A2A/D2 Receptors and G-Protein Interactions

Identification Using Confocal Microscopy in Live Cells

  • Fig. 1: Representative images of N2a cells co-transfected with Adenosin receptor (A2A) labelled with EGFP (A and D) and mCherry (B and E) fluorescence proteins. A2A/EGFP and A2A/mCherry FRET image show the homodimerization of A2A receptors (C and F)Fig. 1: Representative images of N2a cells co-transfected with Adenosin receptor (A2A) labelled with EGFP (A and D) and mCherry (B and E) fluorescence proteins. A2A/EGFP and A2A/mCherry FRET image show the homodimerization of A2A receptors (C and F)
  • Fig. 1: Representative images of N2a cells co-transfected with Adenosin receptor (A2A) labelled with EGFP (A and D) and mCherry (B and E) fluorescence proteins. A2A/EGFP and A2A/mCherry FRET image show the homodimerization of A2A receptors (C and F)
  • Fig. 2: Representative images of N2a cells transfected with split EGFP labelled A2A receptors. When A2A receptors homodimerize, C and N EGFP come together and give a signal in EGFP channel. The effect of binning was tested; binning 2x2 (A), no binning A2A-EGFP
  • Fig. 3: Representative images of N2a cells co-transfected with EGFP labelled G13 protein and A2A receptor lebelled with mCherry chromophores. The images were taken from mCherry channel (A), EPFG channel (B), FRET channel (C), EPFG and mCherry channel (D), mCherry and FRET channel (E)
  • Fig. 4: N2a cells co-transfected with G13/EFGP and A2A/ mCherry. Images were taken from EGFP channel (A), mCherry channel (B) FRET channel (C), and both EGFP and mCherry channel (D and E)

According to the Gα subunit amino acid sequence homology, G proteins divide into four families that can couple to different GPCRs. To investigate the combination of interaction between G proteins (Gs, Gi/Go, q11 and 12/13) and A2A, D2R, or A2A/D2 respectively, Föster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays, with the advantage to analyze protein-protein interaction in live cells using confocal microscopy, has been performed.

Huge Protein Family: GPCR Receptors

G-Protein coupled receptors (GPCRs), composed of seven-transmembrane (7-TM) domain receptors, are large and divergent group of membrane proteins with the estimated total number of 800 in eukaryotes [1]. GPCRs interact with G proteins in the plasma membrane, Gα subunit binds to guanosine diphosphate (GDP) in an inactive form. When a ligand binds to GPCR, however, the receptor activates the bound G-protein α subunit by replacing GTP to GDP. Consequently, Gα divides Gβγ subunit and transmits the signals by interacting with other membrane proteins included signal transduction and secondary messengers enzymes such as cAMP, inositol 1, 4, 5-triphosphate (IP3) and  diacylglycerol (DAG) by inhibiting or activating [6].

Adenosine and Dopamine Receptors

Adenosine 2A (A2A) and Dopamine 2 (D2) receptors are G-protein coupled receptors (GPCRs) known to oligomerize in neuron cells. Like other (GPCRs), transfer extracellular signals through the plasma membrane via activation of heterotrimeric G proteins [9].

These receptors are found heavily in the striatopallidal γ-aminobutyric acid (GABA) containing neurons. The neurons are implicated in basal ganglia functions and that is why both receptors are involved in schizophrenia, Parkinson disease and drug abuse illnesses. Thus, A2A and D2 receptors are main drug targets in these psychiatric disorders. The GPCR oligomerization event is important for the receptor biosynthesis, maturation, trafficking, plasma membrane diffusion, signaling and drug discovery [4]. Dimerization between adenosine receptors at the cell surface has been demonstrated by different methods.

Also it has been shown that Adenosine receptors homo-dimers are the functional form present on the plasma membrane [2]. Likely, different types of Dopamine receptors such as D1, D2, and D3 also form functional homodimers in transfected cell lines [4]. Additionally, when adenosine receptors oligomerize, they can improve the molecular mechanism by signal transferred G proteins in the string of receptor transduction [3].

Following Different Pathways

Upon agonist induced activation of these receptors, which induces the stimulation or inhibition of adenylyl cyclase by Gαs and Gαi proteins, respectively, result in a control of cellular cAMP levels. When A2A and D2R receptors heterodimerize, they initiate the activation of the Gq11 pathway, that generates PIP2 and DAG secondary messengers [7].  Moreover, it has been investigated that A1A and A3A adenosine receptors interact with the Gi and Go family, while A2A and A2B adenosine receptors activate adenylyl cyclase via Gs [8]. On the other hand, in human microvascular cells, A2B receptors stimulate the expression of angiogenic factors via coupling to Gq and via G12/13 [5]. Another signaling pathway of GPCRs is through Gα12/13, which activates the Rho family that helps to control cytoskeletal proteins and cellular motility. This signal pathway is a key to cell migration and hypertension. However, it is not clear if they interact with A2A, D2R and their homo/heterodimers.

Experimental Procedure

A2A and D2R alone carried EGFP and EGFP splits that will form the complete EGFP during heteromer formation and Gα protein (G12/13) carried mCherry chromospheres. That is why commercial Gα protein genes plasmids were tagged with mCherry chromophore from different in frame positions by the insertional PCR method. A2A, D2R receptor genes were also tagged by EGFP fluorophore from C terminus of  receptor simultaneously. To express the tagged Gα protein genes in the N2a mammalian cell line, they were cloned to pcDNA 3.1 (-) plasmid by the PCR method. After the size of the gene has been controlled by gel electrophoresis, in order to express and visualize the tagged receptors, live N2a cells have been transfected with expression vectors, containing receptor and Gα genes, fused to fluorescent protein genes by the use of Lipofectamine TM LTX with PlusTM reagents. Subsequently, the cells were transfected with the desired plasmids and grown for 2 days. The expressed receptors were visualized using a Leica DMI 4000, equipped with Andor DSD2 spinning disk confocal microscope with a 63X oil N1.4 objective.

Result

To investigate the combination of doubled or tripled interaction between G proteins (G12/13) and A2A, D2, A2A/A2A, D2R/D2R or A2A/D2, respectively, Föster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays, with the advantage to analyze protein-protein interaction in live cells using confocal microscopy, has been performed. Firstly A2A receptor homodimerization has been observed. EGFP and mCherry tagged A2A receptors co-transfected to neuroblastoma N2a cell line (fig. 1). Also homodimerization is observed with split EGFP (N and C-EGFP) tagged A2A receptors by using the BIFC method (fig. 2) and dimerization is occurring mostly on the cell membrane.

Additionally, to demonstrate the interaction of G13 with A2A receptor, images have been taken for FRET analysis (fig. 3 and 4), but to show certain interactions, additional PixFRET analysis experiments will be performed. For future experiments, images will be taken for G12/A2A receptor interaction at first, then with D2R, A2A/A2A, A2A/D2R, respectively. 

References
[1] Mei Bai: Dimerization of G-protein-coupled receptors: roles in signal transduction, Cellular Signalling 16, 175–186 (2004) 
[2] Meritxell Canals, Javier Burgueño, Daniel Marcellino, Núria Cabello, Enric I. Canela, Josefa Mallol, Luigi Agnati, Sergi Ferré, Michel Bouvier, Kjell Fuxe, Francisco Ciruela, Carmen Lluis, Rafael Franco: Homodimerization of adenosine A2A receptors: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer, J. Neurochem. 88, 726–734 (2004)
[3] Francisco Ciruela, Maricel Gómez-Soler, Diego Guidolin, Dasiel O. Borroto-Escuela, Luigi F. Agnati, Kjell Fuxec, Víctor Fernández-Dueñas: Adenosine receptor containing oligomers: Their role in the control of dopamine and glutamate neurotransmission in the brain, Biochimica et Biophysica Acta 1808 1245–1255 (2011)
[4] Marta Dziedzicka-Wasylewska, Agata Faron-Górecka, Joanna Andrecka, Agnieszka Polit, Maciej Kuśmider, and Zygmunt Wasylewski: Fluorescence Studies Reveal Heterodimerization of Dopamine D1 and D2 Receptors in the Plasma Membrane, Biochemistry 45, 8751–8759. (2006)
[5] Igor Feoktistov, Anna E. Goldstein, Sergey Ryzhov, Dewan Zeng, Luiz Belardinelli, Tatyana Voyno-Yasenetskaya, and Italo Biaggioni: Differential Expression of Adenosine Receptors in Human Endothelial Cells: Role of A2B Receptors in Angiogenic Factor Regulation, Circ. Res. 90, 531-538 (2002)
[6] Brian K. Kobilka: G Protein Coupled Receptor Structure and Activation, Biochimica et Biophysica Acta, 1768, 794–807 (2006) 
[7] Natalia A. Riobo, David R. Manning: Receptors coupled to heterotrimeric G proteins of the G12 family, Pharmacol. Sci. 26, 146–154 (2005)
[8] Veronika Sexl, Gudrun Mancusi, Christoph Höller, Eva Gloria-Maercker, Wolfgang Schütz and Michael Freissmuth: Stimulation of the Mitogen-activated Protein Kinase via the A2A-Adenosine Receptor in Primary Human Endothelial Cells, J. Biol. Chem. 272, 5792-5799 (1997)
[9] Sandra Siehler: G12/13-dependent signaling of G-protein-coupled receptors: disease context and impact on drug discovery, Expert Opin. Drug Discov 2 (12) 1591-1604 (2007)

Author
Irmak Begum Sahin Kostromin
(email request)
Çağdaş Devrim Son

Biology Department
Middle East Technical University
Çankaya, Ankara, Turkey
bio.metu.edu.tr

Contact

Middle East Technical University
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