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Pharmacological analysis of CCK2 receptor ligands using COS-7 and SK-N-MC cells, expressing the human CCK2 receptor
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
Östergötlands Läns Landsting, LMÖ - Laboratoriemedicin i Östergötland.
Department of Pharmacology, Institute of Physiological Sciences, University of Lund, Lund, Sweden.
Department of Pharmacology, Institute of Physiological Sciences, University of Lund, Lund, Sweden.
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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.

Place, publisher, year, edition, pages
2002. Vol. 103, no 1, 29-37 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28364DOI: 10.1016/S0167-0115(01)00324-XLocal ID: 13499OAI: oai:DiVA.org:liu-28364DiVA: diva2:249169
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13Bibliographically 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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Nilsson, IsabelleMonstein, Hans-JurgSvensson, Samuel

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