Interactions of CD40 and its ligand (CD154, CD40L, gp39) control the development of humoral and cell-mediated immune responses. Given the central and important function of CD40, little is known about CD40 receptor assembly and signal transduction. It is the purpose of this project, to fully explore CD40 receptor complex assembly and regulation, and to discover elements of the CD40 receptor complex that couple receptor assembly with downstream biological responses. The first goal of this application is to identify proteins that are recruited to the CD40 receptor complex upon CD154 binding. Preliminary data are presented that show that in a human B-cell line both TRAF2 and TRAF3 are recruited to the CD40 receptor complex within minutes of ligand binding. Other novel proteins, like GRP75 appear constitutively associated with CD40 in the absence of ligand. These studies will be expanded to examine the recruitment of other TRAF family members and associated molecules to the CD40 receptor complex. In addition, studies in other B-cell lines and defined subsets of ex vivo B-cell will be pursed. Using established methodologies, we will continue to identify novel proteins that bind to the CD40 cytoplasmic domain by protein absorption and microsequencing. Furthermore, specific strategies are presented to evaluate and identify the assembly of kinases into the complex because of the likelihood that CD40 transduces signals through the activation of ser/thr or tyr kinases. The second goal is to evaluate if the TRAF composition of the CD40 receptor complex is causally linked to the biological changes induced by CD154 binding. Data presented show that there is a correlation between the TRAF composition of the CD40 receptor complex and the biologic response to CD154. Genetic approaches are presented that will alter the cellular expression of TRAFs or related proteins, or change the availability of specific TRAFs to assemble into the receptor complex. Cells with altered TRAF composition will be tested for their responsiveness to CD40 signalling. These studies will causally link the function of specific TRAFs to defined CD40-driven biological responses. The third goal is to: 1) study """"""""receptor cross-talk"""""""" between CD40 and the IL4 and membrane Ig receptors; and 2) study receptor desensitization amongst CD40 and other TNFR family members. Both IL4R and mIg signalling differentially alter the TRAF composition of the CD40 receptor complex and the biological response to CD154 binding to CD40. These data establish that signals from other receptors can alter the character of the CD40 receptor complex. Studies are presented to resolve the biochemical basis for how these receptors govern CD40 receptor assembly and how this altered assembly influences CD40 signals. Data also show that CD40 engagement depletes the majority of the cytosolic TRAF2 and TRAF3 by sequestration to the receptor complex. Since other TNFR family members utilize these same signalling molecules, this suggests that receptor desensitization may be occur within this family. Studies are proposed which will evaluate if engagement of one TNFR family member can prohibit the recruitment of TRAFs by other family members; and what the functional consequences of receptor desensitization are to the B-cell.
