Vasopressin (VP) and its non-mammalian homolog vasotocin (VT) are secreted from the posterior pituitary to control various physiological phenomena via G protein-coupled receptors. The six known subtypes of VP/VT receptor family (V1a/bR and V2a/b/c/dR) originate from a single ancestral molecule, that most likely coupled with Gαq and used Ca2+ for signaling. However, among the current VP/VT receptors, V2aR predominantly couples with Gαs and increases intracellular cAMP levels. This receptor is also known to mediate the potent antidiuretic effect of VP/VT, and therefore is clinically important. Although the structure-function relationship of V2R has been of great interest to researchers, an evolutionary perspective was missing; how did the ancestral V2aR switched its G protein selectivity? To address this question, a comparative study between V2aR and its Ca2+-signaling cognates (V2b/c/dR) is needed. The aim of the present study was to identify residues/motifs that are crucial to cAMP signaling by V2aR; chimeric receptors were engineered using the functional V2aR and V2bR of medaka. In vitro assays demonstrated that Gαs-coupling ability of the receptors can be altered by swapping a single residue in the second intracellular loop (ICL2), without affecting Gαq-coupling ability. The three-dimensional models predicted in silico suggested that the interaction between ICL2 and Gαs-αN chain and Gαs-β2/β3 loop contributes to the stabilization of the signaling complex, enhancing the receptor's capacity to stimulate cAMP pathway upon ligand binding. These findings provide new insights into the molecular and functional evolution of V2aR, as well as into the mechanisms of G protein selectivity of GPCR.
Keywords: G protein selectivity; GPCR; V2R; Vasopressin; Vasotocin.
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