Conformational coupling between extracellular and transmembrane domains modulates holo-adhesion GPCR function.

Area of Science:

• Structural biology
• Cellular signaling
• Biochemistry

Background:

• Adhesion G protein-coupled receptors (aGPCRs) are crucial cell-adhesion molecules regulating diverse physiological processes.
• aGPCRs possess large extracellular regions (ECRs) with a conserved GAIN domain preceding their seven-pass transmembrane (7TM) domain.
• The mechanism by which the ECR influences 7TM activity, particularly the orientation and dynamics of the ECR relative to the 7TM, remains poorly understood.

Purpose of the Study:

• To elucidate the structural and dynamic relationship between the ECR and 7TM in aGPCRs.
• To investigate how the GAIN domain's orientation and movement impact aGPCR function.
• To explore the functional consequences of alterations at the GAIN-7TM interface.

Main Methods:

• Cryo-electron microscopy (cryo-EM) for high-resolution structural reconstruction of Latrophilin3/ADGRL3.
• Single-molecule Förster Resonance Energy Transfer (smFRET) to probe ECR-7TM dynamics.
• Functional assays assessing receptor signaling in response to GAIN-targeted antibodies and mutations.

Main Results:

• The cryo-EM structure reveals a parallel orientation of the GAIN domain relative to the 7TM region with restricted mobility.
• smFRET experiments identified three distinct, slowly interconverting conformational states of the ECR relative to the 7TM.
• Modifications targeting the GAIN domain or the GAIN-7TM interface altered these conformational states, cryo-EM structures, and receptor signaling activity.

Conclusions:

• Demonstrates a direct conformational and functional coupling between the ECR and 7TM in aGPCRs.
• Suggests a mechanism where the ECR, specifically the GAIN domain, mediates aGPCR activation.
• Highlights the importance of the GAIN-7TM interface in regulating aGPCR signaling pathways.