Adaptor proteins function to integrate signal transduction pathways by linking key signaling molecules together in a kind of 'regulation by association' or 'molecular glue'. Many adaptors such as Grb2 are broadly distributed and used in a number of cell types. Others such as SLP-76 are relatively restricted to hematopoietic cells and have more specialized functions. We have discovered two lymphocyte-associated adaptor molecules. One called GrpL (Grb2-related protein of the Lymphoid system), is closely related to Grb2 and is expressed at highest levels in lymphoid tissues. GrpL is expressed in naive B cells and not in germinal center (GC) B cells, associates with SLP-76 and interacts with SLP-76 to promote antigen-receptor induced activation of the transcription factor NF-AT. In contrast, BAM-32 (for B cell Adaptor Molecule of 32 kDa), is highly expressed in GC B cells and less so in naive B cells, has an N-terminal SH2 domain followed by a pleckstrin homology (PH) domain, and inhibits activation of NF-AT. We propose to elucidate the roles which GrpL and BAM-32 play in the regulation of B cell development and activation.
Our Specific Aims are: 1) To test the hypothesis that GrpL functions to link antigen receptor signaling to downstream cellular responses. We will transfect either wildtype GrpL and potentially dominant negative (DN) mutants of GrpL into T and B cell lines. The effect of GrpL on the activation NF-AT, NF-kappaB and AP1 dependent transcription and on the Ras and MAP family kinase pathways will be determined; 2) To test the hypothesis that GrpL and Grb2 interact with a unique set of signaling molecules in B and T lymphocytes. We will define the set of molecules with which GrpL interacts in vitro and in vivo through its SH2 domain, its SH3 domains or through its central proline-rich (PR) domain; 3) To test the hypothesis that BAM-32 regulates the NF-AT pathway through interactions with phospholipase C. We will define the molecules with which BAM-32 interacts in B cells via its SH2 domain and PH domain. 4) To test the hypotheses that GrpL and BAM-32 are essential for B and T cell development and specific immune responses to Ags. We will generate first GrpL-/-mice and then BAM32-/- mice, and test if GrpL-/- or BAM-32-/- mice have defective lymphoid development or responses to Ag.
