Ligand-induced selective signalling at the gonadotrophin releasing hormone receptor

dc.contributor.advisorFlanagan, Colleenen_ZA
dc.contributor.authorCoetsee, Marla Catherineen_ZA
dc.date.accessioned2014-07-28T14:54:47Z
dc.date.available2014-07-28T14:54:47Z
dc.date.issued2008en_ZA
dc.descriptionIncludes abstract.
dc.descriptionIncludes bibliographical references (p. 179-202).
dc.description.abstractThe pituitary gonadotrophin releasing hormone (GnRH) receptor regulates reproduction by activation of Gq/11 proteins. In contrast, GnRH receptors at extrapituitary sites induce anti-proliferative effects that do not correlate with Gq/11 activation. We propose that the two endogenous ligands, GnRH I and GnRH II, and certain antagonists selectively activate distinct signalling pathways by stabilisation of distinct active conformations of the GnRH receptor, a concept termed ligand-induced selective signalling (LiSS). This dissertation has investigated LiSS at the GnRH receptor using several approaches. The sequences of GnRH I and II differ in positions 5, 7 and 8. I investigated the interaction of position 5 of GnRH I and GnRH II with Tyr6.58 of the receptor. Compared with the Leu and Ala mutants, the Tyr6.58Phe mutant had higher affinity for native GnRHs, but not Ala5-substituted GnRHs, suggesting that Tyr5 of GnRH I and His5 of GnRH II interact with Tyr6.58 by aromatic interactions. Our molecular models show that GnRHs interact with distinct rotamer conformations of Tyr6.58. This is supported by the Tyr6.58Leu receptor, which makes compensatory interactions that improve binding affinity and receptor activation for GnRH II, but not GnRH I, compared with the Tyr6.58Ala receptor. Together these results suggest that GnRHs stabilise distinct receptor active conformations. To identify the most proximal signalling proteins that mediate GnRH receptordependent anti-proliferative effects, I established a range of [35S]GTPS binding assays. I confirmed that the GnRH receptor activates Gq/11, but in contrast to previous proposals, my results show that the GnRH receptor cannot directly activate Gi. I subsequently identified a novel GnRH receptor signalling partner, the SH2 domaincontaining phosphatase 2 (SHP-2). I propose that SHP-2 mediates the antiproliferative effects of the receptor. I show that the SHP-2 pathway is activated independently of Gq/11 and suggest that signalling occurs by a direct interaction of SHP-2 and src with the GnRH receptor. Furthermore, this pathway is activated by a classical Gq/11 antagonist or by Gq/11-uncoupled GnRH receptor mutants. My results provide convincing evidence supporting LiSS at the GnRH receptor and may facilitate development of therapeutics with increased signalling specificity at this receptor.en_ZA
dc.identifier.apacitationCoetsee, M. C. (2008). <i>Ligand-induced selective signalling at the gonadotrophin releasing hormone receptor</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry. Retrieved from http://hdl.handle.net/11427/3123en_ZA
dc.identifier.chicagocitationCoetsee, Marla Catherine. <i>"Ligand-induced selective signalling at the gonadotrophin releasing hormone receptor."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry, 2008. http://hdl.handle.net/11427/3123en_ZA
dc.identifier.citationCoetsee, M. 2008. Ligand-induced selective signalling at the gonadotrophin releasing hormone receptor. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Coetsee, Marla Catherine AB - The pituitary gonadotrophin releasing hormone (GnRH) receptor regulates reproduction by activation of Gq/11 proteins. In contrast, GnRH receptors at extrapituitary sites induce anti-proliferative effects that do not correlate with Gq/11 activation. We propose that the two endogenous ligands, GnRH I and GnRH II, and certain antagonists selectively activate distinct signalling pathways by stabilisation of distinct active conformations of the GnRH receptor, a concept termed ligand-induced selective signalling (LiSS). This dissertation has investigated LiSS at the GnRH receptor using several approaches. The sequences of GnRH I and II differ in positions 5, 7 and 8. I investigated the interaction of position 5 of GnRH I and GnRH II with Tyr6.58 of the receptor. Compared with the Leu and Ala mutants, the Tyr6.58Phe mutant had higher affinity for native GnRHs, but not Ala5-substituted GnRHs, suggesting that Tyr5 of GnRH I and His5 of GnRH II interact with Tyr6.58 by aromatic interactions. Our molecular models show that GnRHs interact with distinct rotamer conformations of Tyr6.58. This is supported by the Tyr6.58Leu receptor, which makes compensatory interactions that improve binding affinity and receptor activation for GnRH II, but not GnRH I, compared with the Tyr6.58Ala receptor. Together these results suggest that GnRHs stabilise distinct receptor active conformations. To identify the most proximal signalling proteins that mediate GnRH receptordependent anti-proliferative effects, I established a range of [35S]GTPS binding assays. I confirmed that the GnRH receptor activates Gq/11, but in contrast to previous proposals, my results show that the GnRH receptor cannot directly activate Gi. I subsequently identified a novel GnRH receptor signalling partner, the SH2 domaincontaining phosphatase 2 (SHP-2). I propose that SHP-2 mediates the antiproliferative effects of the receptor. I show that the SHP-2 pathway is activated independently of Gq/11 and suggest that signalling occurs by a direct interaction of SHP-2 and src with the GnRH receptor. Furthermore, this pathway is activated by a classical Gq/11 antagonist or by Gq/11-uncoupled GnRH receptor mutants. My results provide convincing evidence supporting LiSS at the GnRH receptor and may facilitate development of therapeutics with increased signalling specificity at this receptor. DA - 2008 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2008 T1 - Ligand-induced selective signalling at the gonadotrophin releasing hormone receptor TI - Ligand-induced selective signalling at the gonadotrophin releasing hormone receptor UR - http://hdl.handle.net/11427/3123 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/3123
dc.identifier.vancouvercitationCoetsee MC. Ligand-induced selective signalling at the gonadotrophin releasing hormone receptor. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry, 2008 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/3123en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Medical Biochemistryen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherMedical Biochemistryen_ZA
dc.titleLigand-induced selective signalling at the gonadotrophin releasing hormone receptoren_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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