The central feature of B cell development is the regulated production of first the pre-B cell receptor and then immunoglobulin, the functional antigen receptor. The subunit composition of receptors, the amount of protein synthesized and the traffic of the receptors are subject to large and regulated changes. Through their ability to associate with individual subunits and assembly intermediates of the antigen receptors, molecular chaperones in the endoplasmic reticulum are, in large part, responsible for the regulation of synthesis and folding. We hypothesize that at least two chaperones, BiP and GRP94, act in concert to mediate efficient assembly of pre-B and B cell receptors and guide the repertoire selection of H and LCs that have undergone somatic mutation. Thus, the focus of this proposal is to determine how BiP recognizes newly synthesized receptor subunits and how GRP94, which we showed to be a different type of chaperone, recognizes its substrates and interfaces with BiP.
The specific aims are: 1) To use a new cell-free assay to map BiP binding sites in Ig variable domains, determine if they are similar in wild type and in pathologic Ig and study how BiP and GRP94 chaperone aggregation-prone Ig. 2) To test the mechanism by which Ig is relayed from BiP to GRP94 using in vivo experiments with a dominant-negative mutant of BiP and in vitro experiments with purified BiP, GRP94 and LCs. 3) To define how BiP and GRP94 associate with the three subunits of the pre-B cell receptor during its assembly and compare the assembly of the surrogate LC when paired with H chains having different VH domains. 4) To ablate expression of GRP94 via homologous recombination in order to assess its role in lymphocyte development. Because immunoglobulins share a great deal of structural homology with many other antigen receptors, co-receptors and adhesion molecules, it is highly likely that the mechanisms defined by this work will have widespread implications for other aspects of immunology.
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