Constitutive activation of the human bradykinin B2 receptor induced by mutations in transmembrane helices III and VI.

We report that mutation of specific residues in the human B2 bradykinin (BK) receptor induces its marked constitutive activation, evaluated through inositol phosphate production in COS-7 cells expressing the wild-type or mutant receptors. We provide evidence for a strikingly high constitutive activation of the B2 receptor induced by alanine substitution of the Asn113 residue, located in the third transmembrane domain. These results are reminiscent of our previous finding that mutation of the homologous Asn111 residue induces constitutive activation of the AT1 angiotensin II receptor. BK overstimulation of the constitutively activated mutant N113A receptor was also observed. Phe replacement of the Trp256 residue, fairly conserved in transmembrane domain VI of G protein-coupled receptors, also induced a less prominent but significant constitutive activation. Interestingly, the peptidic HOE 140 compound and an original nonpeptidic compound LF 16 0335, which both behaved as inverse agonists of the wild-type receptor expressed in COS-7 cells, became potent and efficient agonists of the two constitutively activated mutant N113A and W256F receptors. These parallel changes observed for two chemically unrelated series can serve as a basis for future studies of structure-function relationships and modeling of activation processes, based on a detailed analysis of the network of helix-helix interactions, which stabilize the inactive receptor conformation and undergo rearrangements on transition to activated states.