Cytokine networking plays an important role in the regulation of cell growth, differentiation wound healing, and the inflammatory process. For example, TNFalpha and IL-1 bind to specific cellular receptors and stimulate the secretion of a number of secondary cytokines. One such family of secondary cytokines are the chemokines which play a major role in chemotaxis of neutrophils, lymphocytes, and monocytes and also effect growth and cell functions of non-hematopoietic cells. MGSA/GRO and IL-8 are members of the chemokine-alpha group and these proteins are highly chemotactic for neutrophils, and to a lesser extent for lymphocytes and monocytes. The neutrophil receptors for IL-8 have been characterized as 7-transmembrane 6-protein coupled receptors. Two receptors have been cloned: one binds IL-8 with high affinity but not MGSA/GRO. The other binds MGSA/GRO and IL-8 with high affinity. In addition to hematopoietic cells, melanocytes and keratinocytes respond to MGSA and/or IL-8 with enhanced growth. The proposed study is based upon the hypothesis that there are a number of receptors which bind MGSA/GRO proteins and that epithelial cells and mesenchymal cells respond to these ligands differently than neutrophils, basophils, lymphocytes and monocytes, as a result of different receptors, G-protein coupling, or signal transduction pathways. In work described in this proposal we plan to l) use expression cloning, degenerate PCR, and/or low stringency cDNA library screening to identify mouse receptor homologs and to determine whether there are additional human receptors for the MGSA/GRO chemokines and to compare the affinity of the alpha, beta, gamma isoforms for these receptors; 2) determine which serine residues are phosphorylated in response to MGSA and whether these phosphorylation events enhance or inhibit signaling through the receptor and/or biological response; 3) characterize the MGSA signal transduction pathway in non-lymphoid mesenchymal and epithelial cells as compared to neutrophils. 4) determine the effects of antagonism of MGSA receptor function in vivo using a variety of techniques to eliminate receptor function. These studies will utilize cloning strategies, site- directed mutagenesis and deletion mutagenesis, classical signal transduction methodology, purification/characterization of G-proteins, eucaryotic transfection and over-expression strategies, as well as transgenic models. The ultimate goal of the studies will be to develop a mechanism for antagonizing the MGSA receptor so that we may learn more about the role of this group of chemokines in development, growth regulation, wound healing and inflammation.
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