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  Pharmacological and biochemical characterization of the mouse 5HT5A serotonin receptor heterologously produced in the yeast Saccharomyces cerevisiae

Bach, M., Sander, P., Haase, W., & Reiländer, H. (1996). Pharmacological and biochemical characterization of the mouse 5HT5A serotonin receptor heterologously produced in the yeast Saccharomyces cerevisiae. Receptors and Channels, 4(2), 129-139.

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 Creators:
Bach, Marion1, Author           
Sander, Peter1, Author           
Haase, Winfried1, Author           
Reiländer, Helmut1, Author           
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              

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 Abstract: The cDNA for the mouse 5HT5A receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae. The NH2-terminal end of the receptor gene was fused to the Bacillus macerans (1-3, 1-4)-beta-glucanase signal sequence to ensure proper membrane insertion and to the c-myc epitope to permit immunological detection of the heterologously expressed protein. In the resulting episomal yeast expression plasmid pCNNmm5HT5A the modified 5HT5A gene is under the transcriptional control of the endopeptidase B gene promoter (PRB1). After transformation of the vector into the protease deficient S. cerevisiae strain cI3-ABYS-86, recombinant clones were examined for the presence of functional receptor by radioligand binding using [3H]LSD. Whole cells as well as crude membrane preparations of recombinant clones showed saturable binding of the receptor with a KD of approximately 2.2 nM. The pharmacological properties for the heterologous expressed receptor, estimated by ligand-displacement studies using certain serotonin agonists and antagonists, were comparable to those reported for the receptor expressed in mammalian systems. Western blot analysis of membranes prepared from a recombinant clone using the monoclonal antibody 9E10, directed against the c-myc epitope of the modified receptor, revealed an apparent molecular mass of about 43 kDa for the receptor expressed in S. cerevisiae. Glycosylation of the receptor was analysed by EndoH digestion. A heat shock of recombinant yeast significantly increased the number of specific binding sites per cell and also improved the affinity of the receptor. Immunogold electron microscopy was used to study the localization of the heterologously expressed protein within the yeast cells.

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Language(s): eng - English
 Dates: 1996-02
 Publication Status: Published in print
 Pages: 11
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: PMID: 8865364
 Degree: -

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Title: Receptors and Channels
Source Genre: Journal
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Publ. Info: Yverdon, Switzerland : Harwood Academic Publishers :
Pages: - Volume / Issue: 4 (2) Sequence Number: - Start / End Page: 129 - 139 Identifier: ISSN: 1060-6823
CoNE: https://pure.mpg.de/cone/journals/resource/954925597599