Biochemical signal transmitted by Fc receptor for immunoglobulin G2a of a murine macrophage-like cell line, P388D1: mode of activation of adenylate cyclase mediated by immunoglobulin G2a binding proteins.
The effects of immunoglobulin G2a binding proteins isolated from P388D1 cells on adenylate cyclase of cyc- cells were investigated to explore a potential role of Fc gamma 2a receptor in the activation of the adenylate cyclase system. Immunoglobulin G (IgG) binding proteins obtained from the detergent lysate of P388D1 cells by affinity chromatography on IgG-Sepharose were separated into two fractions (denoted as IgG-B1 and IgG-B2) by Sephadex G-100 gel filtration in the presence of 6 M urea. Polyacrylamide gel electrophoretic analysis in the presence of sodium dodecyl sulfate revealed that the major component in the IgG-B1 fraction was a protein of molecular weight near 50 000, whereas the IgG-B2 fraction contained two major components of molecular weight near 25 000 and 17 000. Both IgG-B1 and -B2 proteins can be inserted into liposome consisting of phosphatidylcholine and phosphatidylethanolamine. Liposomes containing IgG-B1 proteins effectively inhibited EA2a, but not EA2b, rosetting by either S49 or P388D1 cells, suggesting their proper orientation within liposome, whereas IgG-B2-containing liposome failed to do so. Simultaneous fusion of the liposomes containing IgG-B1 and -B2 proteins with guanine nucleotide binding stimulatory (G/F) protein/Fc gamma 2aR-deficient cyc- cells resulted in the formation of the hybrid membrane whose adenylate cyclase responds to immune complex formed with IgG2a-subclass antibody (IC2a) by about a 2.7-fold increase in the activity over the control (hybrid membrane between cyc- cells and liposome containing no protein). The response appeared to be specific, since IC2b failed to stimulate the enzymatic activity of this hybrid membrane. Furthermore, IgG-B1 and -B2 proteins were able to confer their activating effects on the enzyme only in concert, since the fusion of liposomes containing either type of protein alone with cyc- cells did not result in the activation of adenylate cyclase of cyc- membrane. IgG-B1 and -B2 proteins could also confer their activating effects in concert to the enzyme in cholate-solubilized forms. Such activation was dependent on the concentration of IC2a, suppressed by the chelating agent ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and significantly inhibited by trifluoperazine, suggesting potential involvement of Ca2+ and calmodulin in the activating process.(ABSTRACT TRUNCATED AT 250 WORDS)
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008223:Lymphoma / Q000201:enzymology / Q000276:immunology; D008264:Macrophages / Q000201:enzymology / Q000276:immunology*; D051379:Mice; D008811:Mice, Inbred DBA; D011494:Protein Kinases / Q000378:metabolism; D011961:Receptors, Fc / Q000378:metabolism*; D014268:Trifluoperazine / Q000494:pharmacology
D007074:Immunoglobulin G; D008081:Liposomes; D011961:Receptors, Fc; D000246:Adenosine Diphosphate Ribose; D014268:Trifluoperazine; D011494:Protein Kinases; D020558:GTP Phosphohydrolases; D000262:Adenylyl Cyclases