Capture of an activated receptor complex from the surface of live cells by affinity receptor chromatography.
Cell surface receptors and their associated signaling pathways on the plasma membrane are key targets in understanding cellular responses. However, the isolation and identification of receptor complexes has been elusive. The Fc receptor was captured from the surface of live cells using microbeads coated with the receptor's cognate ligand, gamma globulin (IgG), and analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) alongside several controls. Live-cell affinity receptor chromatography (LARC) resulted in a partially nonredundant list of 288 proteins that were specific to the Fc receptor complex. The proteins identified were in close agreement with previously determined factors in the Fc receptor complex as demonstrated by genetic and biochemical methods and revealed novel complex members. Confocal microscopy was used to confirm recruitment of SRC, SYK, PLC, PKC, PI3K, SHIP, TEC, CDC42, RAP, PAK, GAP, GEF, GRP, and CRK to the receptor complex upon activation by the same ligand microbeads. The expression of mutants and silencing RNA against specific isoforms were used to demonstrate a functional role for novel members of the Fc receptor complex, including RHOG (RAS homologue member G), p115 RhoGEF (protein of 115-kDa RAS homologue guanine exchange factor), and CRKL (CRK-like). The recruitment of AKT pleckstrin homology (PH) domain green fluorescent protein (GFP) was used to quantify the production of phosphorylated inositol at the activated receptor complex. We conclude that it is feasible to capture an activated receptor complex from the surface of live cells using ligand-coated microbeads for identification of members of a receptor complex or pathway by LC-MS/MS.
Jankowski
Andy
A
Department of Chemistry and Biology, Faculty of Engineering, Architecture, and Science, Ryerson University, Toronto, Ont., Canada.
Zhu
Peihong
P
Marshall
John G
JG
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008264:Macrophages / Q000737:chemistry; D051379:Mice; D018613:Microscopy, Confocal; D008863:Microspheres; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis / Q000378:metabolism; D011961:Receptors, Fc / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
D007074:Immunoglobulin G; D011961:Receptors, Fc; D051057:Proto-Oncogene Proteins c-akt