Die Rolle der Extrazellulärdomäne beim agonistenabhängigen und agonistenunabhängigen Aktivierungsprozess des LH-Rezeptors

Der Lutropin/Choriongonadotropin-Rezeptor (LHR) sowie die übrigen Glykoproteinhormon-Rezeptoren, Thyreotropin-Rezeptor (TSHR) und Follitropin-Rezeptor (FSHR), stellen Vertreter der rhodopsinähnlichen G-Protein-gekoppelten Rezeptoren dar. Glykoproteinhormon-Rezeptoren spielen eine entscheidende Rolle...

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Bibliographic Details
Main Author: Nurwakagari, Pascal
Contributors: Bünemann, Moritz (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2011
Online Access:PDF Full Text
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Glycoprotein hormone receptors such as the lutropin/chorionic gonadotropin receptor (LHR), the thyrotropin receptor (TSHR) and the follicle-stimulating hormone receptor (FSHR) are members of the rhodopsin-like G-protein coupled receptor superfamily and play a pivotal role in growth and function of the gonads and the thyroid gland. Gain-of-function mutations in glycoprotein hormone receptor genes are frequently associated with endocrine dysfunctions such as testicular disorders. A detailed understanding of the mechanism of glycoprotein hormone receptor activation is mandatory for the development of new pharmacological strategies for therapeutic intervention of such dysfunctions. In the present study, the role of the extracellular domain (EZD) for the activation of the LHR was examined. Genetically modified LHR were designed and functionally analyzed in a heterologous expression system by determining receptor protein expression and insertion into the cell membrane as well as receptor-dependent basal and agonist-induced cAMP accumulation. Functional analysis revealed that the LHR-EZD does not function as an inverse agonist but contributes to an active receptor conformation. Partial or total deletion of the EZD resulted in LHR with decreased basal receptor activity when compared to the wildtype LHR. Furthermore, most naturally occurring TMD mutations which induce high ligand-independent receptor activity in the wildtype LHR, were not active in EZD deficient LHR. Additional experiments revealed that the LHR-EZD cannot fully functionally be replaced by the EZD of the closely related TSHR, since swapping of the EZD in the LHR for the EZD of the TSHR impaired the activity of naturally occurring activating TMD mutations. There may be two reasons for this observation: Firstly, the EZD of the TSHR serves as an inverse agonist constraining the LHR-TMD in an inactive conformation, or secondly, due to primary sequence differences between the LHR- and the TSHR-EZD, the TSHR-EZD is not capable to support an LHR-TMD conformation susceptible to activating mutations. The data observed for ligand-receptor chimeras indicate that an intact LHR-EZD is required for ligand-dependent receptor activation and that the hormone per se is unable to activate the TMD of the LHR. Additional experiments support the notion that the integrity of the EZD, particularly the intactness of the LRR domain, is a prerequisite for the ability of the LHR-EZD to support an active receptor conformation induced by the hormone and activating mutations. Altogether, this study provides new insights in the role of the EZD in the activation process of glycoprotein hormone receptors. The data observed in this study strengthen the concept of a major contribution of the LHR-EZD in the activation mechanism apart from hormone binding and provide evidence for a cooperative model with structural and functional interactions of the EZD and the TMD. Thus, our study challenges the prevailing concept that the hormone-binding EZD and the G-protein-coupling TMD are structurally and functionally independent modules of glycoprotein hormone receptors.