A proteomics strategy for the enrichment of receptor-associated complexes.
Multimeric protein complexes are important for cell function and are being identified by proteomics approaches. Enrichment strategies, such as those employing affinity matrices, are required for the characterization of such complexes, for example, those containing growth factor receptors. The receptor for the macrophage lineage growth factor, macrophage-colony stimulating factor (M-CSF or CSF-1), is the tyrosine kinase, c-Fms. There is evidence that the CSF-1 receptor (CSF-1R) forms distinct multimeric complexes involving autophosphorylated tyrosines in its cytoplasmic region; however, these complexes are difficult to identify by immunoprecipitation, making enrichment necessary. We report here the use of a tyrosine-phosphorylated, GST-fusion construct of the entire CSF-1R cytoplasmic region to characterize proteins putatively associating with the activated CSF-1R. Besides signalling molecules known to associate with the receptor or be involved in CSF-1R-dependent signalling, mass spectrometry identified a number of other molecules binding to the construct. So far among these candidate proteins, dynein, claudin and silencer of death domains co-immunoprecipitated with the CSF-1R, suggesting association. This affinity matrix method, using an entire cytoplasmic region, may have relevance for other growth factor receptors.
Cross
Maddalena
M
University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia.
Nguyen
Thao
T
Bogdanoska
Violeta
V
Reynolds
Eric
E
Hamilton
John A
JA
D048868:Adaptor Proteins, Signal Transducing / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D062445:Claudin-1; D004398:Dyneins / Q000302:isolation & purification / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation / Q000379:methods; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; C487979:Bag4 protein, mouse; D062445:Claudin-1; C568113:Cldn1 protein, mouse; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D016186:Receptor, Macrophage Colony-Stimulating Factor; D004398:Dyneins