Transmembrane-4 superfamily (TM4SF) proteins such as CD9, CD63, CD81 and CD82 regulate cell invasion, motility, fusion, signaling, and proliferation. The mechanisms whereby these proteins function are largely unknown. We hypothesize that TM4SF functions depend on their associations with i) intracellular signaling enzymes and ii) other transmembrane proteins. Preliminary results show that a subset of TM4SF proteins specifically associate with phosphatidylinositol 4-kinase (PtdIns 4-K). Also, several TM4SF proteins associate with members of the integrin family of adhesion receptors, with a region in the integrin alpha3 subunit extracellular domain being particularly important. Preliminary evidence also suggests that TM4SF- integrin complexes act together as a functional unit to support cell migration and fusion, and that TM4SF complexes may be organized into """"""""raft-like"""""""" membrane microdomains. This proposal is aimed at better understanding of the structure, function and organization of TM4SF protein complexes. First, we will determine the critical regions within TM4SF protein intracellular domains needed for association with phosphatidylinositol 4-kinase (PtdIns 4-K). Second, we will determine the specific sequence (or sequences) within integrin chains needed for strong, moderate, and weak interactions with TM4SF proteins. Third, we will utilize mutant proteins to ascertain the importance of TM4SF- Ptdlns 4-K complexes and TM4SF-integrin complexes during cell adhesion, migration, invasion, fusion, signaling, protein turnover, and tumor cell growth in vivo. Fourth, we will use monoclonal antibody screening, and protein microsequencing to identity additional protein components in TM4SF protein complexes, and the organization of TM4SF complexes into """"""""raft- like"""""""" microdomains will be investigated. Fifth, we will prepare a """"""""knock-in"""""""" mouse, containing a mutant alpha3 integrin that lacks a TM4SF association site. This mouse will enable us to evaluate the in vivo consequences of disruption of TM4SF- alpha3beta1 integrin complexes. Together these experiments will provide considerable information regarding TM4SF protein complexes, will begin to establish the biochemical basis for some of their many proposed functions.
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