Structural requirements for V2 vasopressin receptor proteolytic cleavage

Area of Science:

• Molecular Pharmacology
• G Protein-Coupled Receptors (GPCRs)
• Signal Transduction

Background:

• The V2 vasopressin receptor (V2R) mediates the effects of vasopressin, regulating water reabsorption.
• Receptor regulation, including proteolytic cleavage, is crucial for controlling cellular responses.
• Understanding V2R cleavage mechanisms is key to deciphering its role in physiological processes.

Purpose of the Study:

• To investigate the ligand-induced proteolytic cleavage of the V2 vasopressin receptor.
• To identify the specific site of cleavage and the structural requirements for this process.
• To explore the functional consequences of V2R cleavage on receptor signaling and desensitization.

Main Methods:

• Expression of V2 receptors in COS cells.
• Incubation with a photoreactive V2 receptor agonist.
• Analysis of receptor cleavage using biochemical methods.
• Construction and characterization of chimeric V2/oxytocin receptors.

Main Results:

• Agonist binding induced proteolytic cleavage of V2 receptors at a conserved heptapeptide sequence within transmembrane helix 2.
• Approximately 90% of V2 receptors were cleaved, while oxytocin receptors showed resistance to proteolysis.
• Chimeric receptors with oxytocin receptor extracellular domains exhibited altered ligand binding and reduced cleavage.
• Proteolysis-resistant chimeric receptors showed enhanced cAMP accumulation and impaired desensitization in response to vasopressin.

Conclusions:

• Proteolytic cleavage of V2 receptors is an agonist-induced process requiring a specific conformation, particularly of extracellular domains.
• The cleavage site is conserved between V2 and oxytocin receptors, but V2R exhibits susceptibility.
• V2 receptor cleavage may function as a mechanism for signal termination at high hormone concentrations.