Molecular cloning of a novel gene involved in serotonin receptor-mediated signal transduction in rat stomach.
In Xenopus oocytes injected with small size mRNAs (500-700 b), obtained from rat stomach by fractionation, application of 10 microM 5-HT induced a substantial Ca2+-activated Cl- current (I(Cl-Ca)). I(Cl-Ca) was not elicited by 5-HT in native oocytes. Consistent results from this assay in the oocyte expression system motivated cDNA cloning experiments. A novel cDNA (named rat stomach serotonin receptor-related cDNA: RSS cDNA) which encodes a small protein involved in specific 5-HT receptor-mediated I(Cl-Ca) activation was identified. The molecular weight of RSS protein in the reticulocyte lysate translation system (approximately 10 kDa) is identical to that calculated from the amino acid sequence. Computer-aided analysis of the predicted protein does not show any obvious sequence homologies (< 18%) to any other proteins including G protein-coupled receptors. Northern analysis revealed that RSS mRNA is ubiquitously expressed at varying levels in a number of different tissues. Furthermore, the binding of [3H]spiperone, a 5-HT2 receptor antagonist, was examined in CHO cells, which highly expressed RSS transcripts (named CHO-RSS). Specific binding of [3H]spiperone was not clearly observed in native CHO but was detected in CHO-RSS. The dissociation constant was 10.3 nM in CHO-RSS. These results suggest that RSS protein may be a factor which facilitates 5-HT receptor expression or, alternatively, an enhancer of the affinity of native 5-HT receptor to 5-HT.
Ohya
S
S
Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, Mizuhoku, Japan.
Takii
T
T
Yamazaki
H F
HF
Matsumori
M
M
Onozaki
K
K
Watanabe
M
M
Imaizumi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D018492:Dopamine Antagonists / Q000378:metabolism; D005753:Gastric Mucosa / Q000378:metabolism*; D008969:Molecular Sequence Data; D009865:Oocytes; D011485:Protein Binding; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011985:Receptors, Serotonin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D013134:Spiperone / Q000378:metabolism; D014981:Xenopus
D018076:DNA, Complementary; D018492:Dopamine Antagonists; D011506:Proteins; D012333:RNA, Messenger; C104074:RSS protein, rat; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D013134:Spiperone