During the past three decades, the adenosine receptors have been found to interact with opioid receptors, both directly [1–3] or indirectly [4], in clinical therapeutics such as analgesia [2] and cardio protection [3]. Our previous study found that the agonist of adenosine A1 receptor (A1R), N6-cyclohexyl-adenosine (CHA), induced phosphorylation of delta opioid receptor (DOR), along with heterologous desensitization of DOR-mediated inhibition of intracellular cAMP and phosphorylation of Akt in CHO cells stably expressing A1R and DOR [5], thus revealing a mechanism underlying DOR desensitization induced by A1R-DOR interaction. However, the signaling pathway supporting such effect remains unclear. To further study the effect of A1R agonist on DOR signaling and its possible mechanism, we focused on the role of mitogen-activated protein kinase (MAPK) pathways mediated by DOR in our present study.
Both A1R and DOR belong to the G protein-coupled receptor (GPCR) superfamily, the activation of which may give rise to transient activation of mitogen-activated protein kinase (MAPK) pathways, and especially the extracellular signal-regulated kinase (ERK) signaling pathway [6–8], which consists of a three-tier hierarchical signaling cascade of protein kinases: v-raf-1 murine leukemia viral oncogene homolog 1 (Raf), mitogen-activated protein kinase or ERK kinase (MEK) and ERK [9, 10]. As homologs of the v-Raf oncogene, three isoforms of the Raf kinase have been discovered in vertebrates: A-Raf, B-Raf and C-Raf (Raf-1), the most intensively studied of all isoforms [10]. All three isoforms are activated by binding of their upstream regulators, the small G proteins of the rat sarcoma (Ras) family, to their N-terminal regions, and share three common conservative regions (CRs), namely CR1, CR2 and CR3 [10]. CR1 contains the RAS-binding domain (RBD) and the cysteine-rich domain (CRD) that are required for membrane recruitment, whereas the hinge region (CR2) and catalytic region (CR3) contain numerous phosphorylation sites for kinase activity regulation that are targeted by a variety of other kinases and proteins, such as protein kinase A and 14-3-3, through direct binding [10, 11].
As a serine/threonine protein kinase with multiple phosphorylation sites, Raf-1 is regulated via a series of phosphorylation status that contribute to either activation or inhibition of itself, which are induced by a number of kinases and proteins, including upstream and downstream kinases of the MAPK cascade itself, or even other regulating proteins. For example, the Ser338 residue is a more important phosphorylation site for activation of the MAPK cascade, since it is indispensable in introducing negative charges into the 4-amino-acid motif (SSYY), which is essential for mediating Ras-dependent Raf-1 activation. Therefore, the phosphorylation of Raf-1 Ser338 is considered as an index of MAPK activation [12]. Other phosphorylation sites include Ser43, Ser233 and Ser259, which are targets of protein kinases that are responsible for Raf-1 inhibition, and Ser233, Ser259 and Ser621 that may result from binding of 14-3-3, thereby activating Raf-1 [10, 13, 14]. On the other hand, Ser289, Ser296 and Ser301, the three adjacent phosphorylation sites are targets of ERK, the downstream kinase of the MAPK cascade, in the key regulatory step on negative feedback. Phosphorylation of these sites contribute to negative regulation of Raf-1, while phosphorylation of Ser338 may block ERK-induced inhibitory effect [15]. U0126, the specific inhibitor of MEK1/2, was able to inhibit phosphorylation of Ser289/296/301 in COS-7 and NIH3T3 cells after prolonged exposure to platelet derived growth factor (PDGF) and epidermal growth factor (EGF), whereas activation or overexpression of ERK resulted in enhanced Ser289/296/301 phosphorylation of Raf-1 [16, 17].
The negative feedback mechanism induced by Ser289/296/301 phosphorylation has revealed an essential manner of Raf-1 regulation, and thereby the modulation of the entire Raf-1/MEK/ERK cascade upon GPCR activation. However, is remains elusive whether CHA-induced heterologous desensitization of DOR-mediated ERK signaling pathway is associated with the triple phosphorylation sites of Raf-1, although we have previously proved that activation of A1R may induce heterologous desensitization by facilitating DOR phosphorylation, while inhibiting cAMP accumulation and Akt phosphorylation [5]. Thus, in our present study, we focus on the mechanism by which prolonged CHA treatment induce heterologous desensitization of DOR-induced MAPK cascade, along with the role of Raf-1-Ser289/296/301 phosphorylation in such regulation.