Molecular cloning of the human brain and gastric cholecystokinin receptor: structure, functional expression and chromosomal localization

Abstract

The receptors for the brain and gastrointestinal peptide, cholecystokinin, can be classified into CCKA and CCKB subtypes. Having recently cloned the rat CCKB receptor, we used it's cDNA to isolate the human CCKB receptor homologue from brain and stomach which encodes a 447 amino acid protein with 90% identity to both rat CCKB and canine gastrin receptors. Northern hybridization identifies transcripts from stomach, pancreas, brain and gallbladder. The CCKB receptor gene maps to chromosome 11. Expression of the receptor cDNA in COS-7 cells was characteristic of a CCKB receptor subtype pharmacology. These data confirm that we have cloned a novel gene for the human brain and stomach CCKB receptor.

FIG. 1.

Nucleotide and deduced amino acid sequences of the human brain and stomach CCK_B receptor cDNA clone. The solid lines labelled with Roman numerals I-VII delineate the putative transmembrane domains predicted by the Kyte-Doolittle criteria (31) and homology with the rat CCK_B as well as other G-protein-coupled receptor superfamily members. The solid triangles indicate three potential sites for N-linked glycosylation. The solid underlines indicate potential sites for serine and threonine phosphorylation (25). The AATAAA cleavage and polyadenyalation sequence is underlined. Solid circles indicate cysteine residues which are potential sites for either disulfide bridge formation (26,27,28) (residues 127 and 205) or palmitoylation (29,30) (residue 408).

FIG. 2.

Alignment of the human CCK_B receptor (HUCCKBR), rat CCK_B receptor (RATCCKBR) and canine gastrin receptor (CANGASR) deduced protein sequences. Using the “Pileup” program sequence analysis package of the Genetics Computer Group (21) the human CCK_B receptor was aligned for maximal homology with the rat CCK_B receptor and the canine gastrin receptor. Shown here using amino acid acid letter symbols is the result of this alignment with solid lines indicating putative transmembrane domains and boxed letters indicating amino acids from rat and dog not conserved in the human receptor sequence.

FIG. 3.

Northern blot analysis of poly (A)+ RNA from the human organs. Two micrograms of poly (A)+ RNA from human brain, stomach, pancreas, and gallbladder were separated on a 1.5% denaturing/formaldehyde agarose gel and probed under conditions of high stringency with the coding region of the CCK_B receptor cDNA labelled with [³²P] by random primer extension. The blot was exposed for 48 hours and scanned with a phosphorimager (Molecular Dynamics). The lines on the left correspond to the migration Positions of the 28S and 18S ribosomal RNA.

FIG. 4.

Ability of CCK receptor agonists and antagonists to inhibit binding of [¹²⁵I]BH-CCK-8 to COS-7 cells expressing the human CCK_B receptor. COS-7 cells were transfected with the mammalian expression vector, pCDL-SRα, containing the human CCK_B receptor cDNA. Transfected COS-7 cells were incubated with either the tracer alone or increasing concentrations of agonists CCK-8 or gastrin-17-I (left panel) or antagonists L-365,260 and L-364,718 (right panel). Data are presented as percent saturable binding (total binding in the presence of labelled hormone alone minus binding in the presence of 1 μM CCK-8).