Signal transduction studies of G-protein coupled receptors using plasmon waveguide resonance spectroscopy

Persistent Link:
http://hdl.handle.net/10150/280539
Title:
Signal transduction studies of G-protein coupled receptors using plasmon waveguide resonance spectroscopy
Author:
Alves, Isabel M. D.
Issue Date:
2004
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
G-protein coupled receptors (GPCRs) represent one of the largest families of proteins in mammals, with 1--5% of the total cell proteins belonging to this group. Together they constitute one of the principal targets of the drugs currently being used in the market, especially those acting in the central nervous system. A better understanding of the mechanisms of activation and signal transduction of these proteins seems imperative. Despite their crucial interest, however, very little is known about their structure/function relationships, mainly as a consequence of their rather low natural abundance and their integral membrane nature. Due to the low stability of membrane proteins when outside of the cell membrane, methodologies that allow direct studies to be done while they are incorporated into artificial lipid bilayers are advantageous. One such technique is plasmon-waveguide resonance (PWR) spectroscopy, which was developed in our laboratories and has been successfully used to investigate several aspects of signal transduction. Because of its ability to obtain resonances with light polarized both perpendicular ( p-polarization) and parallel (s-polarization) to the resonator surface, PWR can follow changes in conformation accompanying protein-ligand, protein-protein and protein-lipid interactions occurring in GPCRs in real time with high sensitivity and without the need for molecular labeling. In the present work we have used two GPCRs, namely rhodopsin (a GPCR prototype) and the human delta opioid receptor (hDOR), which our laboratory has been actively investigating from the drug design point of view for 25 years. For these studies we have incorporated the detergent-solubilized receptor into a solid-supported lipid bilayer and monitored PWR changes upon activation by ligand (hDOR) or light (rhodopsin), as well as the interaction of these receptors with their G-proteins, one of the first steps in the signaling cascade. We have investigated the lipid modulation of rhodopsin activation and have found that lipids can greatly affect not only the activation process, but the G-protein interaction as well. In addition, we have found that the hDOR adopts different conformations upon binding of different classes of ligands, and that its affinity for G-proteins is highly dependent on the nature of the ligand pre-bound to the receptor. Significant selectivity towards the different G-protein subtypes and subunits was also observed. The results have yielded new insights into GPCR function and have demonstrated that PWR provides a new and direct approach to investigate transmembrane signaling.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Chemistry, Biochemistry.; Chemistry, Pharmaceutical.; Biophysics, General.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Biochemistry and Molecular Biophysics
Degree Grantor:
University of Arizona
Advisor:
Hruby, Victor J.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleSignal transduction studies of G-protein coupled receptors using plasmon waveguide resonance spectroscopyen_US
dc.creatorAlves, Isabel M. D.en_US
dc.contributor.authorAlves, Isabel M. D.en_US
dc.date.issued2004en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractG-protein coupled receptors (GPCRs) represent one of the largest families of proteins in mammals, with 1--5% of the total cell proteins belonging to this group. Together they constitute one of the principal targets of the drugs currently being used in the market, especially those acting in the central nervous system. A better understanding of the mechanisms of activation and signal transduction of these proteins seems imperative. Despite their crucial interest, however, very little is known about their structure/function relationships, mainly as a consequence of their rather low natural abundance and their integral membrane nature. Due to the low stability of membrane proteins when outside of the cell membrane, methodologies that allow direct studies to be done while they are incorporated into artificial lipid bilayers are advantageous. One such technique is plasmon-waveguide resonance (PWR) spectroscopy, which was developed in our laboratories and has been successfully used to investigate several aspects of signal transduction. Because of its ability to obtain resonances with light polarized both perpendicular ( p-polarization) and parallel (s-polarization) to the resonator surface, PWR can follow changes in conformation accompanying protein-ligand, protein-protein and protein-lipid interactions occurring in GPCRs in real time with high sensitivity and without the need for molecular labeling. In the present work we have used two GPCRs, namely rhodopsin (a GPCR prototype) and the human delta opioid receptor (hDOR), which our laboratory has been actively investigating from the drug design point of view for 25 years. For these studies we have incorporated the detergent-solubilized receptor into a solid-supported lipid bilayer and monitored PWR changes upon activation by ligand (hDOR) or light (rhodopsin), as well as the interaction of these receptors with their G-proteins, one of the first steps in the signaling cascade. We have investigated the lipid modulation of rhodopsin activation and have found that lipids can greatly affect not only the activation process, but the G-protein interaction as well. In addition, we have found that the hDOR adopts different conformations upon binding of different classes of ligands, and that its affinity for G-proteins is highly dependent on the nature of the ligand pre-bound to the receptor. Significant selectivity towards the different G-protein subtypes and subunits was also observed. The results have yielded new insights into GPCR function and have demonstrated that PWR provides a new and direct approach to investigate transmembrane signaling.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectChemistry, Biochemistry.en_US
dc.subjectChemistry, Pharmaceutical.en_US
dc.subjectBiophysics, General.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineBiochemistry and Molecular Biophysicsen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorHruby, Victor J.en_US
dc.identifier.proquest3132198en_US
dc.identifier.bibrecord.b46707529en_US
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