Regulation of G-protein subtypes by receptors, guanine nucleotides and Mn2+

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2014-05-29

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Käesoleva töö eesmärgiks oli uurida signaaliülekande mehhanisme, mida vahendavad G-valk seotud retseptorid: rakumembraanides paiknevad valgud, mille kaasabil aktiveerivad raku väljaspoolt tulevad signaalid raku sisemuses olevaid guaniinnukleotiide siduvaid valke e. G-valke. G-valk seotud retseptorid osalevad väga paljudes füsioloogilistes protsessides (olles seega olulisteks ravimimärklaudadeks): alustades embrüonaalsete rakkude diferentseerumisest kuni kesknärvisüsteemi täppisregulatsioonini välja. G-valkude ülesanne on kanda edasi retseptorite poolt vastuvõetud signaal rakusisestele efektorsüsteemidele. Antud töös keskenduti erinevate G-valkude alatüüpide vaheliste erinevuste määramisele, eriti nende nukleotiidide sidumisomadustele ja sellele, kuidas neid mõjutavad G-valk seotud retseptorid (nagu serotoniin 1A retseptor) ja mangaani ioonid. Selle eesmärgi täitmiseks arendati välja mitmeid uudseid meetodeid, mis hõlmas G-valkude puhastamist ja nende aktiivsuse määramist nii puhtal kujul lahuses kui kompleksis retseptoritega bakuloviirusosakestes.
The aim of this thesis was to investigate signal transduction mechanisms that are mediated by G-protein coupled receptors. These receptors are located in the cell membrane, where they receive extracellular signals and relay them to intracellular guanine nucleotide binding proteins (G-proteins). G-protein coupled receptors play a major role in regulating numerous physiological processes – from embryonal cell differentation to fine-tuning the activity of the central nervous system – and are therefore important drug targets. G-proteins are responsible for relaying the signals that receptors detect to various intracellular effector systems. Our objective was to compare how various G-protein subtypes differ in their properties, especially in their ability to bind nucleotides and also how their activity is modulated by receptors (like the serotonin 1A receptor) and manganese ions. In order to meet these objectives, several novel methods were developed for both purifying G-proteins and for determining their activity in either their purified form or in complex with receptors in baculovirus particles.

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