CD4 is active as a signaling molecule on the human monocytic cell line Thp-1.
CD4 is a 56-kDa membrane glycoprotein expressed by a subset of T cells, by cells of the monocyte/macrophage lineage, and by eosinophils and dendritic cells. CD4 serves as a coreceptor for HIV and IL-16. T cell CD4 mediates signal transduction by associating with the protein tyrosine kinase p56(lck); this interaction does not exist in monocytes. We wished to elucidate the mechanism(s) by which monocyte CD4 transduces signals. Stimulation of CD4 on Thp-1 monocytic cells induced a Ca(2+) flux and the time-dependent activation of phosphotyrosine proteins ranging from 35 to 180 kDa. We identified the 140- and 85-kDa proteins as phospholipase C gamma (PLC-gamma) and the regulatory subunit of phosphatidylinositol 3-kinase (PI-3K), respectively. Using immunoprecipitation/Western immunoblotting however, we were unable to show any direct association between CD4 and PLC-gamma, PI-3K, or other known signaling proteins. To identify proteins capable of associating with the cytoplasmic tail of CD4, we fused it with gluthatione S-transferase and used the fusion protein in far Western and pull-down experiments. In both types of experiments, the fusion protein routinely associated with 45- and 55-kDa proteins. Mass spectrometry analysis of the tryptic peptides generated from these two proteins indicated novel sequences.
Graziani-Bowering
Gina
G
Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, K1H 8M5, Canada.
Filion
Lionel G
LG
Thibault
Pierre
P
Kozlowski
Maya
M
D000906:Antibodies / Q000494:pharmacology; D015704:CD4 Antigens / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009000:Monocytes / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051966:Phospholipase C gamma; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
D000906:Antibodies; D015704:CD4 Antigens; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium