Regulation of the human bradykinin B2 receptor expressed in sf21 insect cells: A possible role for tyrosine kinases

The functional regulation of the human bradykinin B2 receptor expressed in sf21 cells was studied. Human bradykinin B2 receptors were immunodetected a s a band of 75-80 kDa in membranes from recombinant baculovirus-infected ce lls and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 recepto rs, detected in membranes by [H-3-bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfec tion. In these cells, bradykinin induced a transient increase of intracellu lar calcium ([Ca2+](i)) in fura 2-AM loaded sf21 cells, and promoted [S-35] -GTP(gamma)S binding to membranes. The effects of bradykinin were dose depe ndent (with an EC50 of 50 nmol/L for calcium mobilization) and were inhibit ed by N-alpha-adamantaneacetyl-D-Arg-[Hyp(3),Thi(5,8),D-phe(7)]-Bk, a speci fic B2 receptor antagonist. When the B2 antagonist was applied at the top o f the calcium transient, it accelerated the decline of the peak, suggesting that calcium mobilization at this point was still influenced by receptor o ccupation. No calcium mobilization was elicited by 1 mu mol/L (Des-Arg(9))- Bk a B1 receptor agonist that did not inhibit the subsequent action of 100 nmol/L bradykinin. No effect of bradykinin was detected in uninfected cells or cells infected with the wild-type baculovirus. Bradykinin-induced [Ca2](i) mobilization was increased by genistein and tyrphostin A51. These tyro sine kinase inhibitors did not modify basal levels of [Ca2+](i). Homologous desensitization of the B2 receptor was observed after repeated application s of bradykinin, which resulted in attenuated changes in intracellular calc ium. In addition, genistein promoted an increased response to a third expos ure to the agonist when applied after washing the cells that had been previ ously challenged with two increasing doses of bradykinin. Genistein did not affect the calcium mobilization induced by activation of the endogenous oc topamine G protein-coupled receptor or by thapsigargin. The B2 receptor, de tected by confocal microscopy in unpermeabilized cells, remained constant a t the surface of cells stimulated with bradykinin for 10 min, in the presen ce or absence of genistein. Agonist-promoted phosphorylation of the B2 rece ptor was markedly accentuated by genistein treatment. Phosphoaminoacid anal ysis revealed the presence of phosphoserine and traces of phosphothreonine, but not phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by bradykinin, could act in a step previous to receptor phosphory lation. Interestingly, genistein prevented agonist-induced G protein uncoup ling from B2 receptors, determined by in vitro bradykinin-stimulated [S-35] -GTP(gamma)S binding, in membranes from bradykinin pretreated cells. Our re sults suggest that tyrosine kinase(s) regulate the activity of the human B2 receptor in sf21 cells by affecting its coupling to G proteins and its pho sphorylation. (C) 2000 Wiley-Liss, inc.