Phagocytosis is a phylogenetically ancient response to particulate stimuli adapted by specialized cells of the immune system. Among the best characterized phagocytic receptors are those that recognize the Fc portion of IgG (Fc1Rs). Phagocytosis via FcyRs requires the recruitment and activation Syk, a 72 kDa tyrosine kinase expressed predominantly in hematopoetic cells. In this proposal we describe experiments to explore the molecular mechanisms of FcyR-mediated phagocytosis in a variety of macrophages, including transformed macrophage cell lines and primary pulmonary alveolar macrophages. We will test a two-compartment model of phagocytosis that assigns separate signal transduction requirements for actin assembly and pseudopod extension.
In Specific Aim 1, we will test the hypothesis that a Syk-binding protein is required for phagocytosis. We will identify those tyrosine residues on Syk that are required for phagocytic signaling and purify potential Syk substrates that associate with these residues. These experiments will utilize site-directed mutagenesis, expression of Syk and various Syk mutants in several complementary model systems of phagocytosis, and protein purification using affinity chromatography.
In Specific Aim 2, we will test the hypothesis that Crk, an SH2/SH3 domain-containing adaptor protein, is recruited to Syk and that Crk is required for phagocytosis. We will isolate phosphotyrosyl-containing Crk-binding proteins that interact with Crk-SH2 and determine if they correspond to known phosphotyrosyl-containing proteins; if not, we will characterize them further. We will test the hypothesis that CrkL and/or CrkII is required specifically for FcyR-mediated phagocytosis and not other forms of phagocytosis; if so, we will attempt to identify CrkL- or CrkllSH3-binding proteins that might participate in phagocytosis. To address the mechanism of P1 3-kinase-dependent pseudopod extension, we have studied myosin X (M10), a PH domain-containing unconventional myosin with high affinity for PIP3 that is expressed in murine macrophages. Truncation fragments of M1O inhibit phagocytosis of IgG-coated targets. In contrast, these mutants do not affect """"""""phagocytic cup"""""""" formation, nor do they inhibit membrane ruffling. This suggests that M10 is required specifically for pseudopod extension and phagocytosis. We will critically test this hypothesis and determine which domains of M10 are required for phagocytosis.
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