Constitutive activation of the human bradykinin B-2 receptor induced by mutations in transmembrane helices III and VI

We report that mutation of specific residues in the human B-2 bradykinin (B K) receptor induces its marked constitutive activation, evaluated through i nositol phosphate production in COS-7 cells expressing the wild-type or mut ant receptors. We provide evidence for a strikingly high constitutive activ ation of the B-2 receptor induced by alanine substitution of the Asn(113) r esidue, located in the third transmembrane domain. These results are remini scent of our previous finding that mutation of the homologous Asn(111) resi due induces constitutive activation of the AT(1) angiotensin II receptor. B K overstimulation of the constitutively activated mutant N113A receptor was also observed. Phe replacement of the Trp(256) residue, fairly conserved i n transmembrane domain VI of G protein-coupled receptors, also induced a le ss prominent but significant constitutive activation. Interestingly, the pe ptidic HOE 140 compound and an original nonpeptidic compound LF 16 0335, wh ich both behaved as inverse agonists of the wild-type receptor expressed in COS-7 cells, became potent and efficient agonists of the two constitutivel y activated mutant N113A and W256F receptors. These parallel changes observ ed for two chemically unrelated series can serve as a basis for future stud ies of structure-function relationships and modeling of activation processe s, based on a detailed analysis of the network of helix-helix interactions, which stabilize the inactive receptor conformation and undergo rearrangeme nts on transition to activated states.