Adhesion G Protein-Coupled Receptors (aGPCRs) are crucial for mediating diverse physiological processes such as brain development, immunity, and organogenesis. Characterized by their large extracellular regions (ECRs) and seven-pass transmembrane domains (7TMs), these receptors convert extracellular stimuli (or signals), such as mechanical forces and ligand binding into downstream intracellular signals. However, the precise mechanisms underlying aGPCR activation have remained elusive.

Our recent work sought to address key gaps in this understanding, focusing on an extracellular region (ECR)-mediated mechanism – an alternative to the conventional tethered agonist (TA)-mediated activation mechanism. Through a combination of structural and functional studies, we provide a more detailed characterization of the ECR-mediated mechanism and suggest that it offers a more versatile mode of receptor activation. Here, we summarize our findings and their implications for aGPCR biology.

Insights and Challenges of the Tethered Agonist Mechanism

For years, the field has been focused on the tethered agonist (TA)-mediated model of aGPCR activation (Fig. 1 – left). This mechanism proposes that autoproteolysis within the conserved GPCR Autoproteolysis Inducing (GAIN) domain allows for the exposure of a TA peptide, which then activates the receptor through its interaction with the transmembrane domain. While compelling and widely studied, this model does not explain all observed phenomena and several findings challenge its universality:

1. Autoproteolysis Independence: Some aGPCRs lack the catalytic residues for autoproteolysis, yet remain functionally active.
2. Reversible Activation: The TA-mediated mechanism is irreversible (“one and done”), requiring de novo receptor synthesis for repeated activation.
3. Biophysical Constraints: The forces required to expose the TA peptide are often larger than typical biological forces, raising questions about the applicability of this mechanism to all physiological scenarios.

Could an alternative mechanism address these issues while offering a more flexible and reversible form of regulation? We hypothesized that the ECR-mediated mechanism (Fig. 1 – right), involving direct conformational coupling between the extracellular and transmembrane regions of aGPCRs, could provide an answer to these challenges. For our study we focused on Latrophilin-3 (ADGRL3), a key player in neurodevelopmental and oncological processes.

Structural Insights into ECR-Mediated Activation – From Domains to Holoreceptor

Studying aGPCRs is challenging, primarily due to their complex structure and the technical difficulties in stabilizing their full-length state for analysis. Many previous studies focused on isolated domains, which, while informative, failed to capture the dynamic interplay between the extracellular and transmembrane regions.

In our work, we used cryogenic electron microscopy (cryo-EM), single-molecule Förster Resonance Energy Transfer (smFRET), and ECR-targeted synthetic antibodies to explore the structural and functional dynamics of Latrophilin-3.

Through cryo-EM, we resolved the model of the Latrophilin-3 holoreceptor, revealing critical features of its organization (Fig. 2a). We found that the GAIN domain adopts a parallel orientation relative to the 7TM region, and displays constrained movement, limited to a ~45° range (Fig. 2b). This configuration suggests a possible interaction between the ECR and 7TM, supporting a potential direct coupling model. Importantly, the TA peptide is tightly packed within the core of the GAIN domain, is not exposed to the solvent, and does not interact with the transmembrane domain. This structural stability contrasts with the previous assumptions of extensive conformational flexibility of the ECR and is consistent with an ECR-mediated mechanism of signal transduction.

Modulation of aGPCR Activity Through ECR-7TM Coupling

To complement our structural data, we used smFRET to measure real-time conformational changes between the ECR and 7TM regions. These experiments revealed three distinct, stable conformational states with slow transitions between them (Fig. 3 – middle).

Synthetic antibody binders targeting the GAIN domain provided further evidence for ECR-mediated signaling. These binders not only aided structural analysis but also allowed us to modulate receptor activity experimentally, providing insights into how extracellular forces and ligands influence aGPCR signaling.

Agonistic antibodies shifted the receptor’s conformational equilibrium, promoting states potentially associated with increased signaling (Fig. 3 – left). Conversely, cancer-associated mutations at the GAIN-7TM interface altered the conformational distribution in the opposite direction, leading to reduced receptor activity (Fig. 3 – right).

These findings underscore the functional relevance of ECR-7TM coupling, correlating it with receptor signaling activity. Our results suggest that the extracellular region communicates directly with the transmembrane region, bypassing the need for TA release in many scenarios, reinforcing the hypothesis of an alternative, ECR-mediated activation mechanism.

 ECR-Mediated Mechanism Offers a Dynamic and Versatile Approach to aGPCR Activation

Our results propose a complementary activation model where the ECR directly modulates the 7TM domain through transient interactions, bypassing the need for TA exposure. This ECR-mediated mechanism offers several advantages:

1. Reversibility: Unlike irreversible TA-mediated activation, ECR-mediated activation can be dynamically regulated by ligand binding or mechanical forces, allowing the receptor to switch between active and inactive states.
2. Sensitivity to Diverse Forces: The ECR-mediated mechanism enables the receptor to respond to a wider range of mechanical stimuli, and respond to both pulling and compression forces.
3. Functional Adaptability: This mechanism accommodates activation in scenarios where autoproteolysis is absent or the TA peptide remains buried within the GAIN domain.

These features suggest that the ECR-mediated mechanism operates alongside TA-mediated activation, providing a versatile toolkit for aGPCRs to adapt to varied biological contexts (Fig. 4). The ECR-mediated activation mechanism has broad implications for the field of GPCR biology. aGPCRs represent a largely untapped class of therapeutic targets, despite their involvement in numerous diseases, including cancer, neurodevelopmental disorders, and immune dysfunction. The correlation between receptor conformation and specific signaling pathways hints at potential for signaling bias, where certain ligands or forces preferentially activate one pathway over another. Understanding aGPCRs dual activation mechanisms could help with the design of drugs that selectively modulate receptor activity, adapting therapeutic strategies to specific signaling pathways.

Conclusion

Each step of our work brought us closer to a holistic understanding of these complex receptors. We describe an ECR-mediated mechanism that complements the established TA-mediated model. By providing structural and functional evidence for direct ECR-7TM coupling, our work highlights the dynamic and versatile nature of aGPCR signaling.

While we made significant progress, many questions remain. How important the interplay between ECR- and TA-mediated mechanisms is for different physiological contexts? What is the generalizability of ECR-mediated activation across the aGPCR family? And what strategies can we exploit to translate this signaling bias for treatment benefit, utilizing the distinct conformational states of aGPCRs?

These insights lay the groundwork for future investigations into the diverse roles of aGPCRs and their potential as therapeutic targets.