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Development, growth and differentiation of cells is regulated by environmental queues which take the form of soluble or cell associated ligands that bind cell surface or intracellular receptors. In some cases, such as in the immune system, receptor mediated regulation of cell biology is very complex, involving many incoming signals that must be properly integrated. Signal integration can be accomplished at a number of molecular levels. For example, distinct receptor types may be coupled via distinct transduction pathways to unique sets of transcription regulators that complement each other. Alternatively, signals can be integrated earlier at the level of molecular events in transduction pathways, with signaling cascades activated by one receptor being modified by those activated by another. We have been interested in transduction and integration of regulatory signals in lymphoid cells in part because aberrancies in these mechanisms may lead to autoimmunity and immunodeficiency. Ongoing efforts in the laboratory address 5 major questions.

The first is the molecular mechanism underlying HIV gp120 inactivation of T cells. Binding of the gp120 virus coat protein to one of its receptors, CD4, renders T cells hypo responsive to antigen receptor stimulation and prone to undergo death by apoptosis. Our studies indicate that this inhibitory signaling is mediated by the CD4 associate tyrosine kinase Lck, via secondary associations with the SH2-containing inositol 5-phophatase SHIP and the linker downstream of Kinase Dok. Dok acts as a linker to rasGAP, a regulator of p21ras. CD4 aggregation by gp120 leads to phosphorylation of these effectors and blockade of Akt and ras activation following TCR stimulation. Despite T cell expression of partially redundant SHIP2 and Dok2, TCCR function is partially restored in SHIP and Dok knockout mice. These findings indicate that SHIP and Dok play important roles in gp120 induced loss of T cell function in AIDs. Further studies address the basis of Lck/SHIP/Dok/rasGAP interaction and downstream function.

In a second series of experiments we are trying to determine the molecular basis of anergy, a particular type of immunologic tolerance. We have discovered that anergy in B cells can be mediated by destabilization of the multi-subunit antigen receptor complex. As a consequence, information is not transduced from the antigen binding substructure (mIg) to the transmembrane transducer substructure (Ig-a/ß of the receptor. This prevents transmembrane transduction of the signal. Ongoing studies seek to determine the physiologic significance of this mechanism, and to determine if it is generalizable to T cell antigen receptors. Finally, based on these findings we are exploring approaches for pharmacologic induction of receptor destabilization. For example, we hypothesize that antibodies against epitopes in the Ig-a/ß-mIg interface may block signal transduction. Such agents might be useful for immunosuppression and for therapy in autoimmunity.

The third area of interest is the molecular basis of integration of signals transduced concurrently by B cell antigen receptors (BCR), the type 2 complement receptors (CR2) and receptors for immunoglobulin G constant regions (Fc?RIIB 1). BCR and CR2 exhibit positive cooperativity wherein receptor co-crosslinking causes as much as 10,000 for increase in BCR signal output. BCR and Fc?RIIB exhibit negative cooperativity wherein co-crosslinking of Fc?RIIB terminates BCR signaling. Our studies address the molecular basis of cooperativity of the operative signaling pathways. Findings demonstrate that both positively and negatively cooperative mechanisms target levels of phosphatidylinositol 3,4,5 triphosphate (PIP3) - a critical signaling intermediary. They do this by affecting synthesis of PIP3 by PI3-kinase and degradation of PIP3 by the inositol phosphatase SHIP. Among other aspects of these studies we are undertaking crystallographic studies on complexes of SHIP, the adaptor Grb2 and the Fc?RIIB receptor tail. Such complexes form in vivo during inhibitory signaling. Finally, the adaptor molecule Downstream of kinase, Dok, is also involved in inhibitory FcR signaling. It is recruited by SHIP and, in turn, recruits rasGAP, an activator of the GTPase activity of p21-ras. The physical basis of interaction of these molecules, as well as their functions is a major focus of our studies. Finally, we have recently extended these studies to FceRI-Fc?RIIB interactions on mast cell.

The fourth area of focus in the laboratory is signal transduction that occurs in B cells during cognate interactions with helper T cells. Of particular interest is signal transduction by MHC class II molecules. Recent studies in the laboratory have revealed that antigen stimulation of resting B cells leads within a few hours to association of MHC class II molecules with the transducers Ig-a and Ig-ß. These molecules were previously thought to associate only with the B cell antigen receptors. Ligation of MHC class II molecules on "primed" B cells by TCR/CD4 during cognate interactions appear to lead to signal transduction via the associated Ig-a Ig-ß dimers. Future studies will define the molecular basis and biological consequences of MHC mediated signaling.

Our fifth area of research focus is the decline in B cell function seen during aging. In many individuals, antibody responses to infectious agents are of decreasing affinity and effectiveness due in part to cessation of B cell production and resultant dominance of the peripheral repertoire with antigen experienced and thus long-lived marginal zone-like cells. Our studies address the dynamics of this process and the molecular basis of B lymphopause. The latter appears to result from decreased responsiveness of progenitor cells to interleukin 7. Current studies explore the possibility that this unresponsiveness results from impaired expression and/or signal transduction by IL-7 receptors.