Is the heterologous expression of metabotropic glutamate receptors (mGluRs) an appropriate method to study the mGluR function? Experience with human embryonic kidney 293 cells transfected with mGluR1.
The cloning of metabotropic glutamate receptors (mgluRs) has initiated a new approach to the study of their function: the introduction of mGluR cDNA into cells that do not normally express mGluRs, thus allowing the heterologous receptor expression. We have transfected human embryonic kidney (HEK) 293 cells with the full length mGluR1a cDNA and with its truncated variant which encodes the receptor termed mGluR1T (a receptor lacking the long intracellular domain and similar to the splice variant mGluR1c). Transient transfection of HEK-293 cells with mGluR1a, but not the mGluR1T cDNA, resulted in a significant increase in inositol phosphate (IP) formation in absence of any mGluR agonists. This effect was completely dependent on the presence of extracellular calcium, and unlike the agonist-stimulated IP formation it was insensitive to pertussis toxin. The prolonged activation of IP formation might affect the cell physiology. In an attempt to obtain stably transfected cells, we transfected about 1.5 x 10(6) HEK-293 cells with the plasmid conveying the full-length mGluR1a cDNA and the neomycin-resistance gene. Only 12 clones survived the antibiotic selection, and only one of these 12 clones continued to divide. The size of mRNA from the clone was smaller than the full-length mGluR1a mRNA. The shortened mRNA, revealed in the clone, apparently encoded a functional mGluR that was sensitive to glutamate, but unlike the mGluR1a, it did not respond to 1S,3R-ACPD (1S,3R-aminocyclopentane-1,3-dicarboxylic acid). A prudent use of the heterologous cell transfection technique is necessary in studying the function and the pharmacology of mGluRs.
Gabellini
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Dipartimento di Chimica Biologica, Padova University, Italy.
Manev
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Manev
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D017398:Alternative Splicing; D015152:Blotting, Northern; D002118:Calcium / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D003515:Cycloleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D004622:Embryo, Mammalian; D015870:Gene Expression; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007668:Kidney; D009498:Neurotoxins / Q000494:pharmacology; D037342:Pertussis Toxin; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D018094:Receptors, Metabotropic Glutamate / Q000096:biosynthesis* / Q000502:physiology; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007295:Inositol Phosphates; D009498:Neurotoxins; D012333:RNA, Messenger; D018094:Receptors, Metabotropic Glutamate; D010566:Virulence Factors, Bordetella; D003515:Cycloleucine; C036361:1-amino-1,3-dicarboxycyclopentane; D005576:Colforsin; D018698:Glutamic Acid; D037342:Pertussis Toxin; D002118:Calcium; D013936:Thymidine