A system for receptor functional analysis based on c-fos mRNA expression: analysis of GnRH receptors as a test system.
This report describes the establishment of a system for assessing receptor activation by RT-PCR-based detection of c-fos mRNA induction. In this system, COS-7 cells were transiently transfected with GnRH receptor expression plasmid, and ligand-induced c-fos expression was quantified by the RT-competitive PCR method. The results were compared with those of a conventional inositol phosphate (IP) assay. Changes in c-fos expression levels were observed in a dose- and ligand-dependent manner. Similar tendencies were observed in ligand selectivity between c-fos expression and IP production. The novel system developed and established in the present study is sensitive by using RT-PCR and convenient because it requires only basic methods of cell culture and molecular biology. It also has the merit that it does not need any specific measuring devices or radioactive substances. Given the ability of c-fos to respond to diverse stimuli, the present system may be applicable for various receptors for bioactive substances in addition to GnRH receptor, and useful for various purposes including screening ligands for orphan receptors.
Ikemoto
Tadahiro
T
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan.
Park
Min Kyun
MK
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D019556:COS Cells; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D016762:Genes, fos; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000032:analysis; D012333:RNA, Messenger / Q000235:genetics*; D011966:Receptors, LHRH / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017931:DNA Primers; D007295:Inositol Phosphates; D012333:RNA, Messenger; D011966:Receptors, LHRH; D011994:Recombinant Proteins; D007987:Gonadotropin-Releasing Hormone