EXPRESSION AND CHARACTERIZATION OF HUMAN KAINATE RECEPTOR SUBUNITS INESCHERICHIA-COLI AND MAMMALIAN-CELLS

Cellular expression systems for human kainate receptor subunits (EAA1 and EAA2) have been developed as tools to support drug screening and r ational drug design. EAA1 and EAA2 sequence-specific polyclonal antibo dies were generated to characterize polypeptide expression on introduc tion of appropriate plasmid expression constructs to Escherichia coil (E. coli), chinese hamster ovary (CHO) and human embryonic kidney (HEK -293) cells. A polypeptide with an apparent molecular mass of similar to 120 kilodaltons (kDa) was identified by Western blot analysis in CH O and HEK-293 cells expressing EAA2. Three major immunoreactive bands of 116, 110, and 90 kDa were identified in HEK-293 cells expressing EA A1. The polyclonal antibodies will allow the direct determination of E AA1 acid EAA2 expression in human brain. Pharmacological characterizat ion of a stable CHO cell line expressing EAA2 revealed a dissociation constant for kainate (K-D) of 1.92 +/- 0.28 nM (n = 3). This is the fi rst report describing a stable cell line expressing EAA2 and the third report describing a stable cell line expressing a human glutamate rec eptor subunit. These studies are an important prelude to discovery of EAA1 and/or EAA2 specific drugs. (C) 1995 Wiley-Liss, Inc.