we can now study these receptors on an unprecedented scale,” says first author Ilana Kotliar, a former graduate student at Rockefeller’s Laboratory of Chemical Biology and Signal Transduction, led 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 initially thought, which opens the door for future research.”
Unexplored territory
This family of receptors is known as GPCRs or G protein-coupled receptors. Their accessory proteins are known as RAMPs, short for receptor activity-modifying proteins. RAMPs help transport GPCRs to the cell surface and can dramatically change the way these receptors transmit signals by changing the shape of the receptor or affecting its location. Because GPCRs rarely exist in a vacuum, identifying a GPCR without considering how RAMPs can affect it is a bit like knowing a restaurant’s menu without checking its hours, address, or delivery options.
“You could have two cells in the body in which the same drug targets the same receptor – but the drug only works in one cell,” says Sakmar, the Richard M. and Isabel P. Furlaud Professor. “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 analyze the effect of each RAMP on each GPCR. Such a comprehensive map of GPCR-RAMP interactions would boost drug development, with the added benefit of potentially explaining why some promising GPCR drugs have mysteriously failed to materialize.
They hoped that such a map would also contribute to basic biology by revealing which natural ligands various so-called “orphan” GPCRs interact with. “We still don’t know what activates many GPCRs in the human body,” says Kotliar. “Screeners may have missed those matches in the past because they were not 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 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 from the Science for Life Laboratory in Sweden and the Swedish Human Protein Atlas Project to create a test capable of detecting GPCR-RAMP -screen interactions.
Hundreds of experiments at the same time
The team started by linking 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 engineered cells expressing different combinations of RAMPs and GPCRs. This setup allowed researchers to screen 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, allowing high throughput of 215 GPCRs and their interactions with the three known RAMPs.
“A lot of this technology already existed. Our contribution was an enabling technology that built on it,” says Sakmar. “We have developed a technique to test hundreds of different complexes simultaneously, which generates a huge amount of data and answers many questions at the same time.”
“Most people don’t think in multiplex terms, but that’s what we did: 500 experiments at the same time.”
While this work is the result of a team effort over a long period of time, Kotliar has made tremendous efforts to get it across the finish line – transporting samples and scarce reagents back and forth from Sweden during rare travel periods during COVID.
It has 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 which antibodies bind to it, whether those antibodies are commercially available, and whether that receptor binds to a RAMP,” says Sakmar.
The findings increase the number of experimentally identified GPCR-RAMP interactions by an order of magnitude and lay the foundation for techniques that can help detect combinations of GPCRs and identify harmful autoantibodies. “Ultimately it is a technology-oriented project,” says Sakmar. “That’s what our lab does. We work on technologies to advance drug discovery.”
