title: Cloning and expression of a human metabotropic glutamate receptor 1 alpha: enhanced coupling on co-transfection with a glutamate transporter.
abstract: We cloned and expressed a human metabotropic glutamate receptor 1 alpha (HmGluR1 alpha) in a novel cell line. The human mGluR1 alpha cDNA was found to be 86% identical to rat mGluR1 alpha, and the predicted protein sequence was found to be 93% identical to rat mGluR1 alpha. We expressed HmGluR1 alpha in AV12-664, an adenovirus-transformed Syrian hamster cell line. To prevent tonic activation of HmGluR1 alpha by glutamate that may be released by these cells into the extracellular medium, HmGluR1 alpha was co-expressed in AV12-664 cells with a rat glutamate/aspartate 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 HmGluR1 alpha was similarly expressed in cells with or without co-expression of GLAST. However, HmGluR1 alpha-mediated phosphoinositide hydrolysis was efficiently elicited only in cells co-expressing rat GLAST. Blockade of glutamate transport by L-trans-pyrrolidine-2,4-dicarboxylic acid resulted in an increase in glutamate levels in the media and an increase in basal HmGluR1 alpha-mediated phosphoinositide hydrolysis. Long-term pretreatment of cells with L-trans-pyrrolidine-2,4-dicarboxylic acid resulted in media glutamate levels similar to those in cells not 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.
mesh_terms: D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D003470:Culture Media; D018076:DNA, Complementary / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism; D015870:Gene Expression; D018698:Glutamic Acid / Q000378:metabolism / Q000493:pharmacokinetics; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D006868:Hydrolysis; D008647:Mesocricetus; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000819:agonists / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D003470:Culture Media; D018076:DNA, Complementary; D006023:Glycoproteins; D010716:Phosphatidylinositols; D018094:Receptors, Metabotropic Glutamate; D018698:Glutamic Acid