The mannose receptor (MR), discovered and characterized in alveolar macrophages, has only recently been recognized as the prototype of a new family of endocytically-and phagocytically-active membrane receptors. The MR appears to play a role in innate immunity and in first-line host defense, especially in the lung and at the interfaces between the immune system and the extracellular environment. Recent work has suggested the presence of a signal transduction pathway associated with ligand binding to the MR. These new data raise fundamental questions about the role of the MR in host defense. Moreover, they raise the generic question of whether scavenger receptors, normally associated with the uptake and disposal of microorganisms and extracellular debris, can initiated signaling pathways leading to cytokine production. The long-range goal of this project is to identify and characterize the MR signaling pathway as a means of delineating the role of the MR, and other family members, in host defense and innate immunity. The proposed work will be carried out as three specific aims. The first specific aim is to follow several leads to identify a MR-activated signal transduction pathway. Three complementary approaches will be used. We will characterize several tyrosine phosphorylated proteins which are phosphorylated in response to MR activation. The response to MR activation is wortmannin sensitive linking MR to PI 3-kinase. Protein kinase B/akt and phosphatidylinositol (4) 5-kinase, kinases linked to PI 3-kinase and to link receptors to the cytoskeleton and to activation of gene expression, will be explored as components of the MR signaling pathway. IL-6 amd IL-12 secretin will be used as readouts. Since expression of the MR is closely regulated by IFN-gamma and IL-4, the effects of these cytokines on MR signal transduction will be monitored. The second approach is based on preliminary data indicating that the MR can be isolated with one or more associated proteins. The goal of this part of the proposed is to purify and characterize MR-associated proteins and to determine their role in MR signal transduction. The third approach is to compare the MR with other members of the MR family to determine the structural requirements for signal transduction. By deleting or swapping MR domains with those of other MR family members, we hope to identify regions of the MR required for signaling. The second specific aim is to identify motifs present on the cytoplasmic tail of the MR required for endocytosis and to determine whether the same motifs are required for phagocytosis and/or signaling. Moreover, we will determine whether internalization motifs present in the cytoplasmic tail mediate activation of Rab5, the GTPase which is rate-limiting for endocytosis and required for phagocytosis. The last specific aim is based on preliminary work suggesting the presence of a phenyl-FITC binding site on the MR. We plan to characterize the phenyl binding site using fluorescence spectroscopy and to map its location on the MR using truncation constructs. The overall goal of this project is to identify the molecular components of the MR signaling pathway as a means of delineating the role of the MR, and other family members, in host defense and innate immunity.
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