CD45-mediated regulation of extracellular calcium influx in a CD4-transfected human T cell line.
Transfection of a CD4- Jurkat leukemic T cell line with the human wild-type CD4 gene resulted in the constitutive mobilization of calcium by these cells. The altered calcium phenotype was dependent on expression of a completely functional CD4 molecule on the cell surface. Transfectants receiving the vector alone or those in which a mutated CD4 gene lacking a functional Lck binding region failed to generate a constitutive calcium response. In addition, CD4 wild-type transfectants over time lost CD4 expression. These CD4- revertants failed to constitutively mobilize calcium. Treatment of CD4 wild-type transfected cells with either anti-CD45 mAb, EGTA, or PMA rapidly restored the cells to basal levels of intracellular calcium. Analysis of CD45 cross-linking on CD4+ and CD4- normal Jurkat lines demonstrated that CD4 expression was required for CD45-mediated inhibition of TCR induced calcium responses. CD45-mediated inhibition affected the duration of the response rather than its magnitude. These results, taken together with the observations obtained with CD4 transfected Jurkats suggested that influx of extracellular calcium was the predominant reason for the elevated levels of calcium within the cell. In support of this hypothesis, we could find no evidence of phosphorylated phospholipase C gamma 1 or constitutive inositol 1,4,5 trisphosphate generation in the CD4 wild-type transfected cells, yet both were detectable after anti-CD3 mAb-induced activation. Immunokinase assays of Lck and Fyn precipitated from untreated or anti-CD45-treated wild-type transfectants demonstrated that CD45 cross-linking dephosphorylated Lck and reduced its capacity to phosphorylate enolase. In contrast, neither Fyn autophosphorylation nor its activity was affected by CD45 cross-linking.
Mittler
R S
RS
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121.
Schieven
G L
GL
Dubois
P M
PM
Klussman
K
K
O'Connell
M P
MP
Kiener
P A
PA
Herndon
V
V
D015704:CD4 Antigens / Q000502:physiology*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine; D011940:Receptor Aggregation; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D015704:CD4 Antigens; D007295:Inositol Phosphates; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D004533:Egtazic Acid; D017493:Leukocyte Common Antigens; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium