The relationship between Fc receptor specific for IgG2b (Fc gamma 2bR) and membrane adenylate cyclase was investigated. The specific binding of IgG2b immune complexes to P388D1 cell surface Fc gamma 2bR was found to inhibit the basal, forskolin-stimulated, and NaF-stimulated activities of membrane adenylate cyclase by 53%, 57%, and 31%, respectively. On the other hand, the binding of IgG2a immune complexes to cell surface Fc gamma 2aR increased the basal activity about 2.5-fold and the forskolin- and NaF-stimulated activities slightly. The fusion of liposomes containing Fc gamma 2bR, which was obtained as phosphatidylcholine (PC) binding protein as previously described, with the cyc- membrane preparations resulted in the marked suppression of membrane adenylate cyclase, whereas the fusion of liposomes containing Fc gamma 2a, which was obtained as IgG-binding protein, led to about a 2.7-fold increase. The Fc gamma 2bR-mediated inhibition of adenylate cyclase may be due to the temporary change of the lipid environment caused by the action of phospholipase A2, which was previously shown to be associated with Fc gamma 2bR, since (1) addition of snake venom phospholipase A2 or cholate-solubilized PC-binding protein to P388D1 membrane was found to inhibit adenylate cyclase in a dose-dependent manner, (2) prior treatment of snake venom phospholipase A2 or PC-binding protein with a specific inhibitor, p-bromophenacyl bromide, significantly reduced their inhibitory action, and (3) a product of phospholipase A2 action, arachidonic acid, was found to be an effective inhibitor of membrane adenylate cyclase, whereas the other product, lysophosphatidylcholine, was much less inhibitory than arachidonic acid. Arachidonic acid appeared to interfere with the functions of both guanine nucleotide-binding stimulatory (Gs) protein and the catalytic subunit of adenylate cyclase, since exogenously added arachidonic acid significantly suppressed the GTPase activity of P388D1 membrane and the forskolin response of the adenylate cyclase activity of Gs protein deficient cyc- membrane. The primary site of action of lysophosphatidylcholine is not clear but may be other than Gs protein and/or the catalytic subunit, since it did not change either GTPase activity of P388D1 membrane or the response to forskolin of adenylate cyclase of cyc- membrane. The Fc gamma 2bR/phospholipase A2 mediated inhibition of adenylate cyclase would be a transient event in viable cells, since phospholipase A2 did not inhibit adenylate cyclase in the presence of microsomal fraction, mitochondria, and coenzyme A, suggesting the occurrence of rapid acylation of CoA and reacylation of lysolecithin.

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)

Biochemical and immunological properties of biosynthetically radiolabeled phosphatidylcholine-(PC-) binding proteins were investigated. The PC-binding proteins were extracted from the detergent lysate of biosynthetically radiolabeled P388D1 cells by affinity chromatography on PC-Sepharose and filtered through a Sephadex G-100 gel column in the presence of 6 M urea. Isoelectric focusing of the gel-filtered materials in the presence of 6 M urea revealed the presence of a major protein component of pIe of 5.8 and minor heterogeneous cellular proteins. The yield of the electrofocused PC-binding proteins based on protein determination by Lowry's method ranged from 0.7 to 4 mg per 10(9) cells. The purified PC-binding proteins appeared to be tightly associated with Triton X-100 and phospholipids in the weight ratio of 0.57 and 0.05 g/g of proteins, respectively. The majority of lipids that could be extracted from the PC-binding proteins by chloroform/methanol (2:1 v/v) are free fatty acids, whereas lipids extracted from Pronase-treated PC-binding proteins contained phosphatidylethanolamine. By amino acid analysis, the purified PC-binding proteins were found to consist of a minimum of 417 amino acid residues, suggesting a minimum molecular weight of about 38 000 for this protein. Results of radiolabeling experiments with [3H]glucosamine and amino acid analysis both showed the presence of a mole of glucosamine per a mole of the PC-binding proteins, suggesting their glycoprotein nature. About 40% of the purified PC-binding proteins coprecipitated with monoclonal anti-Fc gamma 2bR antibody (2.4G2) in detergent-containing buffer, whereas only 6% of the isolated IgG binding proteins reacted with this antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

The properties of basal and prostaglandin (PG)-stimulated adenylate cyclase of membrane preparations of P388D1 cells were investigated. Three partially purified membrane fractions were obtained by sucrose density gradient centrifugation at the final step of purification from crude homogenate. About 96% of the basal and 89% of PGE2-stimulated adenylate cyclase activity in the homogenate were recovered in three membrane fractions. Two lighter membrane fractions (I and II), which were enriched 11-fold and 8.4-fold in adenylate cyclase activity over crude homogenate, were pooled and subjected to various studies. Results suggested that the basal activity of the membrane preparations has, as in many other cell types, a relatively broad pH optimum (pH 7.5 to 8.5), requires Mg2+, which must be present in excess ATP, and is inhibited by Ca2+. Highly reactive sulfhydryl group(s), which may be present in the lipid bilayer, is required for the adenylate cyclase activity. Because both fluoride ions and GTP augment the enzymatic activity, P388D1 cell membrane adenylate cyclase must possess stimulatory guanine nucleotide-binding protein. The membrane preparations respond to exogeneously added PG by 1.5-fold to 3-fold increase in adenosine 3'-5' cyclic monophosphate (cAMP) production. The magnitude of PG-responsiveness was dependent on the types of PG and the order of potency in stimulation was PGE1 greater than PGE2 greater than PGI2. PGA1, B1, B2, F1 alpha, and F2 alpha stimulated adenylate cyclase only at the highest concentration tested.

Properties of prostaglandin (PG) E2 binding sites of a murine macrophage cell line (P388D1) were investigated. The specific binding of [3H]-PGE2 to intact P388D1 cells at 4 degrees C in the presence of cytochalasin D (10 micrograms/ml) approached saturation at concentration greater than 7.5 X 10(-9) M, and could be displaced most effectively by unlabeled PGE2 and less effectively by unlabeled PGI2. The Scatchard analysis of the binding data clearly indicated the heterogeneity with respect to the PGE2 binding affinity and showed the presence of about 3.9 fmol/10(8) cells of the high affinity sites (KD = 1.1 X 10(-9) M) and about 24 fmol/10(8) cells of the low affinity sites (KD = 2 X 10(-8) M). PGE2-binding proteins were isolated from the detergent lysate of the radiolabeled P388D1 cells by affinity chromatography on Sepharose 4B coupled to PGE2. The affinity-isolated materials were further purified by successive use of Sephadex G-100 gel filtration and isoelectric focusing in the presence of dithiothreitol (1 mM) and Triton X-100 (0.5%). The final step yielded about 0.25% of the original radioactivity, which sharply focused as a single peak at pH near 6.5. The electrofocused PGE2-binding proteins migrated as a single band with a m.w. of 95,000 during SDS-PAGE. The electrofocused PGE2-binding proteins bound specifically to [3H]-PGE2 but showed again the heterogeneity with respect to their affinity.