Cell surface GPI-anchoring of CD45 isoforms

We have designed a new cell surface expression plasmid to study the structu ral and membrane-topological requirements for functioning of different isof orms of CD45, a leucocyte specific member of the protein tyrosine phosphata se (PTPase) family of proteins. Use of this vector in cell transfection exp eriments enabled us to produce multiple CD45 isoforms (ABC, B, Null), with their extracellular segment intact,;nd the entire membrane spanning and int racellular C-terminal domain replaced by a GPI-membrane-anchor and VSV-tag. Our strategy facilitated the identification and analysis of chimeric prote ins and selection of cell clones from low transfection efficiency experimen ts. We demonstrate here that simple expression of GPI-anchored CD45 isoform s on transfected Cos-l 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 su rface, is necessary in order to engage in the recognition and/or signalling processes normally used by B- and T-cells.