Agonist-induced Formation of Opioid Receptor-G Protein-coupled Receptor Kinase (GRK)-Gβγ 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 Gβγ 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 δ-opioid receptor (DOR) and Gβγ 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 in cells expressing but not of Gβγ subunits GRK2 binding to DOR, of or the carboxyl of GRK2 study that agonist induced the of DOR, and Gβγ subunits in the and that of the of GRK2 on the membrane and in 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 Gβγ 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 δ-opioid receptor (DOR) and Gβγ 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 in cells expressing but not of Gβγ subunits GRK2 binding to DOR, of or the carboxyl of GRK2 study that agonist induced the of DOR, and Gβγ subunits in the and that of the of GRK2 on the membrane and in G protein-coupled G protein-coupled G G protein-coupled receptor DOR, δ-opioid wild-type δ-opioid the residues mutant of human embryonic modified G protein-coupled G G protein-coupled receptor DOR, δ-opioid wild-type δ-opioid the residues mutant of human embryonic modified of plasma membrane of in the of and have in the human of from and to the of cells and in of important of that agonist that The of receptor receptor phosphorylation kinases (GRKs) of the GRK have to and have on the of and and the receptor and and the and of in the catalyze phosphorylation of the activated the or The of with the activated receptor in The of the membrane and receptor of agonist-induced that the of the of and an association with and that the agonist-induced GRK2 translocation to plasma membrane receptor phosphorylation and using that the subunits of the G with the carboxyl of GRK2 and the association of GRK2 with and interaction the phosphorylation of and in vitro The association of GRK2 and Gβγ using GRK2 in vitro and using in studies the the of the Gβγ agonist of GRK2 to the Gβγ and GRK2 to the activated to be in and the of GRK2 translocation to plasma membrane be that GRK2 the δ-opioid receptor (DOR) and results in of the GRK2 phosphorylation sites in DOR The current study the of phosphorylation in have the the of on the membrane in response to agonist stimulation. the of the agonist-induced of the the membrane association and function of GRK2 in and from from and from and from embryonic kidney 293 cells from the and from from and from and GRK2 and DORs the wild-type δ-opioid receptor the DOR and the mutant (M3) The DOR and and the of the of of and with to with an The from and cells in in or cells in or the cells using the The cells and the of binding The of the of of receptor phosphorylation the cells with of in and or the of the cells to and in and with and the on the to with the cells in the or of The cells in and and the The membrane and an of in and in the or of The in and in binding in the of and binding in the of The of binding and cells in in in the or of the of the the cells with and in of and The and the with of and of The three with The to the in and The of and Gβγ in the in the with or The using an the of the on to of the in the cells on in the or of and The cells with in and with The cells with and with and the with with using the of with of study that agonist-induced phosphorylation of DOR on the of the receptor and GRK2 studies that the of DOR important interaction with interaction GRK2 and DOR not shown in of the cells expressing the wild-type DOR with agonist of DOR, induced DOR of GRK2 phosphorylation of DOR lacking the residues and G failed to be phosphorylated in cells with the agonist GRK2 be with DOR in cells expressing DOR and GRK2 GRK2 in the receptor in the of agonist the association of GRK and DOR and receptor phosphorylation not of GRK2 to the receptor agonist and binding in The binding and not but not the of in cells expressing DOR, and agonist-induced of be the to with the wild-type DOR, GRK2 with the mutant receptor mutant the and all of the GRK2 phosphorylation sites that agonist GRK2 binding to DOR and that the residues in the of the receptor interaction with cells with and DOR, and the of GRK agonist using shown in GRK2 in the cytoplasm in the of agonist to plasma membrane and with DOR with The of in cells and the wild-type DOR showed in response to agonist the membrane-associated the in membrane not agonist-induced translocation to in the cells the mutant receptor of binding GRK2 and of phosphorylated that the of DOR not binding but GRK2 membrane agonist-induced membrane translocation of GRK2 studies from and and that phosphorylation sites and the residues of DOR shown in DOR in which all of the and phosphorylation sites in GRK have failed to be phosphorylated treatment with The and of DOR the acidic residues to GRK phosphorylation sites and showed and mutant (E355Q/D364N) receptor phosphorylation of GRK2 the phosphorylation of and but not have on not and binding showed that mutated to be and to G the phosphorylation of and not to be to in receptor and the mutant of all the GRK phosphorylation sites on DOR in of agonist-induced receptor The results that the acidic residues receptor phosphorylation sites role in receptor of GRK2 with DOR GRK2 membrane cells with or or with or GRK2 and cells with the cells in the or of the cells with or GRK2 in with on and to The of phosphorylation and of three with or receptor cells expressing DOR with or and with binding to the of three with agonist cells in the or of and the of the receptor using using GRK2 and the with of the using GRK2 GRK2 shown the of GRK2 in the DOR and of three with the of the GRK phosphorylation sites and the acidic residues on GRK2 membrane and DOR the wild-type and mutant DORs with GRK2 in cells. shown in treatment membrane of in the cells expressing which all of the GRK phosphorylation sites. the of GRK2 membrane translocation with that in the cells expressing wild-type that in response to agonist with GRK2 the wild-type receptor and not be phosphorylated the GRK2 membrane association and with the receptor in cells expressing the mutant the and mutant showed in agonist-induced GRK2 binding in the mutant showed of in and The results that GRK and phosphorylation sites on DOR not in the interaction with GRK2 and in the membrane association of GRK2 induced the and residues phosphorylation sites The that the interaction not DOR phosphorylation but GRK2 membrane of GRK2 with DOR GRK2 membrane cells with or or with or GRK2 and cells with the cells in the or of the cells with or GRK2 in with on and to The of phosphorylation and of three 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binding of the GRK2 in the to the activated receptor to on the membrane. 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using GRK2 in vitro and using in studies the the of the Gβγ agonist of GRK2 to the Gβγ and GRK2 to the activated to be in and the of GRK2 translocation to plasma membrane be Our that GRK2 the δ-opioid receptor (DOR) and results in of the GRK2 phosphorylation sites in DOR The current study the of phosphorylation in have the the of on the membrane in response to agonist stimulation. the of the agonist-induced of the the membrane association and function of GRK2 in and from from and from and from embryonic kidney 293 cells from the and from from and from and GRK2 and DORs the wild-type δ-opioid receptor the DOR and the mutant (M3) The DOR and and the of the of of and with to with an The from and cells in in or cells in or the cells using the The cells and the of binding The of the of of receptor phosphorylation the cells with of in and or the of the cells to and in and with and the on the to with the cells in the or of The cells in and and the The membrane and an of in and in the or of 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studies that the of DOR important interaction with interaction GRK2 and DOR not shown in of the cells expressing the wild-type DOR with agonist of DOR, induced DOR of GRK2 phosphorylation of DOR lacking the residues and G failed to be phosphorylated in cells with the agonist GRK2 be with DOR in cells expressing DOR and GRK2 GRK2 in the receptor in the of agonist the association of GRK and DOR and receptor phosphorylation not of GRK2 to the receptor agonist and binding in The binding and not but not the of in cells expressing DOR, and agonist-induced of be the to with the wild-type DOR, GRK2 with the mutant receptor mutant the and all of the GRK2 phosphorylation sites that agonist GRK2 binding to DOR and that the residues in the of the receptor interaction with cells with and DOR, and the of GRK agonist using shown in GRK2 in the cytoplasm in the of agonist to plasma membrane and with DOR with The of in cells and the wild-type DOR showed in response to agonist the membrane-associated 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shown in treatment membrane of in the cells expressing which all of the GRK phosphorylation sites. the of GRK2 membrane translocation with that in the cells expressing wild-type that in response to agonist with GRK2 the wild-type receptor and not be phosphorylated the GRK2 membrane association and with the receptor in cells expressing the mutant the and mutant showed in agonist-induced GRK2 binding in the mutant showed of in and The results that GRK and phosphorylation sites on DOR not in the interaction with GRK2 and in the membrane association of GRK2 induced the and residues phosphorylation sites The that the interaction not DOR phosphorylation but GRK2 membrane DOR phosphorylation the and and and of The function of and of DOR in the interaction with of the GRK in cells DORs and or The that to and with the wild-type DOR in response to agonist stimulation. of kinases showed binding to and and of binding to not phosphorylated not The results the the interaction of DOR with and in to that the acidic residues and to phosphorylation sites on DOR in the interaction with in the and of GRK2 to membrane-associated Gβγ subunits the and membrane of GRK2 the of interaction in GRK2 membrane translocation and in the interaction with the receptor GRK2 in cells with the wild-type or mutant DORs and the of GRK2 or shown in of cells GRK2 and the wild-type DOR with GRK2 and Gβγ in the DOR of Gβγ subunits in cells and interaction and the of or the of which interaction and results the role of the Gβγ subunits in the interaction DOR and GRK2 and in the of the mutant receptor which in binding not with the Gβγ not of Gβγ that the of of DOR and Gβγ an interaction DOR and GRK2 GRK2 receptor and Gβγ to current study that agonist GRK2 with Gβγ subunits and receptor on the membrane and that Gβγ subunits role in the interaction of GRK with activated receptor in Our results from and that of the of GRK2 on the membrane and in the of phosphorylation in receptor GRK2 interaction with the Gβγ subunits with the membrane. interaction the binding of the GRK2 in the to the activated receptor to on the membrane. GRK2 to and results in the phosphorylation of the activated of GRK2 membrane translocation in cells and Gβγ not the of on the membrane in the of The agonist-induced GRK2 interaction with Gβγ not be the and be the on the plasma membrane. The which have phosphorylated and have to be with the activated The of the of the on the membrane. of the three of the to receptor the to and the with the Gβγ subunits and to be in GRK membrane translocation Our of in response to receptor with studies on and that the interaction of the of GRK2 with the activated receptor and of the of GRK2 with the Gβγ subunits on the shown that of and that phosphorylation of the receptor to the studies that with activated sites from phosphorylation sites Our current that the residues in the carboxyl of DOR sites GRK2 phosphorylation and interaction and that the DORs with phosphorylation sites mutated with GRK2 and with GRK2 and the Gβγ subunits on the membrane in that the GRK2 residues in DOR not residues in to residues GRK2 phosphorylation sites in and with and that kinases in the GRK2 acidic residues flanking the or with the receptor and receptor have that acidic residues important in the phosphorylation and of have that of and residues to GRK phosphorylation in DOR DOR phosphorylation and receptor and have that acidic residues and Our results have in receptor that the acidic residues flanking phosphorylation sites in the interaction of the receptor with of the and in and receptor phosphorylation and desensitization. The current study that agonist GRK2 with Gβγ subunits and receptor on the membrane and that Gβγ subunits role in the interaction of GRK with activated receptor in Our results from and that of the of GRK2 on the membrane and in the of phosphorylation in receptor GRK2 interaction with the Gβγ subunits with the membrane. interaction the binding of the GRK2 in the to the activated receptor to on the membrane. GRK2 to and results in the phosphorylation of the activated of GRK2 membrane translocation in cells and Gβγ not the of on the membrane in the of The agonist-induced GRK2 interaction with Gβγ not be the and be the on the plasma membrane. The which have phosphorylated and have to be with the activated The of the of the on the membrane. of the three of the to receptor the to and the with the Gβγ subunits and to be in GRK membrane translocation Our of in response to receptor with studies on and that the interaction of the of GRK2 with the activated receptor and of the of GRK2 with the Gβγ subunits on the membrane. Previous shown that of and that phosphorylation of the receptor to the studies that with activated sites from phosphorylation sites Our current that the residues in the carboxyl of DOR sites GRK2 phosphorylation and interaction and that the DORs with phosphorylation sites mutated with GRK2 and with GRK2 and the Gβγ subunits on the membrane in that the GRK2 residues in DOR not residues in to residues GRK2 GRK phosphorylation sites in and with and that kinases in the GRK2 acidic residues flanking the or with the receptor and receptor have that acidic residues important in the phosphorylation and of have that of and residues to GRK phosphorylation in DOR DOR phosphorylation and receptor and have that acidic residues and Our results have in receptor that the acidic residues flanking phosphorylation sites in the interaction of the receptor with of the and in and receptor phosphorylation and desensitization. GRK and of and