The chemokines are a newly discovered class of cytokines that are important in host defense and that function in recruitment and activation of various hematopoietic cells. The sixteen chemokines identified to date are divided into the CXC (alpha) and CC (beta) subfamilies. Many CC chemokines are monocyte chemoattractants, but appear to have distinct activities when assay on cells other than monocytes, as well as distinct binding specificities for a complex array of chemokine receptors. All chemokines share a propensity to bind to glycosaminoglycans, a property thought to be important in their immobilization on cells surface and in the extracellular matrix and subsequent presentation to target leukocytes. However the precise inter-relationships between chemokine structure, receptor binding, glycosaminoglycan binding, and cellular activation have not been resolve. The applicants propose to continue their studies of I-309, a monocyte chemoattractant discovered by them, using mutagenesis to identify amino acid residues that determine biological activity (chemotaxis and Ca++ mobilization) on monocytes. Selective mutagenesis of chemokines MCP-1 and MIP-1-alpha will determine whether results for I-309 are general ones. They will use mutagenesis to identify glycosaminoglycan binding site of MIP-1-alpha RANTES, MCP-1 and I-309 and will use a panel of natural and modified glycosaminoglycans to determine whether the molecules display distinct binding specificities. An intact glycosaminoglycan binding sites appears to be required for post-binding, receptor activation step. They will use receptor-transfected cells with defects in glycosaminoglycan synthesis to determine the relationship between glycosaminoglycan binding and cellular activation by chemokines, and will evaluate the role of glycosaminoglycans in chemokine presentation. They will continue studies of chemokine association states by asking whether the chemokines IL-8, MCP-1 and MIP-1-alpha, which are monomers in solution will form dimers on the cell surface due to interactions with glycosaminoglycans and/or receptors, and will test whether dimer formation is relevant to chemokine biological activity. Finally, they propose to define I-309 receptors on monocytes by performing direct and competition binding experiments and by screening cloned and expressed """"""""orphan receptors"""""""".
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