Involvement of a guanine-nucleotide-binding component in membrane IgM- stimulated phosphoinositide breakdown

• Published in: The Journal of immunology (1950) Vol. 139; no. 11; pp. 3604 - 3613
• Main Authors: Gold, MR, Jakway, JP, DeFranco, AL
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Am Assoc Immnol 01.12.1987; American Association of Immunologists
• Subjects: Adenylate Cyclase Toxin; Animals; Applied sciences; B-Lymphocytes - drug effects; B-Lymphocytes - immunology; B-Lymphocytes - physiology; Calcium - metabolism; Cell Line; cell membranes; Cholera Toxin - pharmacology; Exact sciences and technology; GTP-Binding Proteins - physiology; guanine nucleotide-binding protein; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate - analogs & derivatives; Guanosine Triphosphate - metabolism; immunoglobulin M; Immunoglobulin M - immunology; lymphocytes B; Mice; Other techniques and industries; Pertussis Toxin; phosphatidylinositol 4,5-bisphosphate; Phosphatidylinositols - metabolism; phospholipase; Receptors, Antigen, B-Cell - immunology; Thionucleotides - metabolism; Tumor Cells, Cultured - drug effects; Tumor Cells, Cultured - immunology; Tumor Cells, Cultured - physiology; Virulence Factors, Bordetella - pharmacology
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.139.11.3604

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.