A Dissertation submitted to the Faculty of Health Science, University of the Witwatersrand,
in fulfillment of the requirements for the degree of Master of Science.
Johannesburg, 2017 / Gonadotropin-releasing hormone (GnRH) binds to GnRH receptors (GnRHR) in the pituitary and stimulates release of gonadotropins, which control reproduction. It has been proposed that the congenital Glu2.53(90)Lys GnRHR mutation causes infertility by disrupting a salt-bridge important for GnRHR protein expression. To investigate its role in GnRHR function, Glu2.53(90) was mutated to residues that mimic or remove its side-chain properties. Mutant receptors were assessed for inositol phosphate signaling and radioligand binding. Receptors with small or negatively-charged substitutions for Glu2.53(90) exhibited no measurable function. Stabilizing receptor expression by appending a carboxy-terminal tail recovered function of the Glu2.53(90)Lys and Glu2.53(90)Ala GnRHRs, but not the conservative Glu2.53(90)Asp mutant. Receptors with uncharged (Gln) or hydrophobic (Leu, Phe) substitutions that cannot form salt-bridges with Lys3.32(121) were fully functional. Although the positively-charged Arg substitution decreased binding affinity, it preserved GnRHR function, confirming that interaction with the positively-charged Lys3.32(121) is not required. Comparing the GnRHR with structurally-related G protein-coupled receptors revealed that the equivalent residue of rhodopsin, Met2.53(86), interacts with Trp6.48(265). Mutating Trp6.48(280) of the GnRHR to Ala and Arg disrupted GnRH-stimulated function, confirming a role in expression. The Trp6.48(280)Arg GnRHR with an appended carboxy-terminal tail had decreased GnRH binding affinity. The preserved function of mutant receptors with large hydrophobic or positively-charged amino acid substitutions suggests that the size of the Glu2.53(90) is important for stabilizing GnRHR structure. Decreased affinity of mutant receptors with larger (Arg) substitutions for Glu2.53(90) and Trp6.48(280) suggest that both residues make conserved intramolecular interactions that stabilize receptor protein expression and configure the extracellular GnRHR structure.
(250 words) / MT2017
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/23278 |
| Date | January 2017 |
| Creators | Manilall, Ashmeetha |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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