Neutrophils are critical for host defense against microorganisms and are the cellular mediators of acute inflammation. Neutrophils share with other cells signal transduction pathways, many of which are regulated by ras-related GTPases, including the rho proteins recently shown to regulate the actin cytoskeleton. These molecular switches are targeted to membranes by a series of post-translational modifications that include prenylation, proteolysis, and carboxyl methylation. We have established that reversible carboxyl methylation of rho proteins is GTP-dependent and is associated with neutrophil activation. We now propose to identify myeloid proteins that activate rho GTPases, to clone the gene for human myeloid prenylcysteine-directed carboxyl methyltransferase (pcCMT), and to test the hypothesis that carboxyl methylation regulates protein- protein interactions involving rho GTPases.
Specific Aim 1 - Upstream regulators of rho proteins: identification of guanine nucleotide exchange factors (GEFs) for rho proteins in human neutrophils. We will identify novel myeloid rho GEFs by affinity purification using nucleotide-free (transition state) recombinant GST-rho proteins. We will identify activation-dependent neutrophil rho GEFs as activities that promote release of [3H]GDP from recombinant GST-rho proteins, with or without in vitro prenylation.
Specific Aim 2 - Modification of rho proteins: characterization of neutrophil pcCMT. Multiple approaches will be taken: PCR cloning from an HL60 cDNA library based on homology to conserved regions of two yeast pcCMT genes, expression cloning in E. coli using a novel filter-based pcCMT assay for screening, conventional enzymatic purification using a PC/PA liposomal reconstitution assay to follow pcCMT activity, photoaffinity labeling with an artificial substrate to obviate the need to follow labile enzymatic activity, affinity purification with a crosslinkable biotinylated peptide substrate, and developing an anti- pcCMT monoclonal Ab.
Specific Aim 3 - Downstream targets of rho proteins: identification of neutrophil proteins that interact with prenylated cdc42Hs/rac.GTP. We will characterize three novel proteins that, in preliminary experiments, we have found interact with the GTP-bound form of partially purified, neutrophil cdc42Hs and rac with greater affinity than recombinant, unprocessed cdc42Hs and rac, suggesting that the interactions are affected by prenylation, carboxyl methylation, and/or association with rhoGDI. Elucidation of the signaling pathways of rho proteins in neutrophils may identify targets for novel antiinflammatory drugs and is likely to have wide-ranging biological implications.
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