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Cloning and characterization of 5'-end alternatively spliced human cholecystokinin-B receptor mRNAs
Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
1998 (English)In: Receptors and Channels, ISSN 1060-6823, E-ISSN 1607-856X, Vol. 6, no 3, 165-177 p.Article in journal (Refereed) Published
Abstract [en]

We report here the cloning and characterization of a 5'-end alternatively spliced human cholecystokinin-B (CCK-B) receptor mRNA. The 5'-end of this CCK-B receptor transcript (termed CCK-BRtx) consisted of exon Ia, present in the ordinary full-length CCK-B receptor mRNA (CCK-BRwt), and exon Ib, present in a previously described 5'-end alternatively spliced CCK-B receptor mRNA (CCK-BRt). A short open reading frame preceded the AUG translation initiation codon of the CCK-BRtx. Transfection of COS-7 cells with the CCK-BRtx or CCK-BRt cDNAs did not lead to the appearance of peptidergic and non-peptidergic binding sites. Cell free in vitro translation yielded proteins of approximately 44 kDa (CCK-B receptor) and 40 kDa (CCK-BRt receptor) whereas no 40 kDa product was detected from the cloned CCK-BRtx cDNA. Instead, a protein product of approximately 9 kDa was visualized, the size corresponding to the predicted protein encoded by the short open reading frame. The alternatively spliced CCK-B receptor transcripts were concomitantly expressed with the ordinary full-length CCK-B receptor mRNA in the brain, pancreas, and stomach. The possibility that such transcripts are translated in vivo into truncated CCK-B receptors is discussed.

Place, publisher, year, edition, pages
1998. Vol. 6, no 3, 165-177 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-84493PubMedID: 10100325OAI: oai:DiVA.org:liu-84493DiVA: diva2:559693
Available from: 2012-10-10 Created: 2012-10-10 Last updated: 2017-12-07Bibliographically approved
In thesis
1. The cholecystokinin receptor family: molecular cloning and pharmacological characterization
Open this publication in new window or tab >>The cholecystokinin receptor family: molecular cloning and pharmacological characterization
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cholecystokinin (CCK) and gastrin are hormones/neurotransmittors of the gastrointestinal tract and central nervous system. The receptors for gastrin and CCK are members of the G protein-coupled receptor family. The aim of this study was to clone and pharmacologically characterize vertebrate and invertebrate CCK receptors and splice variants. Three 5'-end alternatively spliced human CCK2 receptor mRNAs were cloned: the CCK-BRwt mRNA, that encodes the ordinary full-length CCK2 receptor, CCK-BRt mRNA that contains exon 1b and that encodes a N-terminally truncated receptor protein, and CCK-BRtx mRNA that contains exon 1a (also present in CCKBRwt mRNA) and exon 1b. The CCK-BRtx mRNA contains two open reading frames: a short open reading frame that precedes the open reading frame of the N-terminally truncated receptor. In vitro transcription/translation of the mRNAs yielded proteins of 44 kDa (CCK-BRwt), 40 kDa (CCK-BRt), and 9 kDa (CCK-BRtx). The 9 kDa product corresponded to the predicted size of the short open reading frame of CCK-BRtx. No 40 kDa product was produced by the cloned CCK-BRtx. Pharmacological analysis of CCK2 receptor ligands was performed using the cloned human CCK2 receptor (CCKBRwt) transiently expressed in COS-7 and SK-N-MC cells. The binding of YF476, YM022, AG041R, L-740,093, JB93182, PD134308, and PD136450 was analyzed by radioligand competition using [3H]L-365,260 as the labeled ligand. The binding data for four ofthe ligands fitted a one-site model (YF476, YM022, L-740,093, and AG041R), while the data for the three others fitted a two-site model (PD134308, PD136450, and JB93182) using COS-7 cell membranes in radioligand binding experiments. The data for YM022 and YF476 fitted a one-site model while the data for JB93182 and PD134308 fitted a two-site model using SK-N-MC cell membranes in the radioligand binding experiments. In the presence of a GTP-analogue, similar results were obtained. The human CCK2 receptor seems to exist in a low and/or high affmity state that does not reflect the degree ofG protein-coupling. A chicken brain CCK receptor, CCK-CHR, was cloned using a polymerase chain reaction (PCR)-based cloning strategy that included an initial PCR with deoxyinosine-containing primers targeting conserved regions in vertebrate CCK receptors, followed by rapid amplification of cDNA ends (RACE) and full-length PCR amplification. The CCK-CHR full-length PCR amplicon contained a short upstream open reading frame (uORF) followed by a long ORF encoding the 436 amino acid long receptor protein. CCK-CHR shared ≈50% amino acid sequence homology with cloned vertebrate receptors. The pharmacological profile of CCK-CHR resembled that of mammalian CCK2 receptors using agonists, but CCK1 receptors using subtype-specific antagonists. A putative cionin receptor (CioR), a new member of the CCK/gastrin receptor family was cloned from the gastrointestinal tract of the urochordate Ciona intestinalis using RACE PCR followed by full-length PCR amplification. The full-length PCR amplicon contained an uORF followed by a long ORF encoding the 526 amino acid long receptor protein. At the amino acid level, CioR shared 35-40% homology with vertebrate CCK receptors.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2003. 61 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 791
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27527 (URN)12183 (Local ID)91-7373-552-3 (ISBN)12183 (Archive number)12183 (OAI)
Public defence
2003-05-27, Berzeliussalen, Hälsouniversitet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-10Bibliographically approved

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Monstein, H.-J.Nilsson, IsabelleEllnebo-Svedlund, KatarinaSvensson, S.P.S.

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