Ligand-induced phosphorylation/dephosphorylation of the endogenous bradykinin B2 receptor from human fibroblasts

We have studied the ligand-induced phosphorylation/dephosphorylation of the bradykinin B2 receptor endogenously expressed in human HF-15 fibroblasts. An antiserum (AS346) to a synthetic peptide (CRS36), derived from the extreme carboxyl terminus of the human B2 receptor, precipitated the receptor from solubilized membranes of HF-15 cells that had been labeled with [32P]orthophosphate. A low basal level of B2 receptor phosphorylation was found in the absence of a ligand. Stimulation of the cells with the B2 receptor agonists bradykinin, [Lys0,Hyp3]bradykinin, kallidin, and T-kinin resulted in a rapid and efficient phosphorylation of the receptor. The B2 receptor antagonist HOE140 and the B1 receptor agonist des-Arg9-bradykinin failed to induce significant phosphorylation of the B2 receptor. Phosphoamino acid analysis revealed that the B2 receptor is phosphorylated on serine and threonine, but not on tyrosine residues. The ligand-induced phosphorylation of the receptor was concentration-dependent, with an apparent EC50 of 33 nM, and peaked at 1 min after challenge. The kinin-stimulated phosphorylation of the B2 receptor was rapid and transient and paralleled the kinetics of desensitization/resensitization of the receptor as followed by [Ca2+]i release and radioligand binding assay, respectively. The ligand-induced phosphorylation of the B2 receptor was independent of the protein kinase C pathway. In vitro experiments suggest betaARK1 (beta-adrenergic receptor kinase) as a candidate kinase that could mediate the homologous B2 receptor phosphorylation. Inhibitors of protein phosphatases 1 and 2A effectively blocked the dephosphorylation, but did not affect the internalization of the B2 receptor, whereas inhibitors of receptor internalization delayed its dephosphorylation. These finding point to a role of ligand-induced phosphorylation in the desensitization and redistribution of the bradykinin receptor in human fibroblasts.