gG Fc receptors (FcgammaR) represent a key link between the humoral and cellular immune systems. Crosslinking of FcgammaR by immune complexes leads to the activation of pleiotropic cell effector functions, including antibody-dependent cytotoxicity, phagocytosis, stimulation of the oxidative burst, and induction of cytokine synthesis. Experiments with knockout mice lacking FcgammaR expression have confirmed their importance in immune complex-medicated tissue injury. Knowledge of the structure and function of FcgammaR is therefore of major importance in understanding the basic pathophysiology of immune complex diseases in man. Previous studies have demonstrated that the human high-affinity FcgammaR, FcgammaRI (CD64), is constitutively associated with other proteins that are likely to play important roles in its function. These are its gamma subunit (FcepsilonRIgamma), which is shared by FcgammaRI as well as other Fc receptors, and the Src family protein tyrosine kinases Hck and Lyn. FcgammaRI and the gamma subunit probably bind directly to each other via their transmembrane domains. However, the mechanism by which Hck and Lyn associate with FcgammaRI is not known. The proposed studies will evaluate two possible mechanisms of binding of Hck to FcgammaRI. First, direct binding of each of the three non- catalytic Hck domains to FcgammaRI or FcepsilonRIgamma will be evaluated in immunoblotting experiments using a panel of GST-hck fusion proteins and in yeast two-hybrid assays. Second, the possibility that Hck binds to additional proteins that associate with FcgammaRI will be evaluated. We will assess whether the constitutive association of Hck with FcgammaRI and FcepsilonRIgamma can be reconstituted in Cos cell transfectants. If not, or if only a weak association is observed, this would suggest that additional proteins are required to mediate the association of Hck with FcgammaRI. A yeast two-hybrid screen of a THP-1 cell cDNA library will be performed to identify and clone Hck-binding proteins that may associate with FcgammaRI. Finally, the function of Hck in FcgammaRI signaling will be addressed in studies on cells transfected with full-length and catalytically silent Hck constructs. The results of these studies will contribute to our understanding of signaling via this important immune recognition receptor and its associated protein tyrosine kinases.
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