A convenient method for the construction and expression of GPI-anchored proteins.

A wide range of proteins are attached to the external face of the plasma membrane of eukaryotic cells via a glycosyl—phosphatidyl-inositol (GPI) anchor (1,2). The function of many GPIanchored proteins is to transport small molecules and ions across the plasma membrane by the process of potocytosis (3). Synthetic GPI-anchored versions of some type I integral membrane proteins and secreted proteins have been generated by replacement of their TM and cytoplasmic domains with a GPI-attachment signal (1,4—8). One of these GPI-proteins has been used as a targeting molecule for liposomes (9); GPI-proteins could also be used for. exploring the function of the transmembrane domain of type I membrane proteins. A simple method for the construction of GPI-anchored proteins is not available. We describe here the construction of a GPIanchored version of FcyRI, the high affinity receptor for IgG (10), by using the signal for GPI-anchor attachment from the cell surface receptor molecule, LFA-3 (11) using the eukaryotic expression vector CDM. We then show how this clone can be used in a simple one step cloning procedure to transform other type I membrane proteins into GPI-anchored proteins. This method should work equally well with secreted proteins. To generate a GPI-anchored version of FcyRI, two clones were constructed. The first chimeric receptor (FC7RI-LFA3/28) comprises the complete extracellular region of FcyRI fused to the carboxy-terminal 28 amino acids of LFA-3 via a BamHI linker (Fig. 1) and includes only part of the predicted signal for GPIanchor attachment (1,11). The second chimeric receptor (FcyRILFA3/31) has an additional three amino acids, which includes the complete signal for GPI-anchor attachment. Plasmid DNA was transiently transfected into COS cells using the DEAE-dextran method (10). Both FcyRI-LFA3/28c and FcyRI-LFA3/31 were expressed at similar levels to the wild type FcyRI (Fig. 2A-C) . To determine whether these proteins were GPI-linked, cells were treated with phosphatidylinositol-specific phospholipase C (PIPLC), an enzyme which hydrolyses GPIanchors and thereby liberates the protein from the cell surface; this loss can be detected by immunofluorescence and FACS analysis (1-3) . Figure 2F shows that only FcyRl-LFA3/31 (renamed FcyRI-GPI) is GPI-linked, demonstrating that the carboxy-terminal 31 amino acids of LFA-3 are necessary and sufficient for GPI-anchor formation.