The Agonist of Adenosine A1 Receptor Induced Desensitization of delta Opioid receptor-mediated Raf-1/MEK/ERK Signaling by Feedback Phosphorylation of Raf-1-Ser289/296/301

Our previous study found that activation of adenosine A1 receptor (A 1 R) induced phosphorylation of delta opioid receptor (DOR) and desensitization of its downstream signaling molecules, cAMP and Akt. To further investigate the effect of A 1 R agonist on DOR signaling and the underlying mechanism, we examined the effect of A 1 R activation upon binding of its agonist N6-cyclohexyl-adenosine (CHA) on DOR-mediated Raf-1/MEK/ERK activation, and found that prolonged CHA exposure resulted in downregulation of DOR-mediated Raf-1/MEK/ERK signaling pathway. CHA-treatment time dependently attenuated Raf-1-Ser338 phosphorylation induced by [D-Pen2,5] enkephalin (DPDPE), a specific agonist of DOR, and further caused downregulation of the Raf-1/MEK/ERK signaling pathway activated by DOR agonist. Moreover, CHA exposure time-dependently induced the phosphorylation of Raf-1-Ser289/296/301, the inhibitory phosphorylation sites that were regulated by negative feedback, thereby inhibiting activation of the MEK/ERK pathway, and this effect could be blocked by MEK inhibitor U0126. Finally, we proved that the heterologous desensitization of the Raf-1/MEK/ERK cascade was essential in the regulation of anti-nociceptive effect of DOR agonists by confirming that such effect was inhibited by pretreatment of CHA. Therefore, we conclude that the activation of A 1 R inhibits DOR-mediated MAPK signaling pathway via heterologous desensitization of the Raf-1/MEK/ERK cascade, which is a result of ERK-mediated Raf-1-Ser289/296/301 phosphorylation mediated by activation of A 1 R.