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 as a band of 75-80 kDa in membranes from recombinant baculovirus-infected cells and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 receptors, detected in membranes by [(3)H-bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfection. In these cells, bradykinin induced a transient increase of intracellular calcium ([Ca(2+)](i)) in fura 2-AM loaded sf21 cells, and promoted [(35)S]-GTP(gamma)S binding to membranes. The effects of bradykinin were dose dependent (with an EC(50) of 50 nmol/L for calcium mobilization) and were inhibited by N-alpha-adamantaneacetyl-D-Arg-[Hyp(3),Thi(5,8),D-phe(7)]-Bk, a specific B2 receptor antagonist. When the B2 antagonist was applied at the top of the calcium transient, it accelerated the decline of the peak, suggesting that calcium mobilization at this point was still influenced by receptor occupation. No calcium mobilization was elicited by 1 micromol/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 [Ca(2+)](i) mobilization was increased by genistein and tyrphostin A51. These tyrosine kinase inhibitors did not modify basal levels of [Ca(2+)](i). Homologous desensitization of the B2 receptor was observed after repeated applications of bradykinin, which resulted in attenuated changes in intracellular calcium. In addition, genistein promoted an increased response to a third exposure to the agonist when applied after washing the cells that had been previously challenged with two increasing doses of bradykinin. Genistein did not affect the calcium mobilization induced by activation of the endogenous octopamine G protein-coupled receptor or by thapsigargin. The B2 receptor, detected by confocal microscopy in unpermeabilized cells, remained constant at the surface of cells stimulated with bradykinin for 10 min, in the presence or absence of genistein. Agonist-promoted phosphorylation of the B2 receptor was markedly accentuated by genistein treatment. Phosphoaminoacid analysis 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 phosphorylation. Interestingly, genistein prevented agonist-induced G protein uncoupling from B2 receptors, determined by in vitro bradykinin-stimulated [(35)S]-GTP(gamma)S binding, in membranes from bradykinin pretreated cells. Our results 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 phosphorylation.
Reyes-Cruz
G
G
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D. F. 04510 México. guadaluper@intra.niddk.nih.gov
Vázquez-Prado
J
J
Müller-Esterl
W
W
Vaca
L
L
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001920:Bradykinin / Q000494:pharmacology; D065168:Bradykinin Receptor Antagonists; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D019833:Genistein / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D009655:Octopamine / Q000494:pharmacology; D019606:Phosphoamino Acids / Q000032:analysis; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000819:agonists / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D020032:Tyrphostins / Q000494:pharmacology
D065168:Bradykinin Receptor Antagonists; D019606:Phosphoamino Acids; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D020032:Tyrphostins; C114975:tyrphostin A51; D009655:Octopamine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019284:Thapsigargin; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins; D001920:Bradykinin; D002118:Calcium