Abstract
Potentiation of initial signal transduction events through the cross-linking of the B cell antigen receptor complex appears to be dependent upon the association of membrane immunoglobulin (mIg) with Ig alpha and Ig beta. We made two groups of mutations within the COOH terminus of mIgM substituting: 1) the spacer, transmembrane, and cytoplasmic domains and 2) the NH2-terminal 2-8 amino acids within the transmembrane domain (NLWTTAST). We then evaluated the ability of the mutated receptors to associate with Ig alpha and Ig beta and to initiate signal transduction events (Ca2+ mobilization and phosphorylation by tyrosine protein kinases) after cross-linking mIgM receptors. Mutant mIgM receptors containing substitutions of gamma 2b (spacer, transmembrane, and cytoplasmic domains), AA for TT, and AAAAA for TTAST bound Ig alpha and Ig beta and initiated signal transduction events after mIgM receptor cross-linking. However, substitutions of I-A alpha (spacer, transmembrane, and cytoplasmic domains) or TTVVCALGL for NLWTTAST blocked association of Ig alpha and Ig beta and initiation of signal transduction events. Results indicate that residues within the first 8 amino acids of the transmembrane domain other than TTAST are necessary for receptor function and association with Ig alpha and Ig beta.
Highlights
We made two groups of mutations within tChOe OH ter- (22)
Activation of the membraneimmunoglobulin' or B cell antigen receptor (BCR) complex initiates a complex series of biochemical events resulting inbiological responses particular to the stagoef B cell differentiation
Mutations within the Dansmembranal Domain of mIgMThe M12.4 B cell lymphoma or the BCL, B cell leukemia cell lines were transfected with plasmids encoding wild type (p-p) or mutant mIgM receptors
Summary
MATERIALSANDMETHODS were washed three times in 0.1% Tween 20in PBS. Primary antibodies. For substitution of the entire COOH terminus (en- conjugated anti-rabbit (Tropix,Inc., Bedford, MA) was incubated with coding spacer,transmembrane, and cytoplasmicdomains),the 1.7-kilo- the membranes at a 15,000 dilution in thebuffer used with the primary base pair KpnI-XhoI fragment from g3R was replaced with the corre- antibody.Membranes were washed three times as described previously. Since the transfected receptor bears the T15 idiotype, cells colleagues(39).Peptide antibodies were affinity-purifiedby chromatogwere stained with monoclonal antibodies (AB1.2, T139.2)that required raphy through peptide coupled to CNBr-activatedSepharose 4B BCL, cell extracts were immunoprecipitated with 9.5 pg of anti-T15 antibody (Sigma) and 1-2 p1 of Protein G-agarose(36).Samples were incubated for approximately 4 h at 4 "C. Protein tyrosine phosphorylationwas detected with rabbit anti-phosphotyrosine antibodies (ICN, Costa Mesa, CA) and the alkaline phosphatase-coupled chemiluminescence Western method as described previously.
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