Conserved polar residues in the transmembrane domain of the human tachykinin NK2 receptor: functional roles and structural implications

We have studied the effects of agonist and antagonist binding, agonist-indu ced activation and agonist-induced desensitization of the human tachykinin NK2 receptor mutated at polar residues Asn-51 [in transmembrane helix 1 (TM 1)], Asp-79 (TM2) and Asn-303 (TM7), which are highly conserved in the tran smembrane domain in the rhodopsin family of G-protein-coupled receptors. Wi ld-type and mutant receptors were expressed in both COS-1 cells and Xenopus oocytes. The results show that the N51D mutation results in a receptor whi ch, in contrast with the wild-type receptor, is desensitized by the applica tion of a concentration of 1 mu M of the partial agonist GR64349, indicatin g that the mutant is more sensitive to agonist activation than is the wild- type receptor. In addition, we show that, whereas the D79E mutant displayed activation properties similar to those of the wild-type receptor, the D79N and D79A mutants displayed a severely impaired ability to activate the cal cium-dependent chloride current. This suggests that it is the negative char ge at Asn-79, rather than the ability of this residue to hydrogen-bond, tha t is critical for the activity of the receptor. Interestingly, the placemen t of a negative charge at position 303 could compensate for the removal of the negative charge at position 79, since the double mutant D79N/N303D disp layed activation properties similar to those of the wild-type receptor. Thi s suggests that these two residues are functionally coupled, and may even b e in close proximity in the three-dimensional structure of the human tachyk inin NK2 receptor. A three-dimensional model of the receptor displaying thi s putative interaction is presented.