Elsevier

Gene

Volume 264, Issue 1, 7 February 2001, Pages 95-103
Gene

The human gastrin-releasing peptide receptor gene structure, its tissue expression and promoter

https://doi.org/10.1016/S0378-1119(00)00596-5Get rights and content

Abstract

The human gastrin-releasing peptide receptor (hGRP-R) is aberrantly expressed in cancers of the colon, lung and prostate and mediates signals of cellular proliferation. However, the underlying mechanisms of aberrant and/or activation of hGRP-R expression are unknown. Therefore, a genomic clone is identified, the hGRP-R gene is characterized, and the hGRP-R promoter is defined. The protein coding region is divided into three exons and exon/intron splice sites occur in the proximal 2nd and distal 3rd intracellular loops of the receptor molecule. The hGRP-R locus extends over more than 27 kb and is assigned to the chromosomal band Xp22 by fluorescence in situ hybridization. With primer extension experiments, we demonstrate two major transcription start sites in gastrointestinal and breast cancer cells, located 43 and 36 bp downstream of a TTTAAA motif which is identified 407 to 402 bp upstream of the ATG start codon. The hGRP-R is found most abundantly expressed in the normal human pancreas, where four gene-specific transcripts can be detected by Northern blot analysis, whereas only two transcripts are detected in the human stomach and, very weakly, in the adrenal cortex and the brain. In contrast, the human GRP-R is not expressed in the normal human colon, lung, and prostate. Steady state hGRP-R mRNA can also be detected in some cultured cells from breast, lung, and duodenal cancer. Robust hGRP-R promoter activity is demonstrated in a duodenal carcinoma cell line that natively expresses the functional hGRP-R. Truncation studies suggest a CRE motif, located 112 bp upstream of the major transcription start site, is required to confer basal hGRP-R promoter activity in duodenal cancer cells. These studies provide the necessary data to further elucidate molecular mechanisms of aberrant hGRP-R expression in human cancers.

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.

Section snippets

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.).

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    GenBank submission Nos.: AF293321, AF293322, AF293323.

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