FUNCTIONAL STUDIES OF HTM4, A CD20/FCE RIB HOMOLOGUE

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DESCRIPTION (Adapted from Investigator's abstract): HTm4 is a newly discovered hematopoietic cell-specific gene encoding a 4-transmembrane protein homologous to the B cell-specific antigen CD20, and the beta-subunit of the high affinity IgE Fc receptor, FcecRIB. Protein structural similarities and the localization of all three genes to the same chromosomal region in II qI2-13.1 provide the first substantial evidence for a distinct family of 4-transmembrane proteins. There are data to suggest that CD20 is a Ca2+ channel. It seems likely that CD20 and HTm4 are associated with membrane proteins since the FceRIB is one chain of the complex which forms the high affinity receptor far IgE on mast cells and functions as an amplifier of signals transduced by the FceRI. These three leucocyte proteins are highly relevant for both basic research and clinical applications: Anti-CD20 antibody is used in clinical trials in the treatment of non-Hodgkin's B-cell lymphoma and genetic and biological data suggest that variants of FceRIB, including HTm4, are one cause of IgE responsiveness. We are conducting genetic association studies, in collaboration with the University of Oxford, to see if HTm4 has a major impact on atopy and asthma. This is a proposal to study the cellular and molecular function of this novel hematopoietic-specific gene identified in our laboratory named HTm4. The gene has the distinctive feature of being expressed in primitive G0 hematopoietic stem cells but is shut off in non-quiescent stem cells that are in the proliferating and differentiating phase. HTm4 codes for a cell surface membrane protein that interacts with molecules (TRAF proteins and KAP) essential to two important cellular pathways, apoptosis/transcriptional activation and cell cycle. HTm4 activates NF-kB. All these evidence indicate that HTm4 is a novel putative receptor that mediates important signaling in hematopoietic stem cells. The role of HTm4 may be different in cycling cells than in GO hematopoietic progenitors. Our aims are to determine if HTm4 is involved in stem cell cycling and differentiation by (i) inhibiting the expression of HTm4 in G0 cells with antisense and dominant negative strategies, using functionally isolated G0 stem cells; (ii) over-expressing wild type or dominant-negative HTm4 in actively cycling CD34+ hematopoietic stem cells. We will further investigate to see if HTm4 has any role in (iii) atopy and asthma and in modulation of the apoptotic program; and (iv) elucidate the nature of its interaction with KAP and other TRAF-related proteins in mammalian cells by coimmunoprecipitation technique. This proposal will not only lead to new findings about HTm4 but also to a better understanding of a novel signaling pathway of emerging importance and likely to fresh insights into mechanisms of immune diseases and their treatment.