Yanamala, Naveena VK
(2009)
Allosteric Modulation of G Protein Coupled Receptors.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Structural coupling between the cytoplasmic (CP), transmembrane (TM) and extracellular (EC) domains of G protein coupled receptors (GPCRs) is crucial for their functioning in signal transfer from the extracellular to the intracellular side of the membrane. The focus of this thesis was to test the hypothesis that ligands can bind in each of the three domains. Depending on the location of the endogenous ligand binding site, the other two sites would become allosteric ligand binding sites. To test this hypothesis, we investigated the binding of accessory ligands to each of the three domains, CP, TM and EC. The major contributions of this thesis are as follows:I. The anthocyanin Cyanidin-3-glucoside (C3G) and the chlorophyll-derivative chlorin e6 (Ce6), were shown to physically interact with rhodopsin. These studies demonstrated the presence of a novel CP allosteric ligand binding site in rhodopsin. Biophysical evidence indicated differential effects of binding of these ligands on rhodopsin function, structure and dynamics. II. The allosteric TM ligand binding pocket in metabotropic glutamate receptors (mGluRs) was shown to be analogous in structure and function to the orthosteric TM retinal ligand binding pocket in rhodopsin. Docking of known allosteric modulators to structural models of mGluRs based on rhodopsin conformations was used to predict allosteric modulatory effects. Structural comparison of the mGluR and rhodopsin binding pockets revealed high overlap and preliminary evidence was obtained showing that an mGluR ligand can bind to rhodopsin.III. Evidence for the existence of an EC ligand binding domain was presented. Rhodopsin was shown to bind the extracellular chemokine ligand, CXCL11, an event which interfered with both rhodopsin and chemokine functions. IV. As part of the above efforts, it became necessary to develop and improve NMR spectroscopic methodology to study ligand binding of membrane proteins such as GPCRs. Thus, 1H and 19F based NMR methods to screen for novel ligands that bind to GPCRs were developed and applied to rhodopsin. Collectively, the studies presented in this thesis enhance the understanding of allosteric modulation of GPCRs in general, and of the molecular mechanism of rhodopsin and mGluR activation in the presence of allosteric ligands in particular. The results could help in the identification of new ligands to allosterically modulate receptor structures and in turn their functions at different binding pockets, thus paving new ways to selectively target this pharmacologically important class of receptors.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
| Creators | Email | Pitt Username | ORCID  |
|---|
| Yanamala, Naveena VK | nay6@pitt.edu | NAY6 | |
|
| ETD Committee: |
| Title | Member | Email Address | Pitt Username | ORCID |
|---|
| Committee Chair | Klein-Seetharaman, Judith | judithks@cs.cmu.edu | | | | Committee Member | DeFranco, Donald B | | | | | Committee Member | Tang, Pei | | | | | Committee Member | Amara, Susan G | | | | | Committee Member | Smithgall, Thomas E | | | |
|
| Date: |
13 May 2009 |
| Date Type: |
Completion |
| Defense Date: |
14 April 2009 |
| Approval Date: |
13 May 2009 |
| Submission Date: |
28 April 2009 |
| Access Restriction: |
5 year -- Restrict access to University of Pittsburgh for a period of 5 years. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Biophysics |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Ligand Binding sites; molecular modeling; structural coupling |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-04282009-142113/, etd-04282009-142113 |
| Date Deposited: |
10 Nov 2011 19:43 |
| Last Modified: |
15 Nov 2016 13:42 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/7716 |
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