The two major classes of antigen receptors on murine B lymphocytes, mIgM and mIgD, are both contained in a complex with two additional molecules, Ig‐alpha and Ig‐beta, which permit signal transduction. Accordingly, early biochemical events after antigen binding to either receptor are similar; biological effects, however, are different. Here, we describe three newly discovered intracellular proteins of 32, 37 and 41 kDa molecular mass, that are non‐covalently associated with mIgM, but not with mIgD. These proteins coprecipitate with mIgM in Triton X‐100 and Nonidet P‐40, but not in digitonin lysates. In addition, mIgM is to some extent associated with 29 and 31 kDa proteins that are predominantly associated with mIgD (see accompanying paper). Amino acid sequencing of p32 and p37 identified p32 as mouse prohibitin; this was corroborated by Western blot analysis with antibodies specific for rat prohibitin. p37 is a newly discovered protein. cDNA clones for both proteins were isolated and sequenced. The deduced amino acid sequence of p32 is identical to that of rat prohibitin. p37 is highly homologous to p32. Since prohibitin was identified as an inhibitor of cell proliferation, its association with mIgM, but not mIgD, could explain the different biological events elicited after engagement of each receptor.

The IgM and IgD classes of antigen receptor can perform different functions on B cells. However, so far no class‐specific components communicating with the cytoplasm have been found in the two antigen receptors. We have employed a new biotinylation protocol to search for intracellular membrane Ig‐associated proteins. Here we describe two proteins of 29 and 31 kDa that are associated with membrane IgD and to some extent with membrane IgM. The membrane IgM molecule is associated specifically with three proteins of 32, 37 and 41 kDa. The purification and sequencing of the two mIgD‐associated proteins revealed that they are novel proteins which are related to each other. These proteins may be the missing link between the antigen receptor and the cytoskeleton and may contribute to functional differences between membrane IgM and membrane IgD.

The B cell antigen receptor is a complex containing the antigen-binding immunoglobulin molecules and the Ig-alpha/Ig-beta heterodimer which presumably connects the B cell antigen receptor to intracellular signaling components. To analyze the functional properties of the cytoplasmic parts of the B cell antigen receptor, we used the K46 B lymphoma line (IgG2a, kappa) to express chimeric molecules composed of the extracellular and transmembrane part of the CD8 alpha molecule and the cytoplasmic sequence of either the Ig-alpha (CD8 alpha/Ig-alpha), the Ig-beta (CD8 alpha/Ig-beta) protein or the membrane-bound gamma 2a heavy chain (CD8 alpha/gamma 2a). From these three types of chimeric molecules only (CD8 alpha/Ig-alpha and CD8 alpha/Ig-beta, but not CD8 alpha/gamma 2a, could transduce signals, thus providing the first evidence that the cytoplasmic tail of Ig-alpha and Ig-beta have a signaling capacity. After cross-linking with anti-CD8 alpha antibodies, both molecules induced a similar increase in intracellular free calcium ion and in MAP kinase phosphorylation. Protein tyrosine kinases, however, were strongly activated via the CD8 alpha/Ig-alpha and only marginally via the CD8 alpha/Ig-beta molecule. This suggests that the Ig-alpha and Ig-beta proteins have distinct roles during signal transduction through the B cell antigen receptor.

SummaryMost mature B lymphocytes coexpress two classes of antigen receptor, immunoglobulin (Ig)M and IgD. The differences in the signal transduction from the two receptors are still a matter of controversy. We have analyzed B cell lines expressing IgM or IgD antigen receptors with the same antigen specificity. Cross-linking of these receptors with either antigen, or class-specific antibodies, results in the activation of protein tyrosine kinases and the phosphorylation of the same substrate proteins. The kinetic and the intensity of phosphorylation, however, was quite different between the two receptors when they were cross-linked by antigen. In membrane IgMexpressing cells, the substrate phosphorylation reached a maximum after 1 minute and diminished after 60 minutes whereas, in the membrane IgD-expressing cells, the substrate phosphorylation increased further over time, reached its maximum at 60 minutes, and persisted longer than 240 minutes after exposure to antigen. As a result, the intensity of protein tyrosine phosphorylation induced by cross-linking of membrane IgD was stronger than that induced by membrane IgM.Studies of chimeric receptors demonstrate that only the membrane-proximal C domain and/or the transmembrane part of membrane-bound IgD molecule is required for the long-lasting substrate phosphorylation. Together, these data suggest that the signal emission from the two receptors is controlled differently.T he B cell antigen receptor (BCK) t is a complex containing the membrane-bound immunoglobulin (mlg) molecule and the Ig-odlg-fl heterodimer (for reviews see references 1-4). Ig-ot and Ig-fl are glycosylated transmembrane proteins encoded by the B cell-specific genes mb-1 and B29, respectively (5, 6). Signal transduction from the cross-linked BCR involves the rapid activation of two types of protein tyrosine kinases (PTK); the src-related PTKs Lyn, Fyn, Lck, and Blk (7-9) (for review see reference 10) as well as the cytoplasmic PTK Syk/PTK72 (11-14). These enzymes phosphorylate several substrate proteins in B cells, including the BCR components Ig-ot and Ig-fl (15). The Ig-odlg-fl heterodimer plays an important role in the activation of these PTKs (16-22) and, after its tyrosine phosphorylation, becomes a target for src-homology-2-carrying proteins (23-25).All classes of mlg are associated with the Ig-ot/Ig-fl heterodimer (26). The molecular weight of the mlgD-associated Ig-ot (IgD-cr) is larger than the mlgM-associated Ig-ot (IgM-c 0 1 Abbreviations used in this paper: BAP, BCR-associated proteins; BCR, B cell antigen receptor; ECL, enhanced chemiluminescence; EGFR, epidermal growth factor receptor; mlg, membrane-bound immunoglobulin; EV, enhancer + V-promotor; NGFR, neuronal growth factor receptor; NIP, 4-hydroxy-3-iodo-5-nitrophenyl; PTK, protein tyrosine kinases; TKR, tyrosine kinase receptors.(27, 28). This size difference is due to an altered glycosylation of the extracellular domains of these proteins (26, 27). The cytoplasmic parts of the Ig-ot/Ig-fl heterodimer, however, are identical in ...