Elsevier

Peptides

Volume 18, Issue 5, 1997, Pages 663-672
Peptides

Location and Characterization of the Human GRP Receptor Expressed by Gastrointestinal Epithelial Cells

https://doi.org/10.1016/S0196-9781(97)00127-7Get rights and content

Abstract

Ferris, H. A., R. E. Carroll, D. L. Lorimer and R. V. Benya. Location and characterization of the human GRP receptor expressed by gastrointestinal epithelial cells. Peptides 18(5) 663-672, 1997.—The exact location of normal gastrin-releasing peptide (GRP) receptor expression by epithelial cells lining the human gastrointestinal (GI) tract is not known; yet this receptor is found on upwards of 50% of GI cancers. Furthermore, the pharmacology reported for GRP receptors expressed by GI cancers varies considerably. Therefore, the purpose of this study was to determine the normal distribution of GRP receptor expression by cells lining the human GI tract, and then determine the normal pharmacology of the human receptor when ectopically expressed by the nonmalignant human colon epithelial cell line NCM460. We obtained endoscopic pinch biopsies of, and extracted the RNA from, epithelial cells lining the esophagus, stomach, jejunum, ileum, and proximal and descending colon. RT-PCR demonstrated that GRP-R expression is limited to cells lining the gastric antrum, indicating that this receptor is aberrantly expressed by GI cancers. To determine the normal pharmacology of this receptor when expressed by nonmalignant human tissues for the first time, we transfected NCM460 cells with the cDNA for the human GRP receptor. By studying three stable NCM460 cell lines expressing varying numbers of receptors, we demonstrate that agonist and antagonist binding affinity, binding kinetics, and G-protein coupling are all independent of receptor number. Finally, by comparing GRP receptors expressed by GI cancers with those on NCM460-transfected cells, we show that the pharmacology of the aberrantly expressed receptors is significantly altered. Thus, these data demonstrate that GI cancers aberrantly express GRP receptors that then behave abnormally.

Section snippets

Materials

Bovine serum albumin and HEPES were obtained from Boehringer Mannheim Biochemicals (Indianapolis, IA), and bombesin, gastrin-releasing peptide, and neuromedin B were obtained from Penninusla Laboratories (Belmont, CA). Dowex AG 1-X8 anion exchange resin, SDS, and 2-mercaptorethanol were from Bio-Rad (Richmond, CA). Hydro-Fluor scintillation fluid, methanol, acetic acid, and hydrochloric acid were from J. T. Baker Chemical Co. (Phillipsburg, NJ). Aminoglycoside G-418, Ham's F-12 media, fetal

GRP Receptor Expression in Human GI Tract Mucosal Epithelium

Initial studies were performed to determine the expression of GRP receptors by mucosal epithelial cells lining the human GI tract. Using RT-PCR we demonstrated that GRP receptors are not expressed by epithelial cells lining the esophagus, duodenum, ileum, or ascending or descending colon (Fig. 1) but are expressed in the antrum. Direct sequencing of the PCR product revealed it to have 100% nucleotide sequence identity with the previously cloned human receptor ([10]). To support the validity of

Discussion

The GRP receptor is widely expressed by many tissues, although the precise location of this receptor within the human GI tract is not known. Unfortunately, studies on other mammals have produced conflicting and confusing results. In autoradiographic studies performed on the dog, high-affinity bombesin/GRP binding sites were only present within the mucosal epithelium lining the gastric antrum ([48]). In contrast, others have demonstrated the presence of bombesin/GRP binding sites in mouse

Acknowledgements

This work was supported in part by an ADHF/American Gastroenterological Association Industry Research Scholar Award and by a Schweppe Foundation Award (both to R. V. B.).

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