My work during this final year has involved characterization of Autotaxin's (ATX) binding to A2058 cells, and elucidation of the signaling pathways stimulated by ATX which lead to the migratory response. Autotaxin is the motility-stimulating factor produced by A2058 cells. To date, the cell surface receptor for ATX has not been identified, although previous work demonstrated a G protein in the signal transduction pathway for ATX-mediated motility. Binding of ATX to the surface of A2058 cells was detected by incubating biotinylated ATX with cells in suspension, with or without excess unlabeled ATX, followed by washing of the cells to remove unbound ATX and extraction of the cell pellet with NP-40. Lysates were Western blotted, and cell-associated ATX was detected with HRP-conjugated avidin. From these experiments, we learned that ATX binds to cells in a specific, and saturable manner. Quantitation of its binding kinetics will require iodination of ATX in a manner which preserves its biological activity. To detect proteins which are tyrosine-phosphorylated in response to ATX, ATX was added to adherent cells at concentrations which stimulate motility. At various times, lysates were prepared, then Western blotted with anti-phosphotyrosine. Initial experiments revealed the increased phosphorylation of bands of 68 kDa and about 125 kDa after 5-10 minutes with ATX, which may include the cytoskeletal protein paxillin and focal adhesion kinase. Immunoprecipitation of lysates with anti-paxillin, followed by probing with anti-phosphotyrosine, has confirmed that paxillin is tyrosine phosphorylated in response to ATX. Future experiments will determine if this tyrosine phosphorylation is required for chemotaxis to ATX. To identify the PT-sensitive G protein which is functionally linked to the ATX receptor, A2058 membranes were initially labeled with PT and [32]P-NAD, followed by electrophoresis to resolve the labeled G protein bands. Two bands were visualized, one which reacted with antibody to Gai2, and the other which is as yet unknown (but not Gai1 or Gai3). Surprisingly, antibody to Gao did not recognize either of the labeled G proteins, although we know that these cells have the PT-sensitive Go. Treatment of membranes with ATX and 35GTPgS will label the PT-sensitive G protein involved in chemotaxis. Subsequently, this G protein will be identified through antibody reactivity or partial sequencing from immobilon.
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