ABSTRACT
Soluble ECDs of type I membrane receptors are often used to study receptor-ligand interactions. In addition, soluble receptors have decided advantages over their native counterparts for drug discovery research. The ability to produce large amounts of homogeneous receptor reagents greatly facilitates the development of efficient drug screening assays. In addition, information derived from structural studies of purified soluble receptors may allow the rational design of drug candidates. Typically, these reagents have been generated using a recombinant, genetic engineering approach by inserting a stop codon into the receptor eDNA sequence immediately upstream from the predicted transmembrane domain sequence. When the gene is expressed in mammalian cells, the ECD is secreted into the extracellular media. We have developed an alternative method of producing soluble ECDs which employs a strategy in which DNA encoding the ECD is fused to DNA encoding a signal sequence of a protein which is normally found on the cell surface in phosphatidylinositol glycan (PI-G) linked form (Lin et aL, 1990; Whitehorn et aL, 1995). The signal sequence directs the expression of the ECD to the cell surface where it is found linked through a PI-G moiety. The ECD can then be released in soluble form by treatment of the cells with the enzyme, PI-PLC. Because the sequences that direct the cell surface anchorage are common for every R-ECD fusion, they provide a common epitope that is recognized by a high-affinity antibody. Cells expressing high levels of ECDs can be identified, FACS-sorted and cloned. Mter cleavage with PI-PLC, the soluble ECDs can be purified, labeled and manipulated in a number
Transfected CHO cells
Soluble Receptor
CHO Cells with High Level of Receptor Expression
Immobilization of Receptor with mAb 179
Receptor Ooning
A generic expression vector, a+KH (Figure lA), has been developed for subcloning of R-ECDs sequences upstream from and in-frame with the PI-G signal sequences of human placental alkaline phosphatase (HPAP), a protein that is normally expressed in a PI-G-linked form. The plasmid contains an SRa promoter (Takebe et al., 1988) to drive the synthesis of the recombinant protein and the neor gene encoding a protein that confers resistance to the mammalian antibiotic, G418. The plasmid has been engineered such that the expressed protein will also contain the "kemptide" sequence, LRRASLG, immediately before the HPAP-derived amino acids. This sequence can serve as a substrate for protein kinase A (PKA) and therefore, facilitates the site-specific radiolabeling in vitro of soluble R-ECDs.
