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 (V_1aR) is palmitoylated, and we show that this modification has an important functional role. Palmitoylation of the V_1aR occurs within the Cys³⁷¹/Cys³⁷² couplet located in the proximal C-terminal tail domain. Substitution of these residues in a [C371G/C372G]V _1aR construct effectively disrupted receptor palmitoylation. Our data also indicate an additional palmitoylation site at another locus in the receptor, as yet undefined. [³H]Palmitate incorporation was agonist-sensitive and increased following exposure to [Arg ⁸]vasopressin (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/depalmitoylation will have a profound effect on the local conformation of this domain. The V_1aR palmitoylation status regulated both phosphorylation and sequestration of the receptor, and furthermore, palmitoylation, phosphorylation, and sequestration were all regulated by AVP. The palmitoylation-defective construct [C371G/C372G]V _1aR exhibited decreased phosphorylation compared to wild-type V _1aR, under both basal and AVP-stimulated conditions, and was sequestered at a faster rate. In contrast, the binding of four different classes of ligand and intracellular signaling were not affected by palmitoylation. This study therefore establishes that there are different conformational requirements for signaling, agonist-induced phosphorylation, and sequestration of the V_1aR.