Agonist-induced formation of opioid receptor-G protein-coupled receptor kinase (GRK)-G beta gamma complex on membrane is required for GRK2 function in vivo.
G protein-coupled receptor kinases (GRKs) catalyze agonist-induced receptor phosphorylation on the membrane and initiate receptor desensitization. Previous in vitro studies have shown that the binding of GRK to membrane-associated G beta gamma subunits plays an important role in translocation of GRK2 from the cytoplasm to the plasma membrane. The current study investigated the role of the interaction of GRK2 with the activated delta-opioid receptor (DOR) and G beta gamma subunits in the membrane translocation and function of GRK2 using intact human embryonic kidney 293 cells. Our results showed that agonist treatment induced GRK2 binding to DOR, GRK2 translocation to the plasma membrane, and DOR phosphorylation in cells expressing the wild-type DOR but not the mutant DOR lacking the carboxyl terminus, which contains all three GRK2 phosphorylation sites. DORs with the GRK2 phosphorylation sites modified (M3) or with the acidic residues flanking phosphorylation sites mutated (E355Q/D364N) failed to be phosphorylated in response to agonist stimulation. Agonist-induced GRK2 membrane translocation and GRK-receptor association were observed in cells expressing M3 but not E355Q/D364N. Moreover, over-expression of G beta gamma subunits promoted GRK2 binding to DOR, whereas over-expression of transducin alpha or the carboxyl terminus of GRK2 blocked binding. Further study demonstrated that agonist stimulation induced the formation of a complex containing DOR, GRK2, and G beta gamma subunits in the cell and that agonist-stimulated formation of this complex is essential for the stable localization of GRK2 on the membrane and for its catalytic activity in vivo.
Li
Jiali
J
National Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai 200032, China.
Xiang
Bin
B
Su
Wenjuan
W
Zhang
Xiaoqing
X
Huang
Yalin
Y
Ma
Lan
L
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D020134:Catalytic Domain; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D054769:G-Protein-Coupled Receptor Kinase 2; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011957:Receptors, Opioid / Q000819:agonists* / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D018076:DNA, Complementary; D011957:Receptors, Opioid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D017868:Cyclic AMP-Dependent Protein Kinases; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2