Characterization of chemokine receptor CXCR2 interacting proteins using a proteomics approach to define the CXCR2 "chemosynapse".
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."
Raman
Dayanidhi
D
Department of Cancer Biology, and Veterans Affairs Medical Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Neel
Nicole F
NF
Sai
Jiqing
J
Mernaugh
Raymond L
RL
Ham
Amy-Joan L
AJ
Richmond
Ann J
AJ
D018922:HL-60 Cells; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D023063:Receptors, Interleukin-8B / Q000235:genetics / Q000378:metabolism*
D011506:Proteins; D023063:Receptors, Interleukin-8B