Visualization of interactions between fluorescently tagged g protein α11, α12/13 subtypes and adenosine 2a, dopamine 2 or homodimer adenosine 2a/2a receptors

Download

index.pdf

Date

2018

Author

Kostromin, İrmak Begüm

Metadata

Show full item record

Item Usage Stats

285
views

104
downloads

G-Protein-coupled receptors (GPCRs) belong to one of the largest family of cell surface receptors, which transmit extracellular signals to intracellular responses by interacting with G- proteins. The G proteins are known as molecular switches that regulates different pathways via control of secondary messengers and signaling proteins. Adenosine 2A (A2A) and Dopamine 2 (D2) receptors belong to G-Protein-coupled receptors (GPCRs) family and are located mostly in striatopallidal γ-aminobutyric acid (GABA) containing neurons. Therefore, problems related to these receptors are associated with physiological disorders such as schizophrenia, and Parkinson disease. A2A receptors regulate neurotransmission, cardiovascular system and immune response; On the other hand, D2 receptors modulate the regulation of locomotion, food intake, learning, emotion and behavior. Studies showed that, A2A and D2 receptors change the cAMP level by stimulating or inhibiting adenylyl cyclase via interacting with Gαs and Gαi proteins. When D1 and D2 receptors heterodimerize, Gαq11 signal pathway was preferred and activate PIP2 and DAG secondary messengers. However, interaction between Gαq11 protein and A2A, D2R or A2A/A2A homodimer is not reported. GPCRs could also signal through Gα12/13 which regulates actin cytoskeleton and cell migration through activation of Rho kinases. It is established that, Gα12/13 mutant cells show neuronal over migration and dysfunction at cerebral cortex. In addition, cell migration is effected positively by A2A receptor activation while it could be negatively regulated by D2R receptor activation. However, it is not clear if Gα12/13 proteins are interacting with A2A and D2 receptors triggering related pathways. The aim of this study is to analyze possible physical interaction between Gαq11-A2A, Gαq11-D2R, Gαq11-A2A/A2A; Gα12/13-A2A, Gα12/13 -D2R and Gα12/13 -A2A/A2A using Fluorescence Resonance Energy Transfer (FRET) method. For this purpose, G-protein α subunits (Gα11,G α12 and Gα13) were labeled with mCherry or EGFP fluorescent proteins from five different internal locations; receptors were labelled with split-EGFP fragments, and full length EGFP fluorophore, and then transfected to Mus musculus Neuroblastoma-2a (N2a) cells as various combinations. To analyze the possible interactions, images were taken via spinning disc confocal microscopy and FRET efficiency was calculated by using pixFRET Plugin for ImageJ software. Images obtained from Gα11+mCherry and D2R+EGFP transfected N2a cells; Gα13+mCherry and A2A+EGFP have high FRET signal mostly located on cell membrane. Gα13-D2R, Gα11A2A and Gα11-A2A/A2A pairs had lower signal than Gα11-D2R and Gα13-A2A pairs. Further experiments are necessary to prove an interaction between these pairs if it ever exists. On the othe hand Gα12-A2A and Gα13-A2A/A2A pairs had very low signal that was impossible to difrentiate from background signal indicating a lack of interaction. Protein interaction studies like the ones detailed in this study could help us understand signal transduction pathways much better, thus will be useful to design new treatment models and discover drug candidates in future.

Subject Keywords

Dopamine, G proteins., Adenosine.

URI

http://etd.lib.metu.edu.tr/upload/12621928/index.pdf
https://hdl.handle.net/11511/27145

Collections

Graduate School of Natural and Applied Sciences, Thesis

Suggestions

OpenMETU
Core

Optimization of internal tagging of inhibitory G-proteins for investigating their interactions with dopamine receptor D2 via fret method Özcan, Gizem; Son, Çağdaş Devrim; Özçubukçu, Salih; Department of Biochemistry (2016) G-Protein Coupled Receptors (GPCRs) constitute a large family of receptors which act by sensing the molecules outside the cell and start a signal transduction inside the cell through interacting with their associated G-proteins. This interaction results in activation or repression of related signaling pathways via associated secondary messengers. Dopamine receptor D2 (D2R) is a member of D2-like Dopamine Receptor group, which also belongs to the GPCR family. It is known that D2R has critical roles in emotio...
Resonctructing signaling pathways from RNAi data using genetic algorithms Ayaz, Eyüp Serdar; Can, Tolga; Department of Bioinformatics (2011) Cell signaling is a list of chemical reactions that are used for intercellular and intracellular communication. Signaling pathways denote these chemical reactions in a systematic manner. Today, many signaling pathways are constructed by several experimental methods. However there are still many communication skills of cells that are needed to be discovered. RNAi system allows us to see the phenotypes when some genes are removed from living cells. By observing these phenotypes, we can build signaling pathway...
Interactions between G-protein Coupled Receptors and Ligand Gated Ion Channels (GPCR-LGIC COUPLING) Son, Çağdaş Devrim(2014-9-30) Dopamine receptors are members of G-protein coupled receptor superfamily. These receptors are the key point of dopaminergic system, which controls the regulation of memory, attention, food intake, endocrine regulation, psychomotor activity and positive reinforcement. To regulate so many critically important neurological events, dopamine receptors have complex interactions with other receptors and ion channels. In this study, a trimeric complex comprising D2 receptor -which is a subtype of dopamine receptors...
Bayesian inference for the MAPK/ERK pathway by considering the dependency of the kinetic parameters Purutçuoğlu Gazi, Vilda (Institute of Mathematical Statistics, 2008-01-01) The MAPK/ERK pathway is one of the major signal transduction systems which regulates the cellular growth control of all eukaryotes like the cell proliferation and the apoptosis. Because of its importance in cellular lifecycle, it has been studied intensively, resulting in a number of qualitative descriptions of this regulatory mechanism. In this study we describe the MAPK/ERK pathway as an explicit set of reactions by combining different sources. Our reaction set takes into account the localization and diff...
Automatic detection of mitochondria from electron microscope tomography images: a curve fitting approach Tasel, Serdar F.; HASSANPOUR, REZA; Mumcuoğlu, Ünal Erkan; Perkins, Guy; Martone, Maryann (2014-02-18) Mitochondria are sub-cellular components which are mainly responsible for synthesis of adenosine tri-phosphate (ATP) and involved in the regulation of several cellular activities such as apoptosis. The relation between some common diseases of aging and morphological structure of mitochondria is gaining strength by an increasing number of studies. Electron microscope tomography (EMT) provides high-resolution images of the 3D structure and internal arrangement of mitochondria. Studies that aim to reveal the c...

Citation Formats

İ. B. Kostromin, “Visualization of interactions between fluorescently tagged g protein α11, α12/13 subtypes and adenosine 2a, dopamine 2 or homodimer adenosine 2a/2a receptors,” M.S. - Master of Science, Middle East Technical University, 2018.