Serpentine G protein-linked receptors regulate leukocyte cell adhesion and motility by conveying signals from activated endothelium and from the surrounding tissues. These signals control proadhesive and chemotactic responses required for leukocyte recruitment from the blood. The PI has used normal leukocytes as well as a model system of chemoattractant receptor-transfected L1/2 lymphoid cells to demonstrate inhibition of chemoattractant and phorbol ester-mediated activation of leukocyte integrins b Clostridium Botulinum toxin C3 transferase. These studies suggest that chemoattractant triggering of leukocyte integrins is mediated by intracellular signaling through the small GTPase rhoA. The goal of this proposal is to characterize the chemoattractant to rho to integrin signaling pathway and to define its role in leukocyte adhesion, chemotaxis, and trafficking.
Aim 1 will define the role of protein and lipid kinases in the rho GTPase pathway by assessing the effects of pharmacologic inhibitors or mutant enzymes in in vitr assays of chemoattractant-mediated adhesion of L1/2 transfectants, neutrophils lymphocytes, and monocytes, as well as biochemical assays of chemoattractant-stimulated rhoA guanine nucleotide exchange. A specific focus will be on a potential role for cAMP-dependent protein kinase A (PKA) as a gating element in chemoattractant to integrin signaling. A role for chelerythrine-sensitive serine-threonine kinases, particularly PKC zeta, in rho-dependent activation of integrins will also be explored.
Aim 2 will use dominant negative and constitutively active mutants of cdc42 and rac1 to determine if these GTPases act in concert with rhoA in chemoattractant to integrin signaling.
Aim 3 will assess the role of the GTPases and kinases implicated as important in Aims 1 and 2 in chemoattractant to integrin signaling and test their importance in regulating leukocyte motility and chemotaxis. Standard transwell and under-agarose assays will be employed.
Aim will address the physiologic importance of rhoA signaling in leukocyte adhesio and trafficking with the development and characterization of gene targeted rhoA-deficient mouse models. These studies should define signaling mechanisms regulating the adhesion of leukocytes and other cells, and may lead to novel therapeutic approaches for the control of neoplastic and inflammatory diseases
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