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STRUCTURE-FUNCTION STUDIES OF THE METABOTROPIC GLUTAMATE RECEPTOR TYPE 6 (mGluR6) AND COMPARISON WITH RHODOPSIN

Tirupula, Kalyan C (2011) STRUCTURE-FUNCTION STUDIES OF THE METABOTROPIC GLUTAMATE RECEPTOR TYPE 6 (mGluR6) AND COMPARISON WITH RHODOPSIN. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    Metabotropic glutamate receptor subtype 6 (mGluR6), a class C G protein coupled receptor (GPCR), plays a key role in visual signal transduction and is also implicated in addiction. Certain mutations in mGluR6 have been reported to cause congenital stationary night blindness. In spite of the importance of mGluR6, knowledge of the molecular basis of its function is lacking. It is imperative to improve the current understanding of its structure-function relationships, so that selective ligands that modulate its activity can be discovered. Furthermore, functional characterization of mGluR6 is also expected to lead to a better understanding of the general principles underlying the activation mechanism of GPCR family. Rhodopsin is the prototypical class A GPCR and serves as a good comparative model to establish general mechanistic patterns of activation of GPCRs. This thesis describes experimental and computational approaches to characterize the structure-function relationship of mGluR6 and its comparison with rhodopsin.Firstly, inducible stable cell lines with high levels of mGluR6 expression were established. Proper trafficking and folding of mGluR6 in these cell lines were verified. To determine mGluR6 function, existing cell-based and novel membrane-based functional assays were optimized and developed, respectively. These efforts led to the establishment of a robust system that expresses properly folded and functional mGluR6 and enabled structure-function studies to be carried out. Several transmembrane cysteine mutants were created and analyzed with the goal to study the role of the transmembrane domain of mGluR6 in activation mechanism. TM6 of mGluR6 like rhodopsin was found to play a key role in its activation supporting the hypothesis that these two GPCRs may share a general mechanism of activation despite the large sequence divergence. Additional support for this hypothesis was obtained from computational sequence analysis which showed that the highly ranking residues involved in long-range interaction in rhodopsin overlap with the allosteric binding pocket of mGluR6. Finally, with the aim to identify selective ligands for mGluR6, an integrated computational-experimental approach was undertaken. Novel allosteric ligands and possibly selective orthosteric ligands for mGluR6 were identified. Further characterization of these ligands may lead to design of selective ligands for mGluR6.


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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmail
    Committee ChairKlein-Seetharaman, Judithjks33@pitt.edu
    Committee MemberDoemling, Alexanderasd30@pitt.edu
    Committee MemberSaxena, Sunilsksaxena@pitt.edu
    Committee MemberMillington, William RWilliam.Millington@acphs.edu
    Title: STRUCTURE-FUNCTION STUDIES OF THE METABOTROPIC GLUTAMATE RECEPTOR TYPE 6 (mGluR6) AND COMPARISON WITH RHODOPSIN
    Status: Unpublished
    Abstract: Metabotropic glutamate receptor subtype 6 (mGluR6), a class C G protein coupled receptor (GPCR), plays a key role in visual signal transduction and is also implicated in addiction. Certain mutations in mGluR6 have been reported to cause congenital stationary night blindness. In spite of the importance of mGluR6, knowledge of the molecular basis of its function is lacking. It is imperative to improve the current understanding of its structure-function relationships, so that selective ligands that modulate its activity can be discovered. Furthermore, functional characterization of mGluR6 is also expected to lead to a better understanding of the general principles underlying the activation mechanism of GPCR family. Rhodopsin is the prototypical class A GPCR and serves as a good comparative model to establish general mechanistic patterns of activation of GPCRs. This thesis describes experimental and computational approaches to characterize the structure-function relationship of mGluR6 and its comparison with rhodopsin.Firstly, inducible stable cell lines with high levels of mGluR6 expression were established. Proper trafficking and folding of mGluR6 in these cell lines were verified. To determine mGluR6 function, existing cell-based and novel membrane-based functional assays were optimized and developed, respectively. These efforts led to the establishment of a robust system that expresses properly folded and functional mGluR6 and enabled structure-function studies to be carried out. Several transmembrane cysteine mutants were created and analyzed with the goal to study the role of the transmembrane domain of mGluR6 in activation mechanism. TM6 of mGluR6 like rhodopsin was found to play a key role in its activation supporting the hypothesis that these two GPCRs may share a general mechanism of activation despite the large sequence divergence. Additional support for this hypothesis was obtained from computational sequence analysis which showed that the highly ranking residues involved in long-range interaction in rhodopsin overlap with the allosteric binding pocket of mGluR6. Finally, with the aim to identify selective ligands for mGluR6, an integrated computational-experimental approach was undertaken. Novel allosteric ligands and possibly selective orthosteric ligands for mGluR6 were identified. Further characterization of these ligands may lead to design of selective ligands for mGluR6.
    Date: 17 May 2011
    Date Type: Completion
    Defense Date: 04 April 2011
    Approval Date: 17 May 2011
    Submission Date: 17 April 2011
    Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
    Patent pending: No
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    URN: etd-04172011-024356
    Uncontrolled Keywords: Europium; Allostery; Anthocyanins; AutoDock Vina; cAMP; dipeptides; Gly-Gln; Gly-Glu; GTP-Eu; GTPgS; homology modelling; L-AP4; L-glutamate; mGluR2; mGluR3; mGluR5; PHCCC; addiction; Chimera; GREMLIN; Immunofluorescence; mGluRs; night blindness; Porphyrins; Virtual Screening; correlated mutation analysis; LY341495
    Schools and Programs: School of Medicine > Molecular Biophysics and Structural Biology
    Date Deposited: 10 Nov 2011 14:38
    Last Modified: 08 May 2012 13:46
    Other ID: http://etd.library.pitt.edu/ETD/available/etd-04172011-024356/, etd-04172011-024356

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