Involvement of the amino terminus of the B(2) 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 B(2) receptor. The binding of the agonist bradykinin to the B(2) receptor endogenously expressed on PC-12 cells led to the formation of receptor dimers, whereas the B(2) antagonist HOE140 did not induce dimerization, suggesting that B(2) receptor dimerization was linked to receptor activation. Addition of a peptide corresponding to the amino terminus of the receptor reduced the amount of detected B(2) 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 B(2) receptor variants with truncated amino termini, B(2)(53) and B(2)(65), starting at amino acids 53 and 65. In contrast to the wild-type B(2) receptor and to B(2)(53), bradykinin did not induce dimerization of the B(2)(65) receptor. Both receptor variants were similar to the wild-type B(2) receptor with respect to agonist binding and signal generation. However, B(2)(65) 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 [(3)H]bradykinin on PC-12 cells. These findings suggest that the amino terminus of the B(2) receptor is necessary for triggering agonist-induced B(2) receptor dimerization, and receptor dimers are involved in receptor-mediated signal attenuation.