The overall goal of this proposal is to study the structure and function of two recently cloned receptors for monocyte chemoattractant protein 1 (MCP-1). MCP-1 is a potent and specific monocyte agonist and chemoattractant and is a member of the chemokine family of chemoattractant proteins. MCP-1 has been implicated as an important mediator of monocytic infiltration of tissues in a wide variety of inflammatory processes including atherosclerosis. The observation that oxidized low density lipoproteins stimulate MCP-1 production in vascular wall cells provides a possible link between atherogenic lipoproteins and the recruitment of monocytes to the artery wall. To elucidate the molecular mechanisms of monocyte activation by MCP-1 we have recently cloned and sequenced two human MCP-1 receptors that differ only in their intracellular carboxyl termini. Both forms of the receptor confer robust and remarkably specific responses to nanomolar concentrations of MCP-1 when expressed in Xenopus oocytes. In this proposal the expression, properties, and origin of the two forms of the MCP-1 receptor will be examined. Stably transfected cells will be used to compare the binding affinities of MCP-1 to both forms of the receptor, and to characterize and compare their signaling pathways and mechanisms of deactivation in mammalian cells. To determine the structural basis of the two forms of the receptor we will clone the gene for the MCP- 1 receptor and identify the intron/exon boundaries. A second goal will be the identification of ligand-binding domains of the MCP-1 receptor and the closely related MIP-1alpha/RANTES receptor by constructing receptor chimeras. The approach will be to create and study chimeras in which the amino-terminal extensions and selected extracellular loops of the two receptors are interchanged. These hybrid receptors will be evaluated for their ability to bind and signal in response to added chemokines to identify regions of the receptor that determine ligand specificity. A third goal of the grant will be to elucidate the signaling pathways of the MCP-1 receptor. Preliminary experiments in COS-7 cells have revealed significant differences in signaling between the MCP-1 receptor and the closely related interleukin 8 (IL-8) receptor. A series of hybrid receptors will be constructed in which the intracellular loops of the IL-8 and MCP-1 receptor are interchanged to identify receptor domains that interact with G-proteins and mediate signal transduction. Experiments proposed in this grant will provide the first information on the relative expression, binding affinities, and mechanisms of signal transduction and deactivation of the two forms of the MCP-1 receptor.
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