Functional expression of rat M5 muscarinic acetylcholine receptor in yeast
We have produced the rat M5 muscarinic acetylcholine receptor, an integral membrane protein, in the yeast Saccharomyces cerevisiae. This was achieved by placing an M5 gene in the yeast vector under the control of the yeast α-factor promoter and leader sequence. Northern blotting revealed the presenc...
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| Veröffentlicht in: | Biochemical and biophysical research communications 1992-02, Vol.182 (3), p.1180-1186 |
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| container_title | Biochemical and biophysical research communications |
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| creator | Huang, Hao-jen Liao, Ching-fong Yang, Bei-chang Kuo, Tsong-teh |
| description | We have produced the rat M5 muscarinic acetylcholine receptor, an integral membrane protein, in the yeast
Saccharomyces cerevisiae. This was achieved by placing an M5 gene in the yeast vector under the control of the yeast α-factor promoter and leader sequence. Northern blotting revealed the presence of M5 transcripts in yeast transformed with the M5 plasmid constructs. Crude extract prepared from the transformant yeasts showed saturable binding of the muscarinic antagonist [
3H]-N-methyl scopolamine ([
3H]NMS) with a k
d of 22.77 nM and B
max of 134.76 fmole per mg protein. Results deduced from saturation binding assay of intact cell demonstrated clearly that the M5 receptor was translocated across the membrane of the endoplasmic reticulum using the secretion signal on α-leader sequence and its binding site was still functional. Yeast expressing M5 receptor did not exhibit cell-cycle arrest in the presence of carbachol, a acetylcholine agonist, indicating that the recombinant M5 receptor could not couple directly to the endogenous yeast pheromone signaling G-protein. |
| doi_str_mv | 10.1016/0006-291X(92)91856-L |
| format | Article |
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Saccharomyces cerevisiae. This was achieved by placing an M5 gene in the yeast vector under the control of the yeast α-factor promoter and leader sequence. Northern blotting revealed the presence of M5 transcripts in yeast transformed with the M5 plasmid constructs. Crude extract prepared from the transformant yeasts showed saturable binding of the muscarinic antagonist [
3H]-N-methyl scopolamine ([
3H]NMS) with a k
d of 22.77 nM and B
max of 134.76 fmole per mg protein. Results deduced from saturation binding assay of intact cell demonstrated clearly that the M5 receptor was translocated across the membrane of the endoplasmic reticulum using the secretion signal on α-leader sequence and its binding site was still functional. Yeast expressing M5 receptor did not exhibit cell-cycle arrest in the presence of carbachol, a acetylcholine agonist, indicating that the recombinant M5 receptor could not couple directly to the endogenous yeast pheromone signaling G-protein.</description><identifier>ISSN: 0006-291X</identifier><identifier>EISSN: 1090-2104</identifier><identifier>DOI: 10.1016/0006-291X(92)91856-L</identifier><identifier>PMID: 1540163</identifier><identifier>CODEN: BBRCA9</identifier><language>eng</language><publisher>San Diego, CA: Elsevier Inc</publisher><subject>Animals ; Base Sequence ; Biological and medical sciences ; Blotting, Northern ; Cloning, Molecular ; Fundamental and applied biological sciences. Psychology ; Gene Expression ; Kinetics ; Molecular and cellular biology ; Molecular genetics ; Molecular Sequence Data ; N-Methylscopolamine ; Plasmids ; Protein Sorting Signals - genetics ; Rats ; Receptors, Muscarinic - genetics ; Receptors, Muscarinic - metabolism ; Recombinant Proteins - metabolism ; Saccharomyces cerevisiae - genetics ; Scopolamine Derivatives - metabolism ; Transcription, Genetic</subject><ispartof>Biochemical and biophysical research communications, 1992-02, Vol.182 (3), p.1180-1186</ispartof><rights>1992</rights><rights>1992 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c386t-9339b586dcb0fef6f937dd61585253aa51237acdf8b344de3590660a1480056a3</citedby><cites>FETCH-LOGICAL-c386t-9339b586dcb0fef6f937dd61585253aa51237acdf8b344de3590660a1480056a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/0006-291X(92)91856-L$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,777,781,3537,27905,27906,45976</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=5168128$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1540163$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Huang, Hao-jen</creatorcontrib><creatorcontrib>Liao, Ching-fong</creatorcontrib><creatorcontrib>Yang, Bei-chang</creatorcontrib><creatorcontrib>Kuo, Tsong-teh</creatorcontrib><title>Functional expression of rat M5 muscarinic acetylcholine receptor in yeast</title><title>Biochemical and biophysical research communications</title><addtitle>Biochem Biophys Res Commun</addtitle><description>We have produced the rat M5 muscarinic acetylcholine receptor, an integral membrane protein, in the yeast
Saccharomyces cerevisiae. This was achieved by placing an M5 gene in the yeast vector under the control of the yeast α-factor promoter and leader sequence. Northern blotting revealed the presence of M5 transcripts in yeast transformed with the M5 plasmid constructs. Crude extract prepared from the transformant yeasts showed saturable binding of the muscarinic antagonist [
3H]-N-methyl scopolamine ([
3H]NMS) with a k
d of 22.77 nM and B
max of 134.76 fmole per mg protein. Results deduced from saturation binding assay of intact cell demonstrated clearly that the M5 receptor was translocated across the membrane of the endoplasmic reticulum using the secretion signal on α-leader sequence and its binding site was still functional. Yeast expressing M5 receptor did not exhibit cell-cycle arrest in the presence of carbachol, a acetylcholine agonist, indicating that the recombinant M5 receptor could not couple directly to the endogenous yeast pheromone signaling G-protein.</description><subject>Animals</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Blotting, Northern</subject><subject>Cloning, Molecular</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression</subject><subject>Kinetics</subject><subject>Molecular and cellular biology</subject><subject>Molecular genetics</subject><subject>Molecular Sequence Data</subject><subject>N-Methylscopolamine</subject><subject>Plasmids</subject><subject>Protein Sorting Signals - genetics</subject><subject>Rats</subject><subject>Receptors, Muscarinic - genetics</subject><subject>Receptors, Muscarinic - metabolism</subject><subject>Recombinant Proteins - metabolism</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Scopolamine Derivatives - metabolism</subject><subject>Transcription, Genetic</subject><issn>0006-291X</issn><issn>1090-2104</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kE1LAzEQhoMotVb_gUIOInpYnWw2aXIRpFg_WPGi4C2k2VmMbHdrsiv237u1pd48DcM8887wEHLM4JIBk1cAIJNUs7dznV5opoRM8h0yZKAhSRlku2S4RfbJQYwfAIxlUg_IgImsj-BD8jjtatf6prYVxe9FwBj7hjYlDbalT4LOu-hs8LV31Dpsl5V7bypfIw3ocNE2gfqaLtHG9pDslbaKeLSpI_I6vX2Z3Cf5893D5CZPHFeyTTTneiaULNwMSixlqfm4KCQTSqSCWytYysfWFaWa8SwrkAsNUoJlmQIQ0vIROVvnLkLz2WFszdxHh1Vla2y6aMap4lyA6sFsDbrQxBiwNIvg5zYsDQOzUmhWfszKj9Gp-VVo8n7tZJPfzeZY_C2tnfXz083c9mqqMtja-bjFBJOKpavr12sMexdfHoOJzmPtsPC9utYUjf__jx-0TYzS</recordid><startdate>19920214</startdate><enddate>19920214</enddate><creator>Huang, Hao-jen</creator><creator>Liao, Ching-fong</creator><creator>Yang, Bei-chang</creator><creator>Kuo, Tsong-teh</creator><general>Elsevier Inc</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19920214</creationdate><title>Functional expression of rat M5 muscarinic acetylcholine receptor in yeast</title><author>Huang, Hao-jen ; Liao, Ching-fong ; Yang, Bei-chang ; Kuo, Tsong-teh</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-9339b586dcb0fef6f937dd61585253aa51237acdf8b344de3590660a1480056a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>Animals</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Blotting, Northern</topic><topic>Cloning, Molecular</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression</topic><topic>Kinetics</topic><topic>Molecular and cellular biology</topic><topic>Molecular genetics</topic><topic>Molecular Sequence Data</topic><topic>N-Methylscopolamine</topic><topic>Plasmids</topic><topic>Protein Sorting Signals - genetics</topic><topic>Rats</topic><topic>Receptors, Muscarinic - genetics</topic><topic>Receptors, Muscarinic - metabolism</topic><topic>Recombinant Proteins - metabolism</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Scopolamine Derivatives - metabolism</topic><topic>Transcription, Genetic</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Huang, Hao-jen</creatorcontrib><creatorcontrib>Liao, Ching-fong</creatorcontrib><creatorcontrib>Yang, Bei-chang</creatorcontrib><creatorcontrib>Kuo, Tsong-teh</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemical and biophysical research communications</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Huang, Hao-jen</au><au>Liao, Ching-fong</au><au>Yang, Bei-chang</au><au>Kuo, Tsong-teh</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Functional expression of rat M5 muscarinic acetylcholine receptor in yeast</atitle><jtitle>Biochemical and biophysical research communications</jtitle><addtitle>Biochem Biophys Res Commun</addtitle><date>1992-02-14</date><risdate>1992</risdate><volume>182</volume><issue>3</issue><spage>1180</spage><epage>1186</epage><pages>1180-1186</pages><issn>0006-291X</issn><eissn>1090-2104</eissn><coden>BBRCA9</coden><abstract>We have produced the rat M5 muscarinic acetylcholine receptor, an integral membrane protein, in the yeast
Saccharomyces cerevisiae. This was achieved by placing an M5 gene in the yeast vector under the control of the yeast α-factor promoter and leader sequence. Northern blotting revealed the presence of M5 transcripts in yeast transformed with the M5 plasmid constructs. Crude extract prepared from the transformant yeasts showed saturable binding of the muscarinic antagonist [
3H]-N-methyl scopolamine ([
3H]NMS) with a k
d of 22.77 nM and B
max of 134.76 fmole per mg protein. Results deduced from saturation binding assay of intact cell demonstrated clearly that the M5 receptor was translocated across the membrane of the endoplasmic reticulum using the secretion signal on α-leader sequence and its binding site was still functional. Yeast expressing M5 receptor did not exhibit cell-cycle arrest in the presence of carbachol, a acetylcholine agonist, indicating that the recombinant M5 receptor could not couple directly to the endogenous yeast pheromone signaling G-protein.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>1540163</pmid><doi>10.1016/0006-291X(92)91856-L</doi><tpages>7</tpages></addata></record> |
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| ispartof | Biochemical and biophysical research communications, 1992-02, Vol.182 (3), p.1180-1186 |
| issn | 0006-291X 1090-2104 |
| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Animals Base Sequence Biological and medical sciences Blotting, Northern Cloning, Molecular Fundamental and applied biological sciences. Psychology Gene Expression Kinetics Molecular and cellular biology Molecular genetics Molecular Sequence Data N-Methylscopolamine Plasmids Protein Sorting Signals - genetics Rats Receptors, Muscarinic - genetics Receptors, Muscarinic - metabolism Recombinant Proteins - metabolism Saccharomyces cerevisiae - genetics Scopolamine Derivatives - metabolism Transcription, Genetic |
| title | Functional expression of rat M5 muscarinic acetylcholine receptor in yeast |
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