The cholecystokinin receptor family : molecular cloning and pharmacological characterization

Sammanfattning: 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.