Nephrogenic syndrome of inappropriate diuresis is a recently-discovered rare disease caused by gain-of-function mutations of the V2 vasopressin receptor gene, AVPR2. To date, mutations of Phe229 and Arg137 have been identified as gain-of-function mutations in V2 vasopressin receptor (V2R). The mutant receptors lead to hyponatremia, which may lead to clinical symptoms in infants. In this study, we present a newly-identified Ile130Asn (I130N) substitution, causing NSIAD in a family. Characterization of the mutation revealed basal constitutive activity of V2R. We have demonstrated that the I130N mutation results in constitutive cAMP generation in HEK293 cells. Basal activity of the mutant receptor could be blocked by the inverse agonist tolvaptan and arginine-vasopressin stimulation enhanced the cAMP production of I130N-V2R. The mutation causes a biased receptor conformation since the basal cAMP generation activity of I130N does not lead to interaction with β-arrestin. The constitutive activity of the mutant receptor causes constitutive dynamindependent and β-arrestin-independent internalization. The inhibition of the basal internalization using dominant-negative dynamin resulted in an increased cell surface expression. In contrast to the constitutive internalization, agonist-induced endocytosis was β-arrestin dependent. Based on our findings, tolvaptan could be used for treatment of hyponatremia, shown in patients with nephrogenic syndrome of inappropriate diuresis who carry the I130N-V2R mutation.

Biased agonism on the type I angiotensin receptor (AT 1 -R) can achieve different outcomes via activation of G protein-dependent and -independent cellular responses. In this study, we investigated whether the biased activation of AT 1 -R can lead to different regulation and intracellular processing of the receptor. We analyzed b-arrestin binding, endocytosis, and subsequent trafficking steps, such as early and late phases of recycling of AT 1 -R in human embryonic kidney 293 cells expressing wild-type or biased mutant receptors in response to different ligands. We used Renilla luciferase-tagged receptors and yellow fluorescent protein-tagged b-arrestin2, Rab5, Rab7, and Rab11 proteins in bioluminescence resonance energy transfer measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. b-arrestin binding and the internalization kinetics of the angiotensin II-stimulated AT 1 -R differed from those stimulated by the biased agonists. Similarly, angiotensin IIstimulated wild-type AT 1 -R showed differences compared with a biased mutant AT 1 -R (DRY/AAY AT 1 -R) with regards to b-arrestin binding and endocytosis. We found that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane phosphatidylinositol 4,5-bisphosphate depletion. Moreover, the stability of the b-arrestin binding is a major determinant of the later fate of the internalized AT 1 -R receptor.