Characterization of LRP1 tyrosine phosphorylation and protein binding.

摘要

The low density lipoprotein receptor-related protein 1 (LRP1) is a member of the LDLR family of internalization receptors. LRP1 binds and internalizes a variety of extracellular molecules that modulate the extracellular environment. These include alpha-2-macroglobulin, a pan-specific proteinase inhibitor, and apolipoprotein E, a component in circulating lipoproteins. In addition, LRP1 has been implicated in the processing of the amyloid beta precursor protein that results in Abeta peptides, the aggregation of which is a possible cause of Alzheimer's Disease. There is growing evidence that in addition to serving as an internalization receptor, LRP1 is involved in signal transduction.; We have found that LRP1 is tyrosine phosphorylated in v-Src transformed cells. Cells expressing v-Src exhibit an oncogenic phenotype. We show that LRP1 is phosphorylated primarily at Tyr4507, which falls within an NPXY motif in its cytoplasmic domain. This is the consensus binding sequence for the Shc PTB domain. We observed that once phosphorylated, LRP1 binds the adaptor protein Shc, which associates using its PTB domain. Shc is also phosphorylated in v-Src transformed cells and it is possible that LRP1 is required for Shc phosphorylation.; It is possible that LRP1 associates with a variety of cytoplasmic proteins. We used a peptide based on the LRP1 phosphorylation site to purify proteins and identified them with mass spectrometry. These potential LRP1 binding proteins include PLCgamma, PI3K, Src, Csk, Shp-2 and Shp-1. All of these proteins are involved in signal transduction and contain at least one SH2 domain that could bind tyrosine phosphorylated LRP1. Shp-2 is a tyrosine phosphatase that, like Shc, is involved in the Ras/MAPK signal transduction pathway. We found that LRP1 bound to Shp-2 modified by tyrosine phosphorylation and SUMO-1. SUMO proteins are small molecules added to proteins that can change their localization or protect them from proteolysis. The observation that LRP1 binds modified Shp-2 in addition to other SH2-containing proteins suggests LRP1 can relay signals in a cascade.