Elsevier

Regulatory Peptides

Volume 101, Issues 1–3, 15 September 2001, Pages 25-33
Regulatory Peptides

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Identification of CCK-B/gastrin receptor splice variants in human peripheral blood mononuclear cells

https://doi.org/10.1016/S0167-0115(01)00281-6Get rights and content

Abstract

There is increasing evidence for a direct interaction of the enteric nervous and immune system. Receptors for neuropeptides such as VIP, somatostatin, and substance P have been characterised in human immuno-haematopoietic cells but little is known about the functional significance and expression of receptors for cholecystokinin (CCK) on cells of the immune system. There are only few studies that describe the expression of CCK receptors on human leukaemia-derived cell lines but the receptor structure and function in normal leukocytes have not been clearly established. We therefore sought to determine CCK receptor expression, structure, and function in nontransformed human peripheral blood mononuclear cells.

Full-length cDNA clones encoding the human CCK-A and CCK-B/gastrin receptor are expressed in peripheral blood mononuclear cells from healthy volunteers without haematopoietic malignancy. In addition to wild-type CCK-B/gastrin receptor cDNAs, we isolated a splice variant with an in frame insertion of 69 amino acids within its putative third intracellular receptor loop. Dideoxy sequence analysis revealed that the cDNA of this splice variant comprises exons 1–4 but retains intron 4 (207 bp) in the absence of mutations within the splice donor sites. Transient expression of this splice variant in COS-7 cells reveals wild-type affinity for CCK-8, Gastrin-17, and antagonist L-365,260. Affinity for glycine-extended gastrin-17 was not increased when compared to the wild-type CCK-B/gastrin receptor. In vitro, gastrin decreased 3H-thymidine labelling in phytohaemagglutinin-pretreated mononuclear cells at a half-maximally effective concentration of 1.5 nM. We also isolated a cDNA encoding another splice variant of the CCK-B/gastrin receptor with a 158 bp deletion of the entire exon 4 sequence.

We conclude that wild-type transcripts of both CCK receptor subtypes and splice variants of the CCK-B/gastrin receptor are expressed in nontransformed human mononuclear cells and that gastrin exhibits antiproliferative effects.

Introduction

The structurally related neuropeptides gastrin and cholecystokinin (CCK) are important regulatory peptides within the gastrointestinal tract, which mediate their effects on target tissues by activating two known G protein-coupled receptors, termed CCK-A and CCK-B/gastrin receptors [1]. While CCK is capable to bind with subnanomolar affinity to both receptor subtypes, gastrin exhibits >1000-fold higher affinity for the CCK-B/gastrin receptor subtype. CCK-A receptors mediate gallbladder contraction and pancreatic amylase release in response to a meal—and as shown previously in CCK-A receptor deficient mice—CCK-A receptors are involved, but not essential, in mediating satiety [2]. Gastrin has been shown to induce gastric acid secretion by direct activation of CCK-B/gastrin receptors on parietal cells and through release of histamin from enterochromaffin-like cells within the gastric mucosa [3]. Besides stimulation of acid secretion, gastrin also mediates growth promoting effects on the acid-producing gastric mucosa and on the colonic epithelium in vivo and in vitro [4], [5], [6].

Recent research on inflammatory processes within the gastrointestinal tract has focused on the interaction of the enteric nervous and immune system. In search for candidate neuropeptides that could modulate T cell function, one recent study showed that somatostatin, calcitonin gene-related peptide, neuropeptide Y, and substance P induced an increase of cytokine secretion from murine lymph node-derived T cell lines [7]. However, the functional significance and molecular structure of CCK receptors on cells of the immune system remain largely undefined. While several groups were able to demonstrate CCK-B/gastrin receptor expression at the protein or mRNA level in the human lymphoblastic Jurkat T cell line [8], [9], [10], [11], [12], [13], gastrin was shown to stimulate growth of human leukaemia cells in an autocrine growth loop by activating functional CCK-B/gastrin receptors [14]. Despite these findings in Jurkat or leukaemia-derived cell lines, there is an ongoing debate on the receptor status, structure, and function in nontransformed human mononuclear cells isolated from peripheral blood or intestinal lamina propria.

We therefore assessed CCK receptor expression in normal human leukocytes by RT-PCR cloning, DNA sequence analysis, and heterologous cDNA expression for radioligand binding studies in transiently transfected COS-7 cells.

Section snippets

Chemicals and culture media

All chemicals were reagent grade and purchased from commercial vendors. Cell culture media, RPMI1640 and Dulbecco's minimal essential medium, and fetal calf serum were from Biochrom, Berlin, Germany. Penicillin and streptomycin were from Sigma, Deisenhofen, Germany. All primers utilised for cDNA cloning were synthesized from Biometra, Göttingen, Germany. 125I-CCK-8 with a specific radioactivity of 2200 Ci/mmol and methyl,1′,2′-3H thymidine 5′-triphosphate (specific radioactivity 90–120 Ci/mmol)

CCK receptor expression in human peripheral blood mononuclear cells

Full-length cDNA clones encoding the CCK-A and CCK-B/gastrin receptors are expressed in peripheral blood mononuclear cells from healthy volunteers without haematopoietic malignancy (Fig. 3). Sequence comparisons with deposited data bank entries revealed that the cloned CCK receptor subtypes are wild-type sequence and identical to the cDNAs isolated from human gallbladder and brain.

Splice variants of the human CCK-B/gastrin receptor are expressed in human peripheral blood mononuclear cells

The human CCK-B/gastrin receptor gene consists of five exons and four introns [15]. The full-length cDNA encodes a

Discussion

In the present study, we demonstrate that wild-type CCK-A and CCK-B/gastrin receptor transcripts are expressed in human peripheral blood mononuclear cells and that gastrin has antiproliferative effects on phytohaemagglutinin-pretreated mononuclear cells in vitro. Besides wild-type CCK-B/gastrin receptor cDNAs, we isolated two splice variants from healthy donors. One CCK-B/gastrin receptor splice variant encodes a CCK-B/gastrin receptor with an in frame insertion of intron 4 sequence. We

Acknowledgements

This work was presented in part at the Digestive Disease Week in San Diego, CA, May 2000. The study was supported by a grant from the Deutsche Forschungsgemeinschaft to F.S. (Schm 10734-1). The authors thank Dr. M.L. Kruse for the careful revision of the manuscript.

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