The human gastrin-releasing peptide receptor gene structure, its tissue expression and promoter☆
Introduction
The gastrin-releasing peptide (GRP) is a member of the mammalian bombesin-like family of regulatory peptides that regulate numerous central nervous and gastrointestinal functions, such as the release of gastrointestinal hormones (Ghatei et al., 1982), stimulation of amylase secretion from the pancreas (Jensen et al., 1988), and smooth muscle cell contraction (Severi et al., 1991).
In normal tissues, bombesin-like peptides (BLP) can stimulate growth of bronchial epithelial cells, gastrointestinal epithelial cells, and cause pancreatic hyperplasia (Jensen et al., 1988, Sunday et al., 1988, Upp et al., 1988, Spindel et al., 1993, Kroog et al., 1995). Furthermore, BLP play an important role in human cancer cells from the lung, colon, stomach, pancreas, breast, and prostate by stimulating cellular proliferation (Cuttitta et al., 1985, Carney et al., 1988, Nelson et al., 1991, Bold et al., 1994, Kroog et al., 1995, Moody et al., 1996, Markwalder and Reubi, 1999). In a series of small cell and non-small cell lung cancer cells it was suggested to have a growth promoting effect via an autocrine loop (Cuttitta et al., 1985, Moody et al., 1988). GRP mediates these important functions by binding to its specific, high-affinity cell surface gastrin-releasing peptide receptor, a member of the heptahelical Gq protein-coupled receptors (Kroog et al., 1995).
Cancers of the colon, prostate, and lung account for significant mortality and morbidity in the United States and represent a major health care problem. Recent studies have demonstrated aberrant human GRP-R (hGRP-R) expression in a majority of tissue samples isolated from these and other cancers suggesting that abnormal hGRP-R regulation can mediate growth signals through ligand-stimulated receptor activation (Pagani et al., 1991, Kroog et al., 1995, Bartholdi et al., 1998, Carroll et al., 1999a, Carroll et al., 1999b, Markwalder and Reubi, 1999, Siegfried et al., 1999). In contrast, the underlying molecular mechanisms of aberrant hGRP-R expression and/or activation in human cancers remain entirely unknown at present.
To gain insight in the molecular mechanisms regulating hGRP-R expression in human cancers, we elucidated the organization of the hGRP-R gene to identify structural elements directing hGRP-R expression. We also examined hGRP-R expression in normal human tissues and demonstrated the minimal DNA sequence required for basal hGRP-R promoter activity in a human duodenal carcinoma cell line. This study provides for the first time data necessary to further examine molecular mechanisms of aberrant hGRP-R (over) expression and its gene activation in human cancers.
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Chemicals and radioactive materials
Restriction and modifying enzymes were purchased from Gibco/BRL (Gaithersburg, MD) unless stated otherwise. Synthetic oligonucleotides were obtained from Midland Certified Reagent Co. (Midland, TX) and Gibco/BRL (Gaithersburg, MD). Luciferase assay buffers are from Pharmigen (San Diego, CA). Emerald™ Luminescence Amplifying material and AMPGD™ chemoluminescence substrate for β-galactosidase were purchased from Tropix (Bedford, MA). 10% (v/v) SDS (sodium dodecyl sulfate) was purchased from
Isolation of the human gastrin-releasing peptide receptor gene
A cDNA clone encoding the hGRP-R has previously been isolated from NIH-H345 cells and characterized (Corjay et al., 1991). To isolate a genomic clone, we designed two gene-specific synthetic oligonucleotides located in the immediate 5′ flanking region of the hGRP-R gene (sense primer from -360 to -341: 5′-GCAGGCCAAAAGTTCTTAG-3′, antisense primer from -62 to -81: 5′-GCAACCGAGTGAAGATGAAG-3′) that amplify a 298 bp fragment from human genomic DNA. This primer pair was used (Incyte Genomics,
Discussion
The hGRP-R was demonstrated aberrantly expressed in gastrointestinal cancers, such as colon and stomach, and also in cancers of the prostate, lung, and breast, where its ligand-activation mediates cellular proliferation. It is therefore believed to play an important role in human carcinogenesis (Roth et al., 1983, Carney et al., 1988, Pagani et al., 1991, Bartholdi et al., 1998, Carroll et al., 1999a, Carroll et al., 1999b, Markwalder and Reubi, 1999, Shriver et al., 2000). Consequently, in
Acknowledgements
Cell lines were kindly provided by Drs R.T. Jensen and T. Moody (NIH, Bethesda) and Dr P. Hildebrandt (Basel University, Switzerland). The plasmid carrying the human GRP-R cDNA was kindly provided by Dr J. F. Battey (NIH, Bethesda). This work was supported in part by a New Investigator Award from the Harcourt General Charitable Foundation (to H.C.W.) and an American Digestive Health Foundation (ADHF)/ American Gastroenterological Association (AGA)/ Industry Research Scholar Award (to H.C.W.).
References (32)
- et al.
Characterization of gastrin-releasing peptide receptors aberrantly expressed by non-antral gastric adenocarcinomas
Peptides
(1999) - et al.
Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells
J. Biol. Chem.
(1991) - et al.
Both the basal and inducible transcription of the tyrosine hydroxylase gene are dependent upon a cAMP response element
J. Biol. Chem.
(1993) - et al.
Comparative mapping of the Grpr locus on the X chromosomes of man and mouse
Genomics
(1993) - et al.
Cloning and characterization of the signal transduction of four splice variants of the human pituitary adenylate cyclase activating polypeptide receptor. Evidence for dual coupling to adenylate cyclase and phospholipase C
J. Biol. Chem.
(1996) - et al.
Evidence for autocrine actions of neuromedin B and gastrin-releasing peptide in non-small cell lung cancer
Pulm. Pharmacol. Ther.
(1999) - et al.
Bombesin-like peptides: of ligands and receptors
Recent Prog. Horm. Res.
(1993) - et al.
Molecular organization of the mouse gastrin-releasing peptide receptor gene and its promoter
Gene
(2000) - et al.
In situ hybridization for gastrin-releasing peptide receptor (GRP receptor) expression in prostatic carcinoma
Int. J. Cancer
(1998) - et al.
Bombesin stimulates the in vitro growth of a human gastric cancer cell line
J. Cell. Physiol.
(1994)
Bombesin: a potent mitogen for small cell lung cancer
Ann. N.Y. Acad. Sci.
Aberrant expression of gastrin-releasing peptide and its receptor by well-differentiated colon cancers in humans
Am. J. Physiol.
Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer
Nature
Basic Methods in Molecular Biology
Bombesin: action on gut hormones and calcium in man
J. Clin. Endocrinol. Metab.
Characterization, in some human breast cancer cell lines, of gastrin- releasing peptide-like receptors which are absent in normal breast epithelial cells
Int. J. Cancer
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GenBank submission Nos.: AF293321, AF293322, AF293323.