| Summary: | The gonadotropin - releasing hormone (GnRH) receptor is a plasma membrane
associated receptor and a member of the GTP - binding protein coupled receptor
family. The interaction of the ligand, GnRH, and the GnRH receptor is a critical
event in the endocrine control of reproduction. This coupling stimulates the
synthesis and release of both luteinizing hormone and follicle stimulating hormone
from the anterior pituitary. In addition, GnRH - GnRH receptor binding acts locally
to regulate human chorionic gonadotropin secretion in the placenta and
steroidogenesis in the ovary. The objective of this thesis was to isolate and
characterize the gene for the GnRH receptor in human. The human GnRH receptor
(GriRH-R) gene was isolated from a human genomic library derived from placental
tissue. The genomic clones obtained encompassed the entire gene including its
coding region (987 bp) as well as substantial 5’ and 3’ sequences. Sequence analysis
revealed a structural organization consisting of three exons and two introns
distributed over 18.9 Kb. Exon II contains only 219 bp and the remainder of the
approximately 4.7 Kb transcript is distributed between exon I (1915 bp) and III
(3321 bp). Sequence analysis and restriction endonuclease mapping revealed the
sizes of intron A and B to be approximately 4.2 and 5.0 Kb, respectively.
Sequencing of the 5’ end of the gene revealed the presence of five consensus TATA sequences clustered within a 700 nucleotide region. Primer extension analysis
detected multiple transcription initiation sites associated with this group of TATA
sequences. Transcription of this region up to the most 5’ initiation site was
demonstrated by the reverse transcription - polymerase chain reaction (RT-PCR)
method. The 5’ nontranslated region has a length between 703 and 1393 bp,
depending on which initiation site is used. Several consensus cis - acting regulatory
sequences were identified within the 5’ end. These include sites for GATA-i,
WAP, PEA-3, AP-1, and Pit-i. In addition, cAMP response element (CRE) - like
and glucocorticoid / progesterone response element (GRE / PRE) - like sequences
were found. The ability of these response elements to bind to their respective
regulatory proteins (CRE binding protein for CRE; progesterone receptor for GRE!
PRE) was investigated by mobility shift assays. No DNA - protein complexes were
observed for these response - like elements suggesting that the mismatches incurred
could not be recognized by the respective regulatory proteins. The 3’ end of the
gene was also sequenced and five classical polyadenylation signals were found
scattered over a region of 800 nucleotides. RT-PCR conducted on the 3’
nontranslated region confirmed transcription up to the most 3’ located
polyadenylation signal. Factoring in the location of the most 5’ initiation site and
the most 3’ polyadenylation signal, the total transcript covers a region of 5455 bp.
The finding of multiple transcription intiation sites and polyadenylation signals raises the possibility of tissue - specific regulation and the existence of variable transcripts
for the human GnRH receptor. Genomic Southern blot analysis indicated the
presence of a single copy of the gene encoding for the GnRH-R gene within the
human genome. Using somatic hybrid analysis, the GnRH-R gene was also assigned
to human chromosome 4. This study represents the first report on the isolation and
characterization of the GnRH-R gene in any species. The characterization of the
human GnRH-R gene should facilitate future investigative efforts on the delineation
of possible genetic disorders for this gene, the mechanisms involved in its regulation,
and on generation of improved GnRH analogues currently in use for several
reproductive disorders and diseases.
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