FK506-Binding Protein 12 Modulatesμ-Opioid Receptor Phosphorylation and Protein Kinase Cε–Dependent Signaling by Its Direct Interaction with the Receptor

Abstract

Protein kinase C (PKC) activation plays an important role in morphine-induced m-opioid receptor (OPRM1) desensitization and tolerance development. It was recently shown that receptor phosphorylation by G protein-coupled receptor kinase regulates agonist-dependent selective signaling and that inefficient phosphorylation of OPRM1 leads to PKC« activation and subsequent responses. Here, we demonstrate that such receptor phosphorylation and PKC« activation can be modulated by FK506-binding protein 12 (FKBP12). Using a yeast two-hybrid screen, FKBP12 was identified as specifically interacting with OPRM1 at the Pro 353 residue. In human embryonic kidney 293 cells expressing OPRM1, the association of FKBP12 with OPRM1 decreased the agonist-induced receptor phosphorylation at Ser 375. The morphine-induced PKC« activation and the recruitment of PKC« to the OPRM1 signaling complex were attenuated both by FKBP12 short interfering RNA (siRNA) treatment and in cells expressing OPRM1 with a P353A mutation (OPRM1P353A), which leads to diminished activation of PKCdependent extracellular signal-regulated kinases. Meanwhile, the overexpression of FKBP12 enabled etorphine to activate PKC«. Further analysis of the receptor complex demonstrated that morphine treatment enhanced the association of FKBP12 and calcineurin with the receptor. The blockade of the FKBP12 association with the receptor by the siRNA-mediated knockdown of endogenous FKBP12 or the mutation of Pro 353 to Ala resulted in a reduction in PKC« and calcineurin recruitment to the receptor signaling complex. The receptor-associated calcineurin modulates OPRM1 phosphorylation, as demonstrated by the ability of the calcineurin autoinhibitory peptide to increase the receptor phosphorylation. Thus, the association of FKBP12 with OPRM1 attenuates the phosphorylation of the receptor and triggers the recruitment and activation of PKC«.

Role of Phosphorylation Sites in Desensitization ofµ-Opioid Receptor

“…Consistent with this, a selective (calphostin C) and nonselective (staurosporine) PKC inhibitor reduced morphine-induced desensitization. By contrast, desensitization by ME was unaffected by PKC inhibition, consistent with evidence that morphine, but not DAMGO, increases PKC activity (Chu et al, 2010;Qiu et al, 2014).…”

Role of FK506 binding protein 12 in morphine-induced μ-opioid receptor internalization and desensitization

“…Whereas FK506 pre-incubation which can dissociate FKBP12 from the receptor increased receptor internalization to 30 ± 5.1% after 4 h of morphine treatment. Similarly, morphine also induced faster internalization of the OPRM1P353A mutant, a receptor mutant that we have shown not to interact with FKBP12 [15]. 42 ± 2.0% of receptor was internalized after 4 h of morphine treatment.…”

“…The transfection of HEK293 cells with PcDNA3 plasmids containing the hemagglutinin (HA) epitope tagged rat OPRM1 (HA-OPRM1) and its mutant at Pro 353 to Ala (HA-OPRM1P353A) and the establishment of stably transfected cells was carried out as described previously [15]. The expression levels of wild type OPRM1 and mutant OPRM1P353A were 1.5 pmol/mg protein and 1.1 pmol/mg protein respectively.…”

“…The interaction of synaptophysin and phospholipase D2 with OPRM1 accelerates receptor internalization [11, 12]; Membrane glycoprotein M6a interacts with OPRM1 and influences receptor endocytosis and recycling [13]; G protein-regulated inducer of neurite outgrowth GRIN1 tethers OPRM1 receptor and G protein in the lipid rafts [14]. In addition to these proteins, previous studies in our lab has demonstrated that FK506 binding protein 12 (FKBP12) specifically interacts with OPRM1 at the Pro 353 residue of OPRM1 [15]. FKBP12, a 12 kDa FK506 binding protein is a peptidyl prolyl cis-trans isomerase (PPIase) [16] belonging to the family of the immunophilins and is highly expressed in the brain [17].…”

“…In our previous study, the interaction of FKBP12 with OPRM1 is demonstrated to reduce receptor phosphorylation and facilitate the recruitment and activation of PKCε [15]. However, whether the interaction of FKBP12 with OPRM1 affects receptor internalization and desensitization is unknown.…”