Involvement of the amino terminus of the B2 receptor in agonist-induced receptor dimerization

The mechanisms and the functional importance of G-protein-coupled receptor dimerization are poorly understood. We therefore analyzed dimerization of the bradykinin B2 receptor. The binding of the agonist bradykinin to the B2 receptor endogenously expressed on PC-12 cells led to the formation of receptor dimers, whereas the B2 antagonist HOE140 did not induce dimerization, suggesting that B2 receptor dimerization was linked to receptor activation. Addition of a peptide corresponding to the amino terminus of the receptor reduced the amount of detected B2 receptor dimers, whereas peptides derived from the extracellular loops had no effect. To further analyze the role of the amino terminus of the receptor in receptor dimerization, we created two different rat B2 receptor variants with truncated amino termini, B253 and B265, starting at amino acids 53 and 65. In contrast to the wild-type B2 receptor and to B253, bradykinin did not induce dimerization of the B265 receptor. Both receptor variants were similar to the wild-type B2 receptor with respect to agonist binding and signal generation. However, B265 was not phosphorylated, did not desensitize, and was not downregulated upon bradykinin stimulation. Likewise, antibodies directed to the amino terminus of the receptor partially reduced internalization of [3H]bradykinin on PC-12 cells. These findings suggest that the amino terminus of the B2 receptor is necessary for triggering agonist-induced B2 receptor dimerization, and receptor dimers are involved in receptor-mediated signal attenuation.