One in three FDA-approved drugs acts on a single superfamily of receptors found on the surfaces of human cells. From beta blockers to antihistamines, these essential, life-saving medications activate 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 initially believed: several of these drugs, in fact, target a complex composed of a receptor and an associated protein. Now, a new study in Scientific advances It presents a new approach to map the interactions between 215 of these receptors and the three proteins with which they form complexes. The findings dramatically expand the understanding of these interactions and their therapeutic potential.
“From a technical standpoint, we can now study these receptors on an unprecedented scale,” said first author Ilana Kotliar, a former graduate student in the Laboratory of Chemical Biology and Signal Transduction at Rockefeller University, led by Thomas P. Sakmar. “And from a biological standpoint, we now know that the phenomenon of these protein-receptor interactions is much more widespread than originally thought, which opens the door to future research.”
Uncharted 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 greatly alter the way these receptors transmit signals by changing the shape of the receptor or influencing its location. Because GPCRs rarely exist in a vacuum, identifying a GPCR without considering how RAMPs might influence it is a bit like knowing a restaurant’s menu without looking at its hours, address, or delivery options.
“There can be two cells in the body where the same drug acts on the same receptor, but the drug only works on one of them,” said 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 his 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 greatly boost drug development, with the added benefit of possibly explaining why some promising GPCR-based drugs have mysteriously failed to pan out.
They hoped this 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. “It’s possible that in the past, such hits have not been detected in analyses because a GPCR-RAMP complex was not being looked for.”
But analyzing 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 at the Science for Life Laboratory in Sweden and the Swedish Human Protein Atlas Project to create an assay capable of detecting GPCR-RAMP interactions.
Hundreds of experiments at once
The team began 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 the researchers to simultaneously analyze hundreds of potential GPCR-RAMP interactions in a single experiment. As each bead passed through a screening instrument, color-coding was used to identify which GPCRs were bound to which RAMP, allowing for high-throughput tracking of 215 GPCRs and their interactions with the three known RAMPs.
“A lot of this technology already existed. Our contribution was an enabling technology based on it,” Sakmar says. “We developed a technique to test hundreds of different complexes at once, which generates a huge amount of data and answers many questions simultaneously.”
“Most people don’t think in terms of multiplexes. 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 get it to the finish line, moving scarce samples and reagents back and forth from Sweden in the rare travel windows during COVID.
And it paid off. The results provide a long-awaited set of resources for GPCR researchers and drug developers: publicly available online libraries of anti-GPCR antibodies, designed 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,” Sakmar says.
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 screen GPCR combinations and identify harmful autoantibodies. “Ultimately, it’s a technology-driven project,” Sakmar says. “That’s what our lab does. We work on technologies to advance drug discovery.”