Mechanism of action of the monosialoganglioside GM1 as a modulator of CD4 expression. Evidence that GM1-CD4 interaction triggers dissociation of p56lck from CD4, and CD4 internalization and degradation.
Analyzing the mechanisms underlying the capability of the monosialoganglioside GM1 to induce CD4 modulation we observed that GM1 has a dual effect on the CD4 molecule. GM1 treatment of the lymphoma cell line MOLT-3 and CD4-transfected HeLa cells for times shorter than 30 min prevented binding of monoclonal antibodies (mAbs) recognizing epitopes located within the first NH2-terminal domains of CD4, but not of the OKT4 mAb, which binds to the region of CD4 proximal to the transmembrane domain. However, no binding of the OKT4 mAb was observed after a few hours of treatment with GM1 in both MOLT-3 cells and HeLa cells transfected with an intact CD4 molecule, but not in HeLa cells transfected with a CD4 molecule lacking the bulk of the cytoplasmic domain, suggesting that modulation of CD4 by GM1 depends on the integrity of the cytoplasmic domain. GM1 treatment blocked binding of several mAbs which recognize epitopes located within the first two NH2-terminal domains of CD4 and did not induce CD4 down-modulation if MOLT-3 cells were preincubated with the OKT4A or the OKT4 mAbs. Immunoprecipitation studies with [35S]methionine-labeled MOLT-3 cells showed that GM1-induced CD4 down-modulation was accompanied by CD4 degradation, and this was preceded by dissociation of p56lck from CD4. GM1-induced CD4 down-modulation, dissociation of p56lck from CD4, and CD4 degradation were unaffected by staurosporine, which, on the contrary, blocked these events in response to phorbol 12-myristate 13-acetate. These observations demonstrate that the first action of GM1 is to mask epitopes located within the first two NH2-terminal domains; then, GM1 triggers protein kinase C-independent signals which cause p56lck dissociation from CD4 and the delivery of the molecule to an intracellular compartment where it is eventually degraded.
Saggioro
D
D
Institute of Oncology, University of Padova, Italy.
Sorio
C
C
Calderazzo
F
F
Callegaro
L
L
Panozzo
M
M
Berton
G
G
Chieco-Bianchi
L
L
D000470:Alkaloids / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015704:CD4 Antigens / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D004220:Disulfides / Q000032:analysis; D000939:Epitopes / Q000032:analysis; D016650:Fluorescein-5-isothiocyanate; D005677:G(M1) Ganglioside / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008214:Lymphocytes; D011487:Protein Conformation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D004220:Disulfides; D000939:Epitopes; D005677:G(M1) Ganglioside; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D011493:Protein Kinase C; D019311:Staurosporine; D016650:Fluorescein-5-isothiocyanate; D013755:Tetradecanoylphorbol Acetate