Characterization of chemokine receptor CXCR2 interacting proteins using a proteomics approach to define the CXCR2 "chemosynapse"

Methods Enzymol. 2009:460:315-30. doi: 10.1016/S0076-6879(09)05215-X.

Abstract

Chemokine-receptor signaling is initiated upon ligand binding to the receptor and continues through the process of endocytic trafficking by the association of a variety of adaptor proteins with the chemokine receptor. In order to define the adaptor proteins that associate with CXCR2 before and after ligand activation, a protocol was developed using differentiated HL-60 cells transfected to express CXCR2 stimulated or not stimulated with ligand for one minute. CXCR2-associating proteins were isolated by immunoprecipitation with CXCR2 antibody and the eluted proteins were electrophoretically run into the separating gel directly without a stacking gel. The stained single band was subjected to in-gel trypsin digestion. The tryptic peptides were subjected to, LC/MS/MS proteomic analysis. Proteins identified in a minimum of three of four separate experiments with multiple peptides were then validated as CXCR2 adaptor proteins by coimmunoprecipitation, GST pull-down studies, and immunocytochemical CXCR2-colocalization experiments using dHL-60-CXCR2 cells. Subsequently, a functional analysis of the interaction between CXCR2 and CXCR2 interacting proteins was performed. This approach can be used to characterize chemokine receptor-associating proteins over time both before and after ligand stimulation, allowing definition of the dynamic spatial and temporal formation of a "chemosynapse."

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • HL-60 Cells
  • Humans
  • Immunohistochemistry
  • Immunoprecipitation
  • Microscopy, Confocal
  • Protein Binding
  • Proteins / metabolism*
  • Proteomics / methods*
  • Receptors, Interleukin-8B / genetics
  • Receptors, Interleukin-8B / metabolism*

Substances

  • Proteins
  • Receptors, Interleukin-8B