Molecular Cloning And Functional Characterization Of A Goldfish Growth Hormone Releasing Hormone Receptor

Author : 孫彩云
Publisher : Open Dissertation Press
ISBN 13 : 9781374676473
Total Pages : pages
Book Rating : 4.6/5 (764 download)

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Download or read book Luteinizing Hormone Receptor and Its Functional Role in Gonadotropin-Induced Growth Hormone Gene Transcription in Grass Carp written by 孫彩云 and published by Open Dissertation Press. This book was released on 2017-01-27 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation, "Luteinizing Hormone Receptor and Its Functional Role in Gonadotropin-induced Growth Hormone Gene Transcription in Grass Carp" by 孫彩云, Caiyun, Sun, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled LUTEINIZING HORMONE RECEPTOR AND ITS FUNCTIONAL ROLE IN GONADOTROPIN-INDUCED GROWTH HORMONE GENE TRANSCRIPTION IN GRASS CARP Submitted by Sun Caiyun for the degree of Doctor of Philosophy at The University of Hong Kong in August 2007 Luteinizing hormone (LH) receptors (LHR) are expressed mainly in the gonad and mediate the reproductive functions of LH released from the anterior pituitary. In the past decade, extragonadal expression of LHR has been reported with exciting findings on the novel functions of LH in non-gonadal tissues. In our recent studies with grass carp as a model for modern day bony fish, endogenous LH was found to act locally at the pituitary level to induce GH secretion and GH gene expression. To further extend our understanding on this unique paracrine interaction within the carp pituitary, the structural identity of carp LHR was established by molecular cloning. The receptor specificity of this newly cloned LHR was confirmed by functional expression in CHO cells. Using RT-PCR, LHR expression was detected in mixed populations of carp pituitary cells as well as in immuno-identified somatotrophs isolated by laser capture microdissection. During our cloning exercise, a splice variant of the full-length LHR (LHR-L) with LRR truncation in N-terminal 1-3 ectodomain was isolated. This LHR variant, namely LHR-S, is unique and has never been reported in other vertebrates. In contrast to LHR-L, which was shown to be 2+ 2+ 2+ functionally coupled to cAMP synthesis, [Ca ]e entry via Ca channels, and [Ca ]i mobilization by IP R and RyR activation, LHR-S stimulation did not trigger cAMP 2+ production nor Ca mobilization. LHR-S co-expression, however, was effective in 2+ attenuating the cAMP responses and Ca signals induced by LHR-L activation, suggesting that this novel variant of LHR may act as a dominant negative LHR in fish model. Based on the LHR sequence obtained, a real-time PCR system was set up to monitor LHR mRNA expression in grass carp pituitary cells. In these studies, dopaminergic stimulation could up-regulate transcript expression of LHR via pituitary D1 receptors coupled to the AC/cAMP/PKA pathway. Furthermore, LH produced locally in carp pituitary cells could suppress basal levels of LHR mRNA whereas exogenous LH was effective in blocking D1-stimulated LHR gene expression. The inhibitory effect of LH on dopamine D1 stimulation was found to occur selectively in the enriched fraction of carp somatotrophs, suggesting that the functional interactions between dopamine and LH may play a role in modulating the sensitivity of somatotrophs to local stimulation by endogenous LH. To shed light on the signaling mechanisms responsible for LH-induced GH gene expression, GH cells with transient expression of carp LHR was co-transfected with luciferase-expressing constructs carrying the 5' promoter of grass carp GH gene. The cell model was used to study the signaling pathways involved in GH gene transcription induced by LHR activation. Using 5' deletion analysis, the LH responsive sequence was mapped to position -616 and -572 of the grass carp GH promoter. Using a combination of both molecular and pharmacological approaches, it has been revealed that GH promoter activity could be up-regulated by LHR activation through the "cross-talk" between the cAMP/PKA p