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"STRUCTURE-FUNCTION STUDIES AND SIGNALING MECHANISMS OF THE DOPAMINE D3 RECEPTOR"

by
KOKILA KOTA
Interdisciplinary Biomedical Sciences Program
B.S., 2002, Jawaharlal Nehru Technological University, India
M.S., 2004, Birla Institute of Technology and Science, Pilani, India

Thesis Advisor: Dr. Eldo V. Kuzhikandathil
Associate Professor,
Department of Pharmacology & Physiology

Tuesday, October 7, 2014
2.30 P.M., MSB- H609b


Abstract

Dopamine is a member of the biogenic amine family of neurotransmitters and improper regulation of dopaminergic transmission results in disorders such as Parkinsonís disease, schizophrenia and addiction. Dopamine receptors are G-protein coupled receptors (GPCRs) and are classified into D1-D5 receptor subtypes. Although the dopamine D3 receptor (D3R) shares high homology with D2, it possesses distinct signaling properties. The D3R exhibits agonist-dependent tolerance and slow response termination (SRT) signaling properties to the native agonist dopamine and to the various agonists like quinpirole, PD128907 and 7-OH-DPAT. We have previously determined that these properties are dependent on the conformational changes induced at the D3R and we also identified novel class of compounds which are D3R agonists but do not elicit tolerance and SRT; cis-8-OH-PBZI being an example of an agonist that does not elicit D3R tolerance and SRT. Structure-function studies from our lab have determined that intracellular loop-2 is necessary but not sufficient for the tolerance property of the receptor and the regions of the D3R contributing to the phenomena of tolerance and SRT are different for different D3R agonists. This led us to hypothesize that there are other regions apart from the intracellular loops of the D3R involved in the tolerance and SRT properties of the D3R. We have identified agonist-specific structural determinants of D3 receptor tolerance property. For example, extracellular loop-2 was identified to be important for the PD128907-elicited tolerance and SRT while extracellular loop-3 was found to be critical for the 7-OH-DPAT-elicited tolerance and SRT properties. Though the currently available crystal structure and molecular modeling studies have identified amino acid residues important for D3R ligand binding, the residues involved in the activation of D3R signaling and induction of the signaling properties have not been determined. In addition, the published D3R crystal structure is antagonist-bound and modeling studies that have used the crystal structure to develop a pharmacophore model to screen D3R ligands have only yielded D3R antagonists but not agonists. Therefore our study was aimed at mapping the critical amino acid residues required for the D3R agonists with an overall goal of contributing to the development of an agonist-bound D3R pharmacophore model. Our site-directed mutagenesis approach coupled with the molecular dynamics simulations studies have identified that the amino-acid residue D187 in the extracellular loop-2 forms a salt-bridge with the residue H354 in the extracellular loop-3 and these interactions determine the PD128907-elicited D3R tolerance property. We used two geometric isomers of D3R agonist, 8-OH-PBZI and the classical tolerance-causing agonist 7-OH-DPAT combined with site-directed mutagenesis to identify key residues involved in D3R signaling function. Our results showed that trans-8-OH-PBZI, but not cis-8-OH-PBZI, elicit the tolerance and SRT properties. We also showed that while both agonists require a subset of residues in the orthosteric binding site of D3R for activation of the receptor, the ability of the two geometric isomers to differentially induce tolerance and SRT is mediated by interactions with specific residues in the sixth transmembrane helix and third extracellular loop of the D3R. The two isomers of 8-OH-PBZI represent novel pharmacological tools for in silico D3R homology modeling and for determining the role of D3R tolerance and SRT properties in signaling and behavior.
In this thesis project we also identified potential molecular mechanisms underlying the D3R signaling properties. Little is known about the interacting protein partners of the D3R and their role in tolerance and SRT. We used mass spectrometry to identify proteins that interact with D3R following agonist activation. The role of some of the D3R interacting proteins in mediating D3R signaling and signaling properties was determined. Our studies indicate that the delay in association of the G-protein beta-gamma subunits with the alpha subunit might be a potential mechanism for the agonist-induced D3R SRT property. Our results also identified a role for the Regulator of G-protein Signaling (RGS) proteins in the D3R tolerance property. Regulation of RGS proteins via PI-3 Kinase or Calcium/Calmodulin Kinase-2 might be one of the regulatory mechanisms of the D3R signaling.
Results from this thesis work provide novel information about residues in the D3 receptor that are important for its activation and signaling properties. These results will help improve D3 receptor pharmacophore models which will facilitate the identification of novel D3 receptor agonists. Also, for the first time our results provide valuable insights into the molecular mechanisms underlying the D3 receptor tolerance and slow response termination signaling properties.


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