Pergamon zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA M olecularImmunology, Vol. zyxwvutsrqponmlkjihgfedcbaZYX 32, No. 10, pp. 753-159, 1995 Elsevier Science Ltd 0161-5890(%)0002&3 Printed in Great Britain THE RABBIT B CELL ANTIGEN RECEPTOR IS NON-COVALENTLY ASSOCIATED WITH UNIQUE HETEROMERIC PROTEIN COMPLEXES: POSSIBLE INSIGHTS INTO THE MEMBRANE IgM/IgD COEXPRESSION PARADOX MATTHEW G. FITTS,* DENNIS W. METZGER,? LINDA M. HENDERSHOTS and ROSE G. MAGE*§ *Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.; tDepartment of Microbiology, Medical College of Ohio, Toledo, OH 43699, U.S.A.; and SDepartment of Tumor Cell Biology, St Jude Children’s Research Hospital, Memphis, TN 38105, U.S.A. (First received 19 May 1994; accepted in revisedform 23 January 1995) Abstract-We describe several proteins that are components of the rabbit B cell receptor complex. Two proteins (37 kDa and 42 kDa) were found in non-covalent association with IgM expressed on B cells from peripheral blood. These proteins were also immunoprecipitated by anti-B29 (Ig-8) and anti-mbl (Ig-cc) monoclonal antibodies. As in the mouse and human, the IgM associated molecules were found as heteromeric structures with non-reduced apparent molecular weights of approximately 7@75 kDa. On rabbit B cells we also found these proteins in a 100-l 35 kDa complex which may represent trimeric or tetrameric structures. By Western blot, the 37 kDa protein was identified as rabbit Ig-8 (B29), suggesting that the 42 kDa protein is rabbit Ig-a. These data suggest that rabbit IgM is associated with both Is-E/P and Ig-(c+)2 or c& complexes. When similar immunoprecipitation studies were performed on lysates made from B cells isolated from appendix follicles, we found two additional IgM associated protein complexes containing 34 kDa and 36 kDa proteins. Key words: immunoglobulin, IgM, IgD, rabbit, B cell receptor. INTRODUCTION Antigen activation of the immune system is manifested through the antigen receptors expressed on the surface of B and T lymphocytes. Mouse IgM and IgD are associated with similar mIg complexes (Hombach zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA et al., 1988; Wienands et al., 1990; Chen et al., 1990; Hombach et al., 1990a; Campbell and Cambier, 1990; Reth et al., 1991). In mice, membrane IgM is associated with a glycoprotein heterodimer consisting of Ig-a (32 kDa) and Ig-fl(37 kDa) chains. Ig-a is the gene product of mb-1 (Sakaguchi et al., 1988) and Ig-/? is the gene product of B29 (Hombach et al., 1990b). Membrane IgD is associated with a similar heterodimer in which Ig-cr is proposed to be a differentially glycosylated mb-1 related protein with a molecular weight of 33 kDa (Campbell et al., 1991). Portions of Ig-cr have been shown to be associated with a third protein, Ig-y (34 kDa), which was reported to be a C-terminally truncated form of B29 (Friedrich et al., 1993). Human IgM was also shown to be associated with heterodimers that were products of, or related to, the human homologs of the B29 and mb-1 genes, but the glycosylated molecular weights for human Ig-a (47 kDa) $ Author to whom all correspondence should be addressed. Abbreviations: mIg, membrane immunoglobulin; pi, intracellu- lar p heavy chain; I,, membrane associated p heavy chain; WBC, white blood cells. and Ig-/? (37 kDa) differed from those reported for the mouse(vanNoeselet al., 1990,199l; Leprinceet al., 1992; Clark et al., 1992a; van Noesel et al., 1992). In addition, an anti-human B29 antibody precipitated multiple forms of the Ig-cr and Ig-/? chains, indicating a molecular heterogeneity among these molecules (Vasile et al., 1994). It was also shown that the same Ig-cr and Ig-B heterodimers could associate with all five immunoglobu- lin classes (Venkitaraman et al., 1991). Both Ig-0:and Ig-fl can be phosphorylated after stimulation with anti-Ig and may therefore be targets of associated kinases (Campbell and Cambier, 1990; Venkitaraman et al., 1991). In fact, the cytoplasmic tails of Ig-a and Ig-/I have been shown to associate with distinct src-related kinases and therefore may be capable of activating independent secondary pathways (Yamanashi et al., 1991; Clark et al., 19926; Choquet et al., 1994). More recently, differences were detected in the association of IgM or IgD with intracellular proteins now called B cell receptor associated proteins or BAPs (Terashima et al., 1994; Kim et al., 1994). These proteins may provide insight into the different biological effects elicited by the engagement of membrane IgM or IgD (reviewed in Ales-Martinez et al., 1991; Roes and Rajewsky, 1993; Cambier and Campbell, 1992). How- ever, recent successful production of IgD deficient mice by gene targeting showed that IgD does not play an