Modulation of A2a receptor antagonist on D2 receptor internalization and ERK phosphorylation

• Published in: Acta pharmacologica Sinica 2013-10, Vol.34 (10), p.1292-1300
• Main Authors: Huang, Li, Wu, Dong-dong, Zhang, Lei, Feng, Lin-yin
• Format: Article
• Language: English
• Subjects: Adenosine A2 Receptor Antagonists - pharmacology; Animals; Arrestins - metabolism; beta-Arrestins; Bioluminescence Resonance Energy Transfer Techniques; Biomedical and Life Sciences; Biomedicine; Cells, Cultured; Corpus Striatum - drug effects; Corpus Striatum - metabolism; ERK; Extracellular Signal-Regulated MAP Kinases - metabolism; HEK293 Cells; HEK293细胞; Humans; Immunology; Internal Medicine; Medical Microbiology; Neurons - drug effects; Neurons - metabolism; Original; original-article; Pharmacology/Toxicology; Phosphorylation - drug effects; Protein Multimerization; Quinpirole - pharmacology; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A - drug effects; Receptor, Adenosine A2A - metabolism; Receptors, Dopamine D2 - drug effects; Receptors, Dopamine D2 - metabolism; src-Family Kinases - metabolism; Triazines - pharmacology; Triazoles - pharmacology; Vaccine; Western印迹法; 内化; 受体拮抗剂; 受体激动剂; 显微镜检测; 磷酸化
• ISSN: 1671-4083; 1745-7254
• DOI: 10.1038/aps.2013.87

Aim： To explore the effects of heterodimerization of D2 receptor/A2a receptor （D2R/A2aR） on D2R internalization and D2R downstream signaling in primary cultured striatal neurons and HEK293 cells co-expressing A2aR and D2R in vitro. Methods： Primary cultured rat striatal neurons and HEK293 cells co-expressing A2aR and D2R were treated with A2aR- or D2R-specific agonists. D2R internalization was detected using a biotinylation assay and confocal microscopy. ERK, Src kinase and β-arrestin were measured using Western blotting. The interaction between A2aR and D2R was detected using bioluminescence resonance energy trans- fer （BRET） and immunoprecipitation. Results： D2R and A2aR were co-localized and formed complexes in striatal neurons, while both the receptors formed heterodimers in the HEK293 cells. In striatal neurons and the HEK293 cells, the D2R agonist quinpirole （1 pmol/L） marked increased Src phosphorylation and β-arrestin recruitment, thereby D2R internalization. Co-treatment with the A2aR antagonist ZM24β85 （100 nmol/L） significantly attenuated these D2R-mediated changes. Furthermore, both ZM24β85 （100 nmol/L） and the specific Src kinase inhibitor PP2 （5 pmol/L） blocked D2R-mediated ERK phosphorylation. Moreover, expression of the mutant β-arrestin （319-418） significantly attenu- ated D2R-mediated ERK phosphorylation in HEK293 cells expressing both D2R and A2aR, but not in those expressing D2R alone. Conclusion： A2aR antagonist ZM24β85 significantly attenuates D2R internalization and D2R-mediated ERK phosphorylation in striatal neurons, involving Src kinase and β-arrestin. Thus, A2aR/D2R heterodimerization plays important roles in D2R downstream signaling.