Xiao D, Wang J, Hampton L L, Weber H C
Boston University School of Medicine, Section of Gastroenterology, 650 Albany Street, EBRC, 5th floor, Boston MA 02118, USA.
Gene. 2001 Feb 7;264(1):95-103. doi: 10.1016/s0378-1119(00)00596-5.
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.
人胃泌素释放肽受体（hGRP-R）在结肠癌、肺癌和前列腺癌中异常表达，并介导细胞增殖信号。然而，hGRP-R表达异常和/或激活的潜在机制尚不清楚。因此，鉴定了一个基因组克隆，对hGRP-R基因进行了表征，并确定了hGRP-R启动子。蛋白编码区分为三个外显子，外显子/内含子剪接位点位于受体分子近端的第二个和远端的第三个细胞内环。hGRP-R基因座延伸超过27 kb，通过荧光原位杂交定位到染色体带Xp22。通过引物延伸实验，我们在胃肠道和乳腺癌细胞中证明了两个主要转录起始位点，位于TTTAAA基序下游43和36 bp处，该基序在ATG起始密码子上游407至402 bp处被鉴定。hGRP-R在正常人类胰腺中表达最为丰富，通过Northern印迹分析可检测到四种基因特异性转录本，而在人类胃中仅检测到两种转录本，在肾上腺皮质和大脑中表达非常微弱。相比之下，人GRP-R在正常人类结肠、肺和前列腺中不表达。在来自乳腺癌、肺癌和十二指肠癌的一些培养细胞中也可检测到稳定状态的hGRP-R mRNA。在天然表达功能性hGRP-R的十二指肠癌细胞系中证明了强大的hGRP-R启动子活性。截短研究表明，位于主要转录起始位点上游112 bp处的CRE基序是在十二指肠癌细胞中赋予基础hGRP-R启动子活性所必需的。这些研究提供了必要的数据，以进一步阐明人类癌症中hGRP-R表达异常的分子机制。
