Reciprocal interactions between adenosine A2A and dopamine D2 receptors in CHO cells co-transfected with the two receptors

Department of Physiology and Neuroscience, Division of Molecular Neurobiology, Wallenberg Neuroscience Centre, University of Lund, S-223 62 Lund, Sweden
Biochemical Pharmacology (Impact Factor: 5.01). 10/1999; 58(6):1035-1045. DOI: 10.1016/S0006-2952(99)00184-7


Human adenosine A2A and rat dopamine D2 receptors (A2A and D2 receptors) were co-transfected in Chinese hamster ovary (CHO) cells to study the interactions between two receptors that are co-localized in striatopallidal γ-aminobutyric acid-(GABA)ergic neurons. Membranes from transfected cells showed a high density of D2 (3.6 pmol per mg protein) and A2A receptors (0.56 pmol per mg protein). The D2 receptors were functional: an agonist, quinpirole, could stimulate GTPγS binding and reduce stimulated adenylyl cyclase activity. The A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680) decreased high-affinity binding of the agonist dopamine at D2 receptors. Activation of adenosine A2A receptors shifted the dose–response curve for quinpirole on adenosine 3′,5′-cyclic monophosphate (cAMP) to the right. However, CGS 21680 did not affect dopamine D2 receptor-induced GTPγS binding, but did cause a concentration-dependent increase in cAMP accumulation. The maximal cAMP response was decreased by the D2 agonist quinpirole in a concentration-dependent manner, but there was no change in ec50 and no effect in cells transfected only with adenosine A2A receptors. A2A receptor activation also increased phosphorylation of cAMP response element-binding protein and expression of c-fos mRNA. These effects were also strongly counteracted by quinpirole. These results show that the antagonistic actions between adenosine A2A and dopamine D2 receptors noted previously in vivo can also be observed in CHO cells where the two receptors are co-transfected. Thus, no brain cell-specific factors are required for such interactions. Furthermore, the interaction at the second messenger level and beyond may be quantitatively more important than A2A receptor-mediated inhibition of high affinity D2 agonist binding to the receptor.

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    • "cAMP ? PKA cascade] (Missale et al., 1998; Arslan et al., 1999; Kull et al., 1999) and prevents phosphorylation of the D2R/D4R epitope (Fig. 2). Thus when Dopamine is the ligand at D2R/D4R, the electrostatic interaction is unaffected, and the heteromer formed is stable, while when Adenosine is the ligand at A2AR activation of the [AC ? "
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    ABSTRACT: Our work suggests that heteromer formation, mainly involves linear motifs (LMs) found in disordered regions of proteins. Local disorder imparts plasticity to LMs. Many molecular recognition of proteins occurs between short linear segments, known as LMs. Interaction of short continuous epitopes is not constrained by sequence and has the advantage of resulting in interactions with micromolar affinities which suit transient, reversible complexes such as receptor heteromers. Electrostatic interactions between epitopes of the G protein-coupled receptors (GPCR) involved, are the key step in driving heteromer formation forward. The first step in heteromerization, involves phosphorylating Ser/Thr in an epitope containing a casein kinase 1/2-consensus site. Our data suggest that dopaminergic neurotransmission, through cAMP-dependent protein kinase A (PKA) slows down heteromerization. The negative charge, acquired by the phosphorylation of a Ser/Thr in a PKA consensus site in the Arg-rich epitope, affects the activity of the receptors involved in heteromerization by causing allosteric conformational changes, due to the repulsive effect generated by the negatively charged phosphate. In addition to modulating heteromerization, it affects the stability of the heteromers' interactions and their binding affinity. So here we have an instance where phosphorylation is not just an on/off switch, instead by weakening the noncovalent bond, heteromerization acts like a rheostat that controls the stability of the heteromer through activation or inhibition of adenylate cyclase by the neurotransmitter Dopamine depending on which Dopamine receptor it docks at.
    Neuroscience 02/2013; 238. DOI:10.1016/j.neuroscience.2013.02.006 · 3.36 Impact Factor
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    • "The reaction was stopped by addition of perchloric acid to a final concentration of 0.4 M and incubation on ice. After lysis and neutralization with KOH in 50 mM Tris, the supernatant was examined for cAMP content as described elsewhere [26]. "
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    Journal of Neuroinflammation 05/2012; 9(1):111. DOI:10.1186/1742-2094-9-111 · 5.41 Impact Factor
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    • "In a second type of A 2A R-D 2 R receptor interaction, D 2 R stimulation prevents A 2A R to signal through the activation of adenylyl cyclase (AC) pathway (Chen et al., 2001; Hakansson et al., 2006; Hillion et al., 2002; Kull et al., 1999). Under normal conditions, the ability of A 2A Rs to activate the AC-PKA cascade is restrained by a tonic inhibitory effect of endogenous dopamine on striatal D 2 R, which efficiently inhibits A 2A R-mediated AC activation (Svenningsson et al., 1999; Karcz-Kubicha et al., 2003a). "
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    Neuropharmacology 10/2011; 61(5-6):967-74. DOI:10.1016/j.neuropharm.2011.06.025 · 5.11 Impact Factor
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