GPI-anchored GFP signals Ca2+ but is homogeneously distributed on the cell surface.
Glycosyl-phosphatidylinositol (GPI)-anchored proteins are unique in that they penetrate only the outer leaflet of the plasma membrane but are still able to mediate intracellular signalling events following antibody-induced ligation. Detergent solubilisation studies suggest that microdomains exist at the cell surface within which are sequestered GPI-linked proteins. Here we report the construction and expression of a fluorescent GPI anchor on the surface of CHO, EL4, and U937 cells by fusing green fluorescent protein (GFP) to the GPI-attachment site of CD59. The resultant GFP-GPI has properties comparable to that of endogenously expressed GPI-anchored molecules as shown by Triton X-114 partitioning. However, sucrose gradient floatation showed that GFP-GPI was only partially resistant to detergent solubilisation. Furthermore confocal scanning laser microscopy revealed a homogeneous distribution of GFP-GPI at the cell surface, which only became clustered following cross-linking of the GPI anchor via an anti-GFP antibody. Surprisingly, GFP-GPI signalled Ca2+ change upon cross-linking demonstrating its signalling competence. Our results suggest that the GPI-anchor itself does not confer a clustered distribution to molecules but that clustering occurs following ligation with antibody, which allows the protein to become Ca2+ signalling competent.
Hiscox
Stephen
S
Department of Pharmacology, Therapeutics and Toxicology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.
Hallett
Maurice B
MB
Morgan
B Paul
BP
van den Berg
Carmen W
CW
D000818:Animals; D016466:CHO Cells; D020013:Calcium Signaling; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D006224:Cricetinae; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000032:analysis* / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020298:U937 Cells
D017261:Glycosylphosphatidylinositols; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins