Exogenous glycosyl phosphatidylinositol-anchored CD59 associates with kinases in membrane clusters on U937 cells and becomes Ca(2+)-signaling competent.
CD59, an 18-20-kD complement inhibitor anchored to the membrane via glycosyl phosphatidylinositol (GPI), can induce activation of T cells and neutrophils upon cross-linking with antibody. GPI-anchored molecules cocluster in high mol wt detergent-resistant complexes containing tyrosine kinases that are implicated in the signaling pathway. Exogenous, incorporated GPI-anchored molecules are initially unable to induce activation, presumably because they are not associated with kinases. Here we demonstrate that erythrocyte-derived CD59 incorporated in a CD59-negative cell line acquires signaling capacity in a time-dependent manner. Confocal microscopy revealed an initial diffuse distribution of CD59 that became clustered within 2 h to give a pattern similar to endogenous GPI-anchored molecules. Gel filtration of detergent-solubilized cells immediately after incorporation revealed that CD59 was mainly monomeric, but after 3 h incubation all was in high mol wt complexes and had become associated with protein kinases. Newly incorporated CD59 did not deliver a Ca2+ signal upon cross-linking, but at a time when it had become clustered and associated with kinase activity, cross-linking induced a large calcium transient, indicating that CD59 had incorporated in a specialized microenvironment that allowed it to function fully as a signal-transducing molecule.
van den Berg
C W
CW
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, United Kingdom.
Cinek
T
T
Hallett
M B
MB
Horejsi
V
V
Morgan
B P
BP
D018957:CD59 Antigens / Q000502:physiology*; D002118:Calcium / Q000502:physiology*; D002784:Cholesterol / Q000378:metabolism; D003902:Detergents; D005372:Filipin / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000502:physiology*; D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse; D018613:Microscopy, Confocal; D010770:Phosphotransferases / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured / Q000502:physiology
D018957:CD59 Antigens; D003902:Detergents; D017261:Glycosylphosphatidylinositols; D005372:Filipin; D002784:Cholesterol; D010770:Phosphotransferases; D002118:Calcium