LATERAL MOBILITY OF THE ANTAGONIST-OCCUPIED V-2 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 V-2 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 V-2 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 [(beta-mercapto-beta,beta-cyclopentamethylene propionic acid)(1),D-Tyr(2),Ile(4),Lys(9)(N-6-fluoresceinylaminothiocarbonyl)]AVP (FL-AVP-anta) and [(beta-mercapto-beta,beta-cyclopentamethylene propionic acid)(1),D-Tyr(2),Ile(4),Lys(9)(N-6-tetramethylrhodamylaminothiocarbonyl)] (TR-AVP-anta) for the V-2 receptor. The latter was used to determine the parameters of lateral mobility of the V-2 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 degrees C. Antagonistic properties of the V-2 receptor ligands are apparently not related to decreased receptor lateral mobility. Photobleaching measurements, however, did show that in contrast to V-2 agonist, V-2 antagonist did not induce receptor immobilization due to aggregation with time at 37 degrees C, indicating that this could be of mechanistic importance in the internalization process.