New findings merge two advanced imaging techniques to explore the entire structure of a common adhesion G protein-coupled receptor (aGPCR). This includes examining how its lengthy and intricate extracellular portion interacts with the transmembrane part situated in the cell membrane. The varying positions and movements of the extracellular section seem to play a crucial role in activating the receptor.
Approximately 35% of drugs approved by the Food and Drug Administration target G protein-coupled receptors (GPCRs), which are proteins located within cell membranes and facilitate communication between cells. Adhesion G protein-coupled receptors (aGPCRs) represent the second largest group of these receptors in humans. As their title implies, they aid cells in adhering to one another and transmitting signals in the body.
These receptors participate in numerous biological processes, including tissue growth, immune responses, and organ development. Malfunctions in aGPCRs can result in various diseases, such as cancer, neurological disorders, and growth abnormalities. Despite their significant role in bodily functions, no drugs have been approved to specifically target aGPCRs due to their large size, complexity, and the challenges associated with studying them.
A research team at the University of Chicago has successfully combined two advanced imaging techniques to investigate the full structure of a common aGPCR. This includes understanding the interaction between its extended extracellular region and the transmembrane section embedded in the cell membrane. It appears that the different positions and movements of the extracellular segment are critical for activating the receptor.
“This opens new doorways for drug targeting of adhesion GPCRs, as we are demonstrating that the extracellular region communicates with the transmembrane section,” stated Demet Araç, PhD, Associate Professor of Biochemistry and Molecular Biology at UChicago, and senior author of the study published in this month’s Nature Communications.
Documenting new images and configurations
The extracellular part of an aGPCR stretches from the cell membrane into the surrounding environment, enabling it to interact with molecules and receptors from other cells. It comprises several domains, including the GPCR Autoproteolysis INducing (GAIN) domain, which can split itself into two pieces.
Conventionally, it is understood that a ligand from outside the cell binds to one of the extracellular domains, applying a force that separates the GAIN domain from its corresponding tethered agonist (TA) that remains attached to the transmembrane section. When the TA is detached, it can maneuver and engage with the transmembrane region to trigger signaling. However, growing biochemical evidence indicates that many functions of aGPCRs do not depend on this cleavage process. Separating the GAIN domain is also an irreversible action, leaving the receptor in a constant activated state, which may be detrimental to the cell. In certain instances, a cell may require toggling a receptor on and off, suggesting a need for alternate activation mechanisms.
Araç’s lab has dedicated 11 years to uncovering the structures of full-length aGPCRs, aiming to understand how signals are relayed from the external environment to the interior of the cell. These receptors are notoriously difficult to study fully due to the complex and various configurations of their extracellular regions. Graduate student Szymon Kordon, PhD, spearheaded this research, continuing from a previous student’s work to capture images of the complete structure of Latrophilin3, an aGPCR crucial for the development of brain synapses and associated with attention deficit hyperactivity disorder and certain cancers.
Kordon and Araç enhanced the generation and purification processes for Latrophilin3 and obtained initial electron microscopy images, although they encountered several obstacles in capturing clear images of the receptor. They collaborated with Antony Kossiakoff, PhD, a distinguished professor of Biochemistry and Molecular Biology at UChicago, to create a synthetic antibody that could attach to the aGPCR. This antibody provided stability to the extracellular region and granted it a unique shape, allowing Kordon to capture the complete receptor structure using cryo-electron microscopy (cryo-EM), an advanced imaging technique that freezes cells and molecules for a momentary image. The resulting images represent the first known structure of a complete aGPCR.
The cryo-EM images indicated that the GAIN domain of the receptor adopted several distinct positions relative to the cell membrane. Each of these positions created different contact points with the transmembrane region. The researchers speculated whether these varied configurations might serve as alternative means of communication to the cell, without necessarily separating the GAIN domain. Consequently, they collaborated with Reza Vafabakhsh, PhD, an Associate Professor of Molecular Biosciences at Northwestern University, and Kristina Cechova, PhD, a postdoctoral researcher at Northwestern, for a second series of experiments to monitor the movements of the extracellular regions.
Cechova and her team applied Förster resonance energy transfer (FRET) imaging, which quantifies the energy transfer between nearby molecules. By attaching fluorescent markers to specific points on both the extracellular and transmembrane regions of the aGPCR, they could track the movements in response to various forces acting upon it. Their observations confirmed their hypothesis regarding the function of the different configurations.
“Different conformational states correlated with varying signaling activities of the receptor,” Kordon stated. “This demonstrates the functional significance of these conformations in cellular signaling.” Kordon, who graduated in 2024, was awarded the Best Dissertation Award from the Department of Biochemistry and Molecular Biology at UChicago for his contributions to this research.
A novel method for activating receptors
Araç noted that with a clearer understanding of aGPCR structures and functions, there is potential to target them with drugs similarly to other receptors. Researchers could engineer antibodies, like those utilized in this study, to stabilize the receptors for imaging purposes, but designed to manipulate their activity instead. Given that aGPCRs have unique shapes and structures, these antibodies could be exceptionally targeted. With 33 distinct aGPCRs identified in humans, this opens up numerous avenues for exploration.
“This could represent the future of drug targeting for adhesion GPCRs,” Araç explained. “The benefit lies in the fact that extracellular regions are significantly different from one another, enabling drug targeting that minimizes unwanted interactions with other receptors and reduces side effects.”