Cell surface GPI-anchoring of CD45 isoforms.
We have designed a new cell surface expression plasmid to study the structural and membrane-topological requirements for functioning of different isoforms of CD45, a leucocyte specific member of the protein tyrosine phosphatase (PTPase) family of proteins. Use of this vector in cell transfection experiments enabled us to produce multiple CD45 isoforms (ABC, B, Null), with their extracellular segment intact, and the entire membrane spanning and intracellular C-terminal domain replaced by a GPI-membrane-anchor and VSV-tag. Our strategy facilitated the identification and analysis of chimeric proteins and selection of cell clones from low transfection efficiency experiments. We demonstrate here that simple expression of GPI-anchored CD45 isoforms on transfected Cos-1 cells does not facilitate binding to CD22+ lymphoid cells. This suggests that not only the mere presence of CD45 extracellular domains but also their assembly into higher order structures at the cell surface, is necessary in order to engage in the recognition and/or signalling processes normally used by B- and T-cells.
ten Dam
G B
GB
Department of Cell Biology and Histology, Faculty of Medical Sciences, University of Nijmegen, The Netherlands.
Poels
L G
LG
Wieringa
B
B
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008214:Lymphocytes / Q000502:physiology; D008969:Molecular Sequence Data; D014066:Palatine Tonsil / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; D008024:Ligands; D020033:Protein Isoforms; D012313:RNA; D017493:Leukocyte Common Antigens; D010738:Type C Phospholipases