The LDL receptor-related protein-1 (LRP-1 or LRP) is a large member of the LDL receptor family that recognizes more that 30 different ligands including tissue-type plasminogen activator (tPA) and is widely expressed in several tissues including vascular smooth muscle cells. Both in vivo and in vitro studies reveal that LRP is a physiological modulator of the PDGF signaling pathway and in the vasculature LRP suppresses the levels and activation state of PDGF receptor-beta and thus protects against the development of atherosclerosis. Mechanisms by which LRP modulates the PDGF signaling pathway are not well understood, and are the focus of studies in this grant. We hypothesize that LRP affects the trafficking of the PDGF receptor-beta leading to its degradation. In order to be effectively degraded, the PDGF receptor-beta must first be mono-ubiquitinated, a process that is catalyzed by the Cbl family of ubiquitin-ligases. Cbl binds to the cytoplasmic domain of LRP, and we discovered that the LRP ligand, tPA, promotes the tyrosine phosphorylation of Cbl leading to its activation. These findings raise the possibility that LRP and its ligands may cooperate to modulate PDGF receptor-beta function by promoting its ligand-induced degradation. The central hypothesis of this application is that LRP (and perhaps other LDLR family members as well) regulate the PDGF signaling pathway by modulating the function of the PDGF receptor-beta. The specific hypotheses to be tested are: 1) that LRP modulates the trafficking and degradation of activated forms of the PDGF receptor-beta by mechanisms that include sequestering of key adaptor proteins, such as Cbl, that facilitate delivery of the PDGF receptor-beta to degradative compartments;2) that certain LRP ligands, especially tissue- type plasminogen activator, modulate the degradation of the PDGF receptor-beta by promoting the phosphorylation of Cbl;and 3) that LDLR family members form a signaling complex with the PDGFR in endosomal compartments. These hypotheses will be tested in the following specific aims: 1) Identify mechanisms by which LRP modulates PDGF receptor-beta levels and activity;2) Determine if LRP ligands, such as tissue-type plasminogen activator, modulate the ability of LRP to regulate PDGF receptor-beta levels and activation;and 3) Determine if the PDGF receptor-beta forms a signaling complex with LRP in endosomes.
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