Groundbreaking Imaging Technique Reveals Promising New Drug Targets
Nearly 35% of drugs approved by the Food and Drug Administration (FDA) target G protein-coupled receptors (GPCRs), a group of proteins embedded in cell membranes that enable cells to communicate. Among these, adhesion G protein-coupled receptors (aGPCRs) form the second-largest family in humans. As their name implies, aGPCRs facilitate cell adhesion, or the ability of cells to stick together, while transmitting signals within the body, the journal Nature Communications reported.
aGPCRs play crucial roles in processes like tissue growth, immune system function, and organ development. However, disruptions in their function can lead to serious conditions such as cancer, neurological disorders, and growth abnormalities. Despite their significance, no drugs have been approved to target aGPCRs. This is largely because these receptors are exceptionally large, complex, and challenging to study.
New research from the University of Chicago combines two powerful imaging techniques to study the complete structure of a common aGPCR, including how its long and complex extracellular region interacts with the transmembrane region embedded in the cell surface. The different positions and movements of the extracellular region appear to be an important way to activate the receptor.
“This opens up new opportunities for drugging adhesion GPCRs, because now we are showing that the extracellular region is communicating with the transmembrane region,” said Demet Araç, PhD, Associate Professor of Biochemistry and Molecular Biology at UChicago and senior author of the new study.
The extracellular region of an aGPCR extends from the cell membrane into space outside the cell, where it can bind to molecules and receptors from other cells. It consists of several domains, including the GPCR Autoproteolysis INducing (GAIN) domain, which can cleave itself into two pieces.
The common understanding of how to activate an aGPCR is that a ligand from outside the cell attaches to one of the extracellular domains and exerts a force that separates the GAIN domain from its other piece, a peptide called the tethered agonist (TA) that remains attached to the transmembrane region. When the TA is separated, it can move and interact with the transmembrane region to initiate signaling, but a growing body of biochemistry research shows that many aGPCR functions don’t rely on this cleavage-dependent mechanism. Separating the GAIN domain is also irreversible, leaving the receptor in a constant “on” state, which may be harmful for the cell. Sometimes a cell may need to toggle a receptor on and off, so there must be some other way of doing it.
Araç’s lab has been working for 11 years to reveal the structure of full-length aGPCRs, hoping to learn how incoming signals are transmitted from outside to inside the cell. These receptors are notoriously difficult to fully understand because the extracellular regions have many complex and distinct configurations. Graduate student Szymon Kordon, PhD, led the new study, picking up the work of a previous student to capture images of the complete structure of Latrophilin3, an aGPCR involved in developing brain synapses that have also been linked to attention deficit hyperactivity disorder and several cancers.
Kordon and Araç optimized generation and purification of Latrophilin3 and captured initial electron microscopy images, but they faced numerous challenges to get a good picture of the receptor. They then worked with Antony Kossiakoff, PhD, the Otho S.A. Sprague Distinguished Service Professor of Biochemistry and Molecular Biology at UChicago, to create a synthetic antibody that could attach to the aGPCR. This antibody stabilized the extracellular region and lent it a distinctive shape that allowed Kordon to capture the full receptor structure using cryo-electron microscopy (cryo-EM), an imaging technique that freezes cells and molecules for a snapshot. The resulting images became the first known structure of a complete aGPCR.
The cryo-EM images showed that the GAIN domain of the receptor assumed several different positions in relation to the cell surface. Each different position of the GAIN domain created a different contact point between it and the transmembrane region. The researchers wondered if these different configurations could be a different means of communicating to the cell, without separating the GAIN domain completely. So, they partnered with Reza Vafabakhsh, PhD, Associate Professor of Molecular Biosciences at Northwestern University, and Kristina Cechova, PhD, a postdoctoral researcher at Northwestern, to run a second series of experiments that tracked the movements of the extracellular regions.
Cechova and the team used Förster resonance energy transfer (FRET) imaging, which can measure the energy transfer between molecules that are close to each other. After attaching fluorescent markers to different points on both the extracellular and transmembrane regions of the aGPCR, they could track its movements as it responded to adhesion forces pulling and pushing on it. What they saw confirmed their suspicion about the function of the different configurations.
“Different conformational states correlated to different signaling activity of the receptor,” Kordon said. “That shows the functional relevance of these conformations on the downstream signaling in the cell.” Kordon, who graduated in 2024, later received the Best Dissertation Award from the Department of Biochemistry and Molecular Biology at UChicago for his work on this project.
Araç said that now that they have a better understanding of the structure of aGPCRs and how they work, they can see the potential for targeting them with drugs in the same way as other receptors. Researchers could engineer antibodies like the ones used in this study to stabilize them for imaging but designed to manipulate their activity instead. Since aGPCRs have distinct shapes and structures, these antibodies could be very precise as well. With 33 different aGPCRs already identified in humans, there are plenty of opportunities.
“This could be the future of drugging adhesion GPCRs,” Araç said. “The advantage of this is that extracellular regions are very different from each other, so you can target them with a drug that doesn’t bind to other receptors and cause unwanted side effects.”
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