LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome

J Proteome Res. 2010 Dec 3;9(12):6689-95. doi: 10.1021/pr1008288. Epub 2010 Oct 28.

Abstract

LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • CHO Cells
  • Cell Line
  • Cell Membrane / metabolism*
  • Chromatography, Liquid
  • Cricetinae
  • Cricetulus
  • Immunoblotting
  • Immunoprecipitation
  • Liver / metabolism
  • Macrophages / cytology
  • Macrophages / metabolism
  • Mass Spectrometry
  • Membrane Microdomains / metabolism
  • Membrane Proteins / analysis*
  • Membrane Proteins / isolation & purification
  • Membrane Proteins / metabolism
  • Mice
  • Nerve Tissue Proteins / metabolism
  • Neuropilin-1 / metabolism
  • Protein Binding
  • Proteomics / methods*
  • RNA Interference
  • Receptors, Cell Surface / metabolism
  • Receptors, LDL / genetics
  • Receptors, LDL / metabolism*
  • Semaphorins / metabolism
  • Signal Transduction
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism*

Substances

  • Lrp1 protein, mouse
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Plxna1 protein, mouse
  • Receptors, Cell Surface
  • Receptors, LDL
  • Sema4d protein, mouse
  • Semaphorins
  • Tumor Suppressor Proteins
  • Neuropilin-1