Activation mechanism of human oxytocin receptor: a combined study of experimental and computer-simulated mutagenesis.

The aim of this study was to investigate the molecular changes associated with the transition of the human oxytocin receptor from its inactive to its active states. Mutation of the conserved arginine of the glutamate/aspartate-arginine-tyrosine motif located in the second intracellular domain gave rise to the first known constitutively active oxytocin receptor (R137A), whereas mutation of the aspartic acid located in the second transmembrane domain led to an inactive receptor (D85A). The structural features of the constitutively active and inactive receptor mutants were compared with those of the wild type in its free and agonist-bound states. The results suggest that, although differently triggered, the activation process induced by the agonist and the activating mutation are characterized by the opening of a solvent exposed site formed by the 2nd intracellular loop, the cytosolic extension of helix 5, and the 3rd intracellular loop; on the contrary, the D85A mutation prevents oxytocin from triggering the opening of a cytosolic site. On the basis of these findings, we hypothesize that this cytosolic crevice plays an important role in G protein recognition. Finally, comparative analysis of the free- and agonist-bound forms of the wild-type oxytocin receptor and alpha1B adrenergic receptor suggests that the highly conserved polar amino acids and the seven helices play similar mechanistic roles in the different G protein-coupled receptors.