Pro258 in transmembrane domain (TMD) 6 of the bradykinin (BK) B-2 receptor (B2R) is highly conserved among G-protein coupled receptors (GPCRs). Using mutagenesis, we show that Pro258 is required for normal trafficking of the receptor to the plasma membrane and that mutation of Pro258 to Ala or Leu but not Gly, enhances BK efficacy to induce receptor activation. Furthermore, P258A mutation suppresses the constitutive activity of a constitutively activated N113A-B2R mutant but preserves the antagonist to agonist efficacy shift previously observed on the N113A single mutant. Our data suggest that Pro258 in TMD6 is required for agonist-independent activation of the B2R and that straightening of TMD6 at the Pro-kink might favor G-protein coupling. It is also shown that Asn113 is a contact point of BK interaction and it is proposed that the release of a TMD3-TMD6 interaction involving Asn113 is crucial for the efficacy shift from antagonism toward agonism. (c) 2007 Elsevier Inc. All rights reserved.