Continued support is requested for analysis of integrin a431 (VLA-4) signaling. a4|31 plays an important role in several autoimmune and inflammatory diseases. The applicant found that a4B1 -dependent cell migration requires cAMP-dependent Protein Kinase (PKA)-mediated phosphorylation of the a4 cytoplasmic domain localized to the leading edge of migrating cells. He hypothesizes that spatially restricted PKA activation leads to this localized phosphorylation of a4. He will use live cell microscopy with a PKA activation biosensor, to examine the topography of PKA activity in migrating cells and examine the role of integrins in this localized activation. The applicant hypothesizes that if integrins do mediate the local activation of PKA they may do so by a direct or an A Kinase Anchoring Protein (AKAP)-mediated interaction with a PKA holoenzyme. He will therefore seek physical associations of integrins with PKA holoenzymes and AKAPs. The applicant found that integrin-associated paxillin inhibits Rac1 activation, thus maintaining the polarity of migrating cells. He hypothesizes that this Rac inhibition is mediated by a paxillin binding """"""""effector"""""""" distinct from those that regulate CAS/Crk complex formation and suspects a paxillin-binding Arf- GAP. He will examine the effects of paxillin mutations that disrupt associations with specific """"""""effectors"""""""" on the capacity of integrin-associated paxillin to block Rac activation. The role of potential effectors (e.g. Arf- GAPs) will be tested by analysis of downstream targets (e.g. Arf6) and by blocking the interaction of the effector with paxillin. Once an effector is identified, he will evaluate the mechanism by which it regulates Rac activity. Finally, the applicant hypothesizes that altering a4 integrin signaling will modify the behavior of mononuclear cells. He will therefore characterize the cellular and organismal phenotypes of previously produced knock-in mice bearing a mutation that blocks paxillin binding (a4(Y991A)). He proposes to generate mice bearing non-phosphorylatable (a4(S988A)) or pseudo phosphorylated (a4(S988D)) mutations for comparison with the a4(Y991A) mice. He will then examine these mice for the same phenotypes as the a4(Y991 A) mice. These fundamental studies will provide new insight into the regulation of inflammation and will test and advance paradigms that could lead to novel therapies for Rheumatic Diseases.
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