Cloning and characterization of the human GnRH receptor

GnRH receptors belong to the family of G protein-coupled receptor proteins and have been localized to the anterior pituitary, brain and reproductive organs as well as many steroid-dependent tumor tissues. Recently, cDNAs for the GnRH receptors of several species including the human have been cloned. To determine the structure of the gene encoding the human GnRH receptor, we isolated the receptor gene clones from the human genomic libraries. Comparison of the genomic and cDNA sequences revealed that the human GnRH receptor gene is composed of three exons and two introns and spans over 20 kb in size. Exon 1 encodes the 5 0 untranslated sequence and nucleotide þ 1 to þ 522 in the open reading frame, exon 2 encodes nucleotide þ 523 to þ 742 and exon 3 encodes nucleotide þ 743 to þ 987 in the open reading frame as well as the 3 0 untranslated sequence. Southern blot analysis of genomic DNA and localization of the GnRH receptor gene to a single site on human chromosome 4 (4q13) indicate the presence of a single copy of the gene in the human genome. Several regulatory sequences for various hormones and other regulatory factors were identified, including PEA-3, AP-1, AP-2, and Pit-1 sites. In addition, glucocorticoid/progesterone response element, thyroid hormone response element, and cAMP response element sequences were identified. Reverse transcriptase-primer extension and 5 0 RACE analysis of the human pituitary RNA demonstrated the presence of multiple transcriptional start sites upstream of the translational start site. Analysis of the 5 0 flanking region of the gene also revealed the presence of multiple TATA and CAAT sequences. The finding of multiple transcriptional start sites raises the possibility of tissue-specific regulation and the existence of variable size transcripts. Chimeras containing 1.26 kb (¹534 to 728) of the 5 0 flanking region of the receptor gene and the luciferase (Luc) gene expressed a significant luciferase activity when transfected into a human endometrial tumor cell line (HEC-1A) and a breast tumor cell line (MCF-7) but not in a mouse pituitary gonadotrope cell line (aT3-1), suggesting the existence of multiple promoter elements in the gene. These findings indicate a multiplicity of regulation of expression of the GnRH receptor and provide the substrate for detailed investigation in the reproductive system.

Section: Isolation Of Genomic Clones and Sequencingsupporting

confidence: 86%

“…2) and antisense 5 0 -TGTGCATAGACTGCAGGTTA-3 0 (gene position, 13 111-13 130, Fig. 2) from the human GnRH receptor cDNA sequence (15), and human leukocyte genomic DNA as a template. PCR generated DNA of size 1.3 kb which was then subcloned into pBlueScript SK þ (Stratagene).…”

Section: Isolation Of Genomic Clones and Sequencingmentioning

confidence: 99%

Molecular structure of the human gonadotropin-releasing hormone receptor gene

Kakar

1997

“…data have been obtained relating to the presence of GnRH receptors (5 -7). Northern blot analysis failed to detect GnRH receptor mRNAs in any of the non-pituitary tissues examined (24). However, using RT-PCR, these mRNAs were recently identified in granulosa luteal cells (16).…”

Section: Hypothalamic Gonadotropin-releasing Hormonementioning

confidence: 90%

“…The latter is comparable to the pituitary GnRH receptor (4, 46,47). In 1992, cloning, sequencing and expression of the human pituitary GnRH receptor were reported (23,24). These findings stimulated intensive research, leading to the demonstration of GnRH receptor gene transcripts in ovarian and endometrial cancer cell lines and in about 80% of the respective primary tumors (23, 48 -50) (Table 1).…”

Section: Expression Of Gnrh and Gnrh Receptorsmentioning

confidence: 99%

Biology of the gonadotropin-releasing hormone system in gynecological cancers

Gründker

Gunthert

Westphalen

et al. 2002

155

139

The expression of GnRH and its receptor as a part of an autocrine regulatory system of cell proliferation has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary and endometrium. Dose-dependent antiproliferative effects of GnRH agonists in cell lines derived from these cancers have been observed by various investigators. GnRH antagonists also have marked antiproliferative activity in most breast, ovarian and endometrial cancer cell lines tested, indicating that the dichotomy of GnRH agonists and antagonists might not apply to the GnRH system in cancer cells. The classical GnRH receptor signal-transduction mechanisms, known to operate in the pituitary, are not involved in the mediation of antiproliferative effects of GnRH analogs in cancer cells. Rather, the GnRH receptor interacts with the mitogenic signal transduction of growth factor receptors and related oncogene products associated with tyrosine kinase activity, via activation of a phosphotyrosine phosphatase, resulting in downregulation of cancer cell proliferation. In addition, GnRH activates nuclear factor kB and protects the cancer cells from apoptosis. Furthermore, GnRH induces activation of the c-Jun N-terminal kinase/activator protein-1 (AP-1) pathway independent of the known AP-1 activators, protein kinase or mitogen activated protein kinase.

“…The human GnRHR consists of 328 amino acids. cDNAs encoding a functional pituitary GnRHR have been obtained in mice (1,2), rats (3), humans (4,5) and sheep (6), and predicted amino acid sequences were highly conserved among the species. The coding region is distributed among three exons separated by two large introns (7,8).…”

Section: Introductionmentioning

confidence: 99%

Tissue-specific pattern of variant transcripts of the human gonadotropin-releasing hormone receptor gene

Kottler

Bergametti²,

Carre³

et al. 1999

The expression pattern of the GnRH receptor was investigated in a variety of normal and neoplastic human tissues by RT-PCR-Southern blotting. In addition to the full-length cDNA (sb1), we identified two other transcripts: the first (sb2) was characterized by a 128 bp deletion as previously described; the second was an unexpected finding composed of a shorter cDNA (sb3), the sequence of which revealed a 220 bp deletion corresponding in size to exon 2. These three transcripts were found in normal pituitary and pituitary adenomas, and in granulosa tumors, but not in testis, where sb2 was lacking. Only sb1 was expressed in normal, fibrocystic and malignant breast tissue. No transcript with a full-length region was found in endometrium, intestine or lymphocytes. This is the first report that shows that splicing of the gonadotropin-releasing hormone receptor gene is tissue dependent.We also determined the intron-exon nucleotide sequence of the gene and identified an MaeIII polymorphic site in exon 1 created by a silent C453T transition found in 10% of unrelated French whites.