Abstract

The highly conserved “Asp-Arg-Tyr” triplet in the distal region of the third transmembrane region of most G-protein-coupled receptors is implicated in their activation process and mediation of G-protein signaling. The aim of this study was to determine whether specific features at this locus are important for the vasopressin V_1a receptor (V_1aR) by performing site-directed mutagenesis. In transfected HEK 293T cells, mutation of Asp (D148A) resulted in a misfolded receptor that was nonfunctional, localized intracellularly, and not constitutively active. Nonconservative (D148R) substitution was not expressed, whereas asparagine (D148N) partially restored cell surface expression, although no specific ligand-binding or inositol phosphate signaling was detected. In contrast, conservative (D148E) substitution was expressed moderately higher, bound ligands, and signaled similarly to a hemagglutinin epitope-tagged wild-type receptor. However, D148E showed a greater tendency to be internalized once it was delivered to the membrane. Individual replacements of the conserved arginine and tyrosine (R149A, Y150A) led to decreased signal transduction without affecting surface expression, agonist affinity, or internalization or increasing basal signaling activity. Incorporation of aspartate (R149D) or reversal of charges (D148R/R149D) were nonfunctional, localized intracellularly, and indicated the absence of an ionic interaction between Asp-148 and Arg-149. It is noteworthy that an important role of arginine was identified for regulating agonist-mediated internalization when a histidine (R149H) was present. This mutant was expressed on the cell surface but was rapidly internalized after agonist treatment. This study highlights the importance of specific charged residues within this motif that provide important determinants for cell surface delivery, internalization and for normal V_1aR function.