Involvement of a guanine-nucleotide-binding component in membrane IgM-stimulated phosphoinositide breakdown.
Cross-linking of membrane immunoglobulin, the B cell receptor for antigen, activates the phosphoinositide signal transduction pathway. The initial event in this pathway is the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) by phospholipase C. This reaction yields two intracellular second messengers, diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which causes an increase in cytoplasmic Ca2+. The experiments reported here demonstrate that activation of phospholipase C by membrane IgM (mIgM) involves a guanine nucleotide-dependent step. Saponin was used to permeabilize WEHI-231 B lymphoma cells and permit direct manipulation of nucleotide and Ca2+ concentrations. Very high levels of Ca2+ (greater than 100 microM) activated the phospholipase maximally without a requirement for cross-linking of mIgM. However, at much lower, physiologically relevant Ca2+ concentrations (100 to 500 nM), receptor-stimulated PtdInsP2 hydrolysis could be demonstrated. The ability of anti-IgM antibodies to activate phospholipase C in permeabilized WEHI-231 cells was greatly increased by nonhydrolyzable guanosine 5'-triphosphate (GTP) analogues (guanosine-5'-O-(3-thiotriphosphate) or 5'-guanylylimidodiphosphate), but not by guanosine diphosphate or guanosine diphosphate analogues or by a nonhydrolyzable analogue of adenosine triphosphate. This specificity for GTP analogues is consistent with the hypothesis that a GTP-binding regulatory protein analogous to those that couple receptors to adenylate cyclase is involved in the activation of phospholipase C by mIgM in WEHI-231 B lymphoma cells. In order to characterize this putative GTP-binding component, we examined the ability of pertussis toxin and cholera toxin to affect anti-IgM-stimulated inositol phosphate production. These bacterial toxins covalently modify and modulate the activity of various GTP-binding regulatory proteins and in some cell types can block receptor-stimulated PtdInsP2 breakdown. In WEHI-231 B lymphoma cells, neither toxin blocked signaling by mIgM. Thus mIgM appears to be coupled to the phosphoinositide signaling pathway by a GTP-dependent component that is insensitive to both pertussis toxin and cholera toxin.
Gold
M R
MR
Department of Microbiology and Immunology, University of California, San Francisco 94143.
Jakway
J P
JP
DeFranco
A L
AL
D037361:Adenylate Cyclase Toxin; D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007075:Immunoglobulin M / Q000276:immunology*; D051379:Mice; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000276:immunology*; D013873:Thionucleotides / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000276:immunology / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D007075:Immunoglobulin M; D010716:Phosphatidylinositols; D011947:Receptors, Antigen, B-Cell; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium