title: Lateral membrane protein associations of CD4 in lymphoid cells detected by cross-linking and mass spectrometry.
abstract: Interactions of membrane proteins are important in various aspects of cell function. However, weak membrane protein-protein interactions are difficult to study using techniques such as co-immunoprecipitations. CD4 is a cell surface protein involved in T cell activation and the binding of the human immunodeficiency virus to HIV target cells. Here we report the use of cross-linking followed by affinity purification of CD4 in combination with mass spectrometry for identification of proteins that are in the proximity of CD4. Besides the components of the CD4 receptor complex, CD4 and lck, we have identified by tandem mass spectrometry 17 tryptic peptides from transferrin receptor CD71, three peptides from protein phosphatase CD45, and one peptide from 4F2 lymphocyte activation antigen CD98. The efficiency of the cross-linking did not correlate with the level of cell surface expression of the detected molecules, excluding a possible bias of the cross-linking toward the most abundant cell surface molecules. Whereas the association of CD4 with CD45 has been reported, the associations with CD71 and CD98 have not been previously described. We used small-scale immunoprecipitation after cross-linking in combination with fluorescence resonance energy transfer (FRET) measurements to investigate the association between CD4 and CD71. Our data show that CD71 self-associates on the cell surface, that a small fraction of CD4 can be detected by copurifying it with CD71 after cross-linking, and that the level of association between CD4 and CD71 significantly increases after phorbol 12-myristate 13-acetate-induced endocytosis of CD4. This suggests that a small fraction of CD4 associates with clusters of CD71. As both molecules undergo endocytic recycling, the association and cross-linking result from their clustering in the same pit and/or vesicle. The CD4-CD98 association probably results from nonspecific cross-linking.
authors.affiliation: Centre for Virus Research, Westmead Millennium Institute, National Centre for HIV Virology Research, Westmead Hospital and The University of Sydney, Darcy Road, Westmead, NSW 2145, Australia.
authors.forename: Oliver K
authors.initials: OK
authors.lastname: Bernhard
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Sheil
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Cunningham
mesh_terms: D015703:Antigens, CD / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D000944:Antigens, Differentiation, B-Lymphocyte / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015153:Blotting, Western; D015704:CD4 Antigens / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004705:Endocytosis / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D027261:Fusion Regulatory Protein-1 / Q000096:biosynthesis; D006801:Humans; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D008214:Lymphocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D011990:Receptors, Transferrin; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013388:Succinimides / Q000737:chemistry; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D015704:CD4 Antigens; C068126:CD71 antigen; D003432:Cross-Linking Reagents; D027261:Fusion Regulatory Protein-1; D008565:Membrane Proteins; D011990:Receptors, Transferrin; D013388:Succinimides; D017493:Leukocyte Common Antigens; C011240:dithiobis(succinimidylpropionate); D013755:Tetradecanoylphorbol Acetate