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 wi ld-type CD4 gene resulted in the constitutive mobilization of calcium by these cells. The altered calcium phenotype was dependent on express ion of a completely functional CD4 molecule on the cell surface. Trans fectants receiving the vector alone or those in which a mutated CD4 ge ne lacking a functional Lck binding region failed to generate a consti tutive calcium response. In addition, CD4 wild-type transfectants over time lost CD4 expression. These CD4(-) revertants failed to constitut ively mobilize calcium. Treatment of CD4 wild-type transfected cells w ith either anti-CD45 mAb, EGTA, or PMA rapidly restored the cells to b asal levels of intracellular calcium. Analysis of CD45 cross-linking o n CD4(+) and CD4(-) normal Jurkat lines demonstrated that CD4 expressi on was required for CD45-mediated inhibition of TCR induced calcium re sponses. CD45-mediated inhibition affected the duration of the respons e rather than its magnitude. These results, taken together with the ob servations 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 c ould find no evidence of phosphorylated phospholipase C gamma 1 or con stitutive inositol 1,4,5 trisphosphate generation in the CD4 wild-type transfected cells, yet both were detectable after anti-CD3 mAb-induce d activation. Immunokinase assays of Lck and Fyn precipitated from unt reated or anti-CD45-treated wild-type transfectants demonstrated that CD45 cross-linking dephosphorylated Lck and reduced its capacity to ph osphorylate enolase. In contrast, neither Fyn autophosphorylation nor its activity was affected by CD45 crosslinking.