Lateral mobility of the antagonist-occupied V₂ vasopressin receptor in membranes of renal epithelial cells

The lateral mobility of membrane integral receptors has been implicated as playing a significant role in signal transduction. The adenylate cyclase-coupled vasopressin V₂ receptor has been shown to be highly laterally mobile in membranes of LLC-PK₁ renal epithelial cells at physiological temperature using a fluorescent vasopressin agonist, with lateral mobility of the V₂ receptor proposed to play a role in both adenylate cyclase activation and ligand induced receptor internalization and down-regulation. This study reports the synthesis and characterization of two new fluorescent antagonists [(β-mercapto-β,β-cyclopentamethylene propionic acid)¹,d-Tyr²,Ile⁴,Lys⁹(N⁶-fluoresceinylaminothiocarbonyl)]AVP (FL-AVP-data) and [(β-mercapto-β,β-cyclopentamethylene propionic acid)¹,d-Tyr²,Ile⁴,Lys⁹(N⁶-tetramethylrhodamylaminothiocarbonyl)]AVP (TR-AVP-anta) for the V₂ receptor. The latter was used to determine the parameters of lateral mobility of the V₂ receptor in the non-activated antagonist-occupied form. Using fluorescence photobleaching techniques, results were largely comparable to those for agonist-occupied receptor, indicating high mobility at 37°C. Antagonistic properties of the V₂ receptor ligands are apparently not related to decreased receptor lateral mobility. Photobleaching measurements, however, did show that in contrast to V₂ agonist, V₂ antagonist did not induce receptor immobilization due to aggregation with time at 37°C, indicating that this could be of mechanistic importance in the internalization process.