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The cholecystokinin receptor family: molecular cloning and pharmacological characterization
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-27527Local ID: 12183ISBN: 91-7373-552-3 (print)OAI: oai:DiVA.org:liu-27527DiVA: diva2:248079
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
List of papers
1. Cloning and characterization of 5'-end alternatively spliced human cholecystokinin-B receptor mRNAs
Open this publication in new window or tab >>Cloning and characterization of 5'-end alternatively spliced human cholecystokinin-B receptor mRNAs
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84493 (URN)10100325 (PubMedID)
Available from: 2012-10-10 Created: 2012-10-10 Last updated: 2017-12-07Bibliographically approved
2. Pharmacological analysis of CCK2 receptor ligands using COS-7 and SK-N-MC cells, expressing the human CCK2 receptor
Open this publication in new window or tab >>Pharmacological analysis of CCK2 receptor ligands using COS-7 and SK-N-MC cells, expressing the human CCK2 receptor
Show others...
2002 (English)In: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 103, no 1, 29-37 p.Article in journal (Refereed) Published
Abstract [en]

A series of CCK2 receptor ligands were analysed with respect to their interaction with binding sites in the membranes of COS-7 cells and SK-N-MC cells transiently expressing the human CCK2 receptor (short isoform). The ligands were YF476, YM022, AG041R, L-740,093, JB93182, PD134308, and PD136450. Their binding was analysed by radioligand competition using [3H]L-365,260 as the labelled ligand. Saturation binding analysis indicated that [3H]L-365,260 interacted with a single class of binding sites. In competition binding experiments using COS-7-cell membranes, all seven ligands were incubated together with 2 nM [3H]L-365,260. The data for four of the compounds fitted a one-site model (pKi values: YM022: 9.2±0.02; YF476: 9.6±0.04; L-740,093: 9.2±0.01; and AG041R: 8.3±0.06), while the data for the three others fitted a two-site model (pKi values: JB93182: 8.8±0.04 and 6.0±0.15; PD134308: 9.0±0.04 and 6.1±0.15; and PD136450: 9.0±0.02 and 5.4±0.41). SK-N-MC cell membranes and 2 nM [3H]L-365,260 were incubated together with YM022, YF476, JB93182, and PD134308. The data for YM022 and YF476 fitted a one-site model (pKi values: YM022: 9.3±0.06; YF476: 9.4±0.02), while the data for JB93182 and PD134308 fitted a two-site model (pKi values: JB93182: 8.7±0.06 and 6.2±0.06; PD134308: 9.1±0.06 and 7.0±0.17). Competition binding experiments in the presence of the GTP-analogue guanylylimidodiphosphate, using either of the two cell types, produced similar binding data for PD134308 and JB93182 as in the absence of GTP-analogue. The human receptor seems to exist in a low and/or high affinity state. The shift from low to high affinity does not seem to reflect the degree of G protein coupling.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-28364 (URN)10.1016/S0167-0115(01)00324-X (DOI)13499 (Local ID)13499 (Archive number)13499 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13Bibliographically approved
3. Molecular cloning of an unusual bicistronic cholecystokinin receptor mRNA expressed in chicken brain: a structural and functional expression study
Open this publication in new window or tab >>Molecular cloning of an unusual bicistronic cholecystokinin receptor mRNA expressed in chicken brain: a structural and functional expression study
2003 (English)In: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 114, no 1, 37-43 p.Article in journal (Refereed) Published
Abstract [en]

This report describes the molecular cloning and pharmacological characterization of a transiently expressed chicken brain cholecystokinin receptor (CCK-CHR) in COS-7 cells. A polymerase chain reaction (PCR)-based cloning strategy was applied using: (1) an initial PCR with deoxyinosine-containing primers designed to target conserved regions in CCK receptors, followed by (2) rapid amplification of cDNA ends (RACE), and (3) full-length PCR of the CCK-CHR cDNA. The full-length cloned bicistronic CCK-CHR cDNA contained a short upstream open reading frame (uORF) coding for a putative six-amino-acid-long peptide of unknown function, followed by a long open reading frame (lORF) encoding the 436-amino-acid-long CCK-CHR receptor protein. At the amino acid level, the CCK-CHR shared ∼50% homology with mammalian and Xenopus laevis CCK receptors. The pharmacological profile of CCK-CHR resembled that of CCK-B receptors using agonists (CCK-8, CCK-4, gastrin-17), whereas CCK-CHR showed higher affinity for the CCK-A receptor antagonist, devazepide, than for the CCK-B receptor antagonist, l-365,260. To the best of our knowledge, this is the first description and functional expression study of a cloned chicken CCK receptor cDNA.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27051 (URN)10.1016/S0167-0115(03)00068-5 (DOI)11696 (Local ID)11696 (Archive number)11696 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
4. Molecular cloning of a putative Ciona intestinalis cionin receptor, a new member of the CCK/gastrin receptor family
Open this publication in new window or tab >>Molecular cloning of a putative Ciona intestinalis cionin receptor, a new member of the CCK/gastrin receptor family
2003 (English)In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 323, no 1-2, 79-88 p.Article in journal (Refereed) Published
Abstract [en]

Cionin, a peptide showing similarities with cholecystokinin and gastrin has been shown to be expressed in the gut and neural ganglion of the protochordate Ciona intestinalis. The present report describes the cloning of a putative cionin receptor (CioR), a new member of the CCK/gastrin family from the gastrointestinal tract of C. intestinalis. mRNA from the stomach of C. intestinalis was isolated using a modified RNA extraction procedure and, subsequently, reverse-transcribed into single-stranded cDNA by means of rapid amplification of 5′- and 3′-cDNA ends (RACE-PCR), followed by full-length PCR amplification. The cloned full-length PCR amplicons contained a short upstream open-reading frame (uORF) coding for a putative 16 amino acid long peptide, followed by a long open reading frame encoding a 526 amino acid putative CioR protein. At the amino acid level, the putative CioR protein shared 35–40% homology with cloned mammalian, chicken, and Xenopus laevis CCK receptors. Phylogenetic analysis revealed that the chicken and X. laevis CCK receptors are orthologues of the mammalian CCK2 receptors whereas CioR protein forms a clade with vertebrate cholecystokinin receptors. Moreover, we found that the CioR cDNA and deduced amino acid sequences were found to correspond to the annotated CCK/gastrin-like receptor gene on Scaffold 117 (C. intestinalis draft genome project, Joint Genome Institute database; http://www.jgi.doe.gov).

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27050 (URN)10.1016/j.gene.2003.09.002 (DOI)11695 (Local ID)11695 (Archive number)11695 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved

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