CLONING AND EXPRESSION OF A HUMAN METABOTROPIC GLUTAMATE-RECEPTOR 1-ALPHA - ENHANCED COUPLING ON COTRANSFECTION WITH A GLUTAMATE TRANSPORTER

We cloned and expressed a human metabotropic glutamate receptor Icr (H mGluR1 alpha) in a novel cell line. The human mGluR1 alpha cDNA was fo und to be 86% identical to rat mGluR1 alpha, and the predicted protein sequence was found to be 93% identical to rat mGluR1 alpha. We expres sed HmGluR1 alpha in AV12-664, an adenovirus-transformed Syrian hamste r cell line. To prevent tonic activation of HmGluR1 alpha by glutamate that may be released by these cells into the extracellular medium, Hm GluR1 alpha was co-expressed in AV12-664 cells with a rat glutamate/as partate transporter (GLAST). This allowed investigation of the effect that clearance of glutamate from the extracellular space would have on HmGluR1 alpha function. A comparison of mRNA levels revealed that HmG luR1 alpha was similarly expressed in cells with or without co-express ion of GLAST. However, HmGluR1 alpha-mediated phosphoinositide hydroly sis was efficiently elicited only in cells co-expressing rat GLAST. Bl ockade of glutamate transport by L-trans-pyrrolidine-2,4-dicarboxylic acid resulted in an increase in glutamate levels in the media and an i ncrease in basal HmGluR1 alpha-mediated phosphoinositide hydrolysis. L ong-term pretreatment of cells with L-trans-pyrrolidine-2,4-dicarboxyl ic acid resulted in media glutamate levels similar to those in cells n ot expressing GLAST. However, this resulted in a dramatic decrease in 1-aminocyclopentane-1S,3R-dicarboxylic acid- and glutamate-stimulated phosphoinositide hydrolysis. These studies suggest that co-expression of mGluR1 alpha with a glutamate transporter prevents desensitization of the receptor, thus achieving optimal coupling of the receptor with its effector system.