Palmitoylation of the vasopressin V-1a receptor reveals different conformational requirements for signaling, agonist-induced receptor phosphorylation, and sequestration

In this study, we establish that the V-1a vasopressin receptor (V1aR) is pa lmitoylated, and we show that this modification has an important functional role. Palmitoylation of the V1aR occurs within the Cys(371)/Cys(372) coupl et located in the proximal C-terminal tail domain. Substitution of these re sidues in a [C371G/C372G]V1aR construct effectively disrupted receptor palm itoylation. Our data also indicate an additional palmitoylation site at ano ther locus in the receptor, as yet undefined. [H-3]Palmitate incorporation was agonist-sensitive and increased following exposure to [Arg(8)]vasopress in (AVP). Given the hydrophobic nature of the acyl chain, palmitoylation of the C terminus of G-protein-coupled receptors has been proposed to form an additional intracellular loop. Consequently, palmitoylation/depalmitoylati on will have a profound effect on the local conformation of this domain. Th e V1aR palmitoylation status regulated both phosphorylation and sequestrati on of the receptor, and furthermore, palmitoylation, phosphorylation, and s equestration were all regulated by AVP. The palmitoylation-defective constr uct [C371G/C372G]V1aR exhibited decreased phosphorylation compared to wild- type V1aR, under both basal and AVP-stimulated conditions, and was sequeste red at a faster rate. In contrast, the binding of four different classes of ligand and intracellular signaling were not affected by palmitoylation. Th is study therefore establishes that there are different conformational requ irements for signaling, agonist-induced phosphorylation, and sequestration of the V1aR.