Abstract
A new role of G protein-coupled receptor (GPCR) phosphorylation was demonstrated in the current studies by using the μ-opioid receptor (OPRM1) as a model. Morphine induces a low level of receptor phosphorylation and uses the PKCε pathway to induce ERK phosphorylation and receptor desensitization, whereas etorphine, fentanyl, and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) induce extensive receptor phosphorylation and use the β-arrestin2 pathway. Blocking OPRM1 phosphorylation (by mutating Ser363, Thr370 and Ser375 to Ala) enabled etorphine, fentanyl, and DAMGO to use the PKCε pathway. This was not due to the decreased recruitment of β-arrestin2 to the receptor signaling complex, because these agonists were unable to use the PKCε pathway when β-arrestin2 was absent. In addition, overexpressing G protein-coupled receptor kinase 2 (GRK2) decreased the ability of morphine to activate PKCε, whereas overexpressing dominant-negative GRK2 enabled etorphine, fentanyl, and DAMGO to activate PKCε. Furthermore, by overexpressing wild-type OPRM1 and a phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that receptor phosphorylation contributes to the differential effects of agonists on dendritic spine stability. Phosphorylation blockage made etorphine, fentanyl, and DAMGO function as morphine in the primary cultures. Therefore, agonist-dependent phosphorylation of GPCR regulates the activation of the PKC pathway and the subsequent responses.
Highlights
Agonist-dependent or agonist-biased signaling is a new concept in understanding the signaling of the G protein-coupled receptor (GPCR)4 [1]
By overexpressing wild-type OPRM1 and a phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that receptor phosphorylation contributes to the differential effects of agonists on dendritic spine stability
The binding of an agonist to a GPCR leads to receptor phosphorylation, which subsequently increases the affinity of the agonist-receptor complex for the cytosolic protein -arrestin
Summary
Cell Culture and Materials—HEKOPRM1, HEK3A, and mouse embryonic fibroblast cells (MEF, wild type, and -arrestin2Ϫ/Ϫ from Dr Lefkowitz, Duke University) were cultured in MEM supplied with 10% FBS and 200 ng/ml G418. PKC subtype antibodies were from Santa Cruz Biotechnology, Inc. Intracellular Calcium Measurement—Intracellular calcium was measured using the FLIPR calcium assay kit (Molecular Devices Corp.) as described previously [8]. PKC Subtype Activity Assay—Activity of the PKC subtypes was determined by using the PKC activity assay kit from Cell Signaling as described previously [12]. PKC⑀, PKR␣, or PKC␥ was immunoprecipitated from the supernatant from total cell lysate by subtype-specific antibodies and protein G-agarose beads. Reaction solution (Cell Signaling) containing biotinlinked PKC substrate was added to the beads. The PKC subtype activity was determined by measuring the fluorescence intensity using a ␣-Fusion plate reader (PerkinElmer Life Sciences, Boston, MA). Lipid Raft Separation and Continuous Sucrose Gradient— Continuous sucrose gradient was used to separate the microdomains on the cell membrane according to their densities as described previously [20]. The error bars present the standard derivations and * indicates a significant difference between the marked result and the basal or control result (indicated in the legends of the x-axis)
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