Structure function studies on prostanoid receptors: Thromboxane A2 receptor (TP) and Prostacyclin receptor (IP)

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dc.contributor.authorChakraborty, Raja
dc.contributor.examiningcommitteeBhullar, Raj (Oral Biology) McNicol, Archie (Oral Biology) Dakshinamurti, Shyamala (Physiology) Klein, Judith (University of Warwick)en_US
dc.contributor.supervisorChelikani, Prashen (Oral Biology)en_US
dc.date.accessioned2014-08-08T15:52:33Z
dc.date.available2014-08-08T15:52:33Z
dc.date.issued2011en_US
dc.date.issued2012en_US
dc.date.issued2012en_US
dc.date.issued2013en_US
dc.date.issued2014en_US
dc.degree.disciplineOral Biologyen_US
dc.degree.levelDoctor of Philosophy (Ph.D.)en_US
dc.description.abstractCell membrane receptors help to mediate communication between the cell and its environment. The largest group of these membrane receptors belong to the family of G protein-coupled receptors (GPCRs). GPCRs contain seven transmembrane (TM) helices and signal predominantly through heterotrimeric G proteins in response to diverse extracellular stimuli. Previously, three levels of amino acid conservation were proposed to understand the structure and function of a GPCR. This includes “signature” amino acids, “group –conserved” amino acids and amino acids conserved only within a specific subfamily. The group-conserved residues in class A GPCR family involve amino acid conservation of up to 99% when considered as a group of small and weakly polar residues (Ala, Gly, Ser, Cys and Thr). These group-conserved residues have been proposed as key determinants in helix-helix interactions. Therefore, I selected these residues for structure-function analysis in the amine and the prostanoid receptor sub-families of class A GPCRs. Molecular and biochemical assays clearly demonstrate the importance of group-conserved residues in β2-adrenergic receptor and thromboxane A2 receptor (TP) structure and function. These studies led to the identification of a non-synonymous single nucleotide polymorphic variant (nsSNP) A160T in TP to be a constitutively active mutant (CAM). Further, the TP-CAM was used as a pharmacological tool that enabled classification of well-known TP-blockers, into neutral antagonists and inverse agonists. The role of TP-A160T in prostanoid receptors, TP- Prostacyclin receptor (IP) heterodimerization and signaling was investigated. Activation of a GPCR ultimately leads to structural changes in its intracellular loops (ICLs), which in turn activates G-protein. TP activates its cognate G protein (Gαq), while IP mediates signaling, through Gαs. Using TP-IP chimeric receptors, molecular modelling, and site directed mutagenesis studies I determined the specific ICL regions required for G protein coupling in TP and IP. Significant challenges exist in expressing and purifying GPCR-CAMs in amounts required to pursue biophysical studies. Using tetracycline inducible HEK293S system, A160T was expressed at high-levels and CD spectropolarimetry studies were successfully pursued on the purified A160T. The CD spectra showed that the loss of thermal stability of the A160T mutant is due to the subtle changes in the secondary structure of the A160T protein. These studies involving molecular, biochemical and pharmacological approaches provide novel insights into the structure and function of prostanoid receptors TP and IP.en_US
dc.description.noteOctober 2014en_US
dc.identifier.citationPalmitoylation of Gαq Determines Its Association with the Thromboxane Receptor in Hypoxic Pulmonary Hypertension, American Journal of Respiratory Cell and Molecular Biology, Vol. 50, No. 1 (2014), pp. 135-143.en_US
dc.identifier.citationMolecular basis of agonist docking in a human GPR103 homology model by site-directed mutagenesis and structure-activity relationship studies, BJP 2014en_US
dc.identifier.citationThe third intracellular loop plays a critical role in bitter taste receptor activationBiochimica et Biophysica Acta 1838 (2014) 231–236en_US
dc.identifier.urihttp://hdl.handle.net/1993/23744
dc.language.isoengen_US
dc.publisherElsevier Ltden_US
dc.publisherPloS Oneen_US
dc.publisherAmerican Society for Microbiologyen_US
dc.publisherPloS Oneen_US
dc.publisherPloS Oneen_US
dc.rightsopen accessen_US
dc.subjectThromboxane A2 receptoren_US
dc.subjectProstacyclin receptoren_US
dc.subjectConstitutively active mutantsen_US
dc.subjectSingle nucleotide polymorphismen_US
dc.subjectInositol-1, 4, 5-trisphosphateen_US
dc.subjectCyclic AMPen_US
dc.titleStructure function studies on prostanoid receptors: Thromboxane A2 receptor (TP) and Prostacyclin receptor (IP)en_US
dc.typedoctoral thesisen_US
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