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Mapping the putative G protein-coupled receptor (GPCR) docking site on GPCR kinase 2: insights from intact cell phosphorylation and recruitment assays.

Beautrait A, Michalski KR, Lopez TS, Mannix KM, McDonald DJ, Cutter AR, Medina CB, Hebert AM, Francis CJ, Bouvier M, Tesmer JJ, Sterne-Marr R - J. Biol. Chem. (2014)

Bottom Line: We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment.We suggest that a surface of GRK2, including Leu(4), Val(7), Leu(8), Val(11), and Ser(12), directly interacts with receptors, whereas residues such as Asp(10), Tyr(13), Ala(16), Met(17), Gly(475), Val(477), and Ile(485) are more important for kinase domain closure and activation.Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biochemistry and the Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

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Time course and titration of RlucII-GRK2 recruitment to NOR-activated α2AAR-GFP2 as measured by BRET.A, time course. HEK293T cells transfected with α2AAR-GFP2 (1 μg) and WT RlucII-GRK2 (250 ng) were treated with 100 μm NOR at the indicated times. Data are expressed as NOR-promoted BRET and are the mean ± S.E. of three independent experiments carried out in triplicate. The dashed line represents the time (6 min) at which BRET data in Fig. 8 were collected. B, BRET titration. Cells transfected with RlucII-GRK2 and increasing amounts of α2AAR-GFP2 were incubated with or without NOR for 6 min. Data are expressed as net BRET values that correspond to BRET measurements that have been subtracted from the background BRET signal originating from luciferase alone. Values obtained from three independent experiments were pooled and plotted as individual data points. Dashed lines represent the time and acceptor/donor ratio, respectively, at which BRET experiments for Fig. 8 were performed.
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Figure 7: Time course and titration of RlucII-GRK2 recruitment to NOR-activated α2AAR-GFP2 as measured by BRET.A, time course. HEK293T cells transfected with α2AAR-GFP2 (1 μg) and WT RlucII-GRK2 (250 ng) were treated with 100 μm NOR at the indicated times. Data are expressed as NOR-promoted BRET and are the mean ± S.E. of three independent experiments carried out in triplicate. The dashed line represents the time (6 min) at which BRET data in Fig. 8 were collected. B, BRET titration. Cells transfected with RlucII-GRK2 and increasing amounts of α2AAR-GFP2 were incubated with or without NOR for 6 min. Data are expressed as net BRET values that correspond to BRET measurements that have been subtracted from the background BRET signal originating from luciferase alone. Values obtained from three independent experiments were pooled and plotted as individual data points. Dashed lines represent the time and acceptor/donor ratio, respectively, at which BRET experiments for Fig. 8 were performed.

Mentions: One of the primary goals of this work was to distinguish residues on GRK2 that are important for direct interaction with GPCRs. This interaction is difficult to measure in vitro because GRK2 is expected to bind weakly to GPCRs and binds strongly to negatively charged phospholipids, which are required along with Gβγ for GRK2 activity (40). Thus, we turned to a cell-based BRET assay to examine recruitment of GRK2 to an activated GPCR. Although we were able to measure phosphorylation of the β2AR(Y326A) by GRK2 in intact cells (Fig. 6B), we have thus far been unable to detect a significant ISO-stimulated BRET signal between β2AR-GFP, β2AR-Venus, β2AR-GFP10, and luciferase-GRK2 or between β2AR-luciferase and GRK2-GFP10. The β2AR(Y326A) variant also did not yield a measurable ISO-promoted BRET response. However, we were able to detect agonist-dependent recruitment of GRK2 to the α2AAR, as indicated by the NOR-induced BRET signal observed between α2AAR-GFP2 and luciferase-GRK2 in a time course experiment (Fig. 7A). Co-expression of a constant level of RlucII-GRK2 with increasing concentrations of α2AAR-GFP2 led to an increase in basal and agonist-induced BRET signals in a saturable fashion (Fig. 7B), indicating a specific interaction between the two proteins. For all subsequent BRET experiments, acceptor/donor ratios yielding maximal BRET were used, and the readings were collected 6 min following receptor activation. These conditions provided stable signals and a good dynamic range.


Mapping the putative G protein-coupled receptor (GPCR) docking site on GPCR kinase 2: insights from intact cell phosphorylation and recruitment assays.

Beautrait A, Michalski KR, Lopez TS, Mannix KM, McDonald DJ, Cutter AR, Medina CB, Hebert AM, Francis CJ, Bouvier M, Tesmer JJ, Sterne-Marr R - J. Biol. Chem. (2014)

Time course and titration of RlucII-GRK2 recruitment to NOR-activated α2AAR-GFP2 as measured by BRET.A, time course. HEK293T cells transfected with α2AAR-GFP2 (1 μg) and WT RlucII-GRK2 (250 ng) were treated with 100 μm NOR at the indicated times. Data are expressed as NOR-promoted BRET and are the mean ± S.E. of three independent experiments carried out in triplicate. The dashed line represents the time (6 min) at which BRET data in Fig. 8 were collected. B, BRET titration. Cells transfected with RlucII-GRK2 and increasing amounts of α2AAR-GFP2 were incubated with or without NOR for 6 min. Data are expressed as net BRET values that correspond to BRET measurements that have been subtracted from the background BRET signal originating from luciferase alone. Values obtained from three independent experiments were pooled and plotted as individual data points. Dashed lines represent the time and acceptor/donor ratio, respectively, at which BRET experiments for Fig. 8 were performed.
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Figure 7: Time course and titration of RlucII-GRK2 recruitment to NOR-activated α2AAR-GFP2 as measured by BRET.A, time course. HEK293T cells transfected with α2AAR-GFP2 (1 μg) and WT RlucII-GRK2 (250 ng) were treated with 100 μm NOR at the indicated times. Data are expressed as NOR-promoted BRET and are the mean ± S.E. of three independent experiments carried out in triplicate. The dashed line represents the time (6 min) at which BRET data in Fig. 8 were collected. B, BRET titration. Cells transfected with RlucII-GRK2 and increasing amounts of α2AAR-GFP2 were incubated with or without NOR for 6 min. Data are expressed as net BRET values that correspond to BRET measurements that have been subtracted from the background BRET signal originating from luciferase alone. Values obtained from three independent experiments were pooled and plotted as individual data points. Dashed lines represent the time and acceptor/donor ratio, respectively, at which BRET experiments for Fig. 8 were performed.
Mentions: One of the primary goals of this work was to distinguish residues on GRK2 that are important for direct interaction with GPCRs. This interaction is difficult to measure in vitro because GRK2 is expected to bind weakly to GPCRs and binds strongly to negatively charged phospholipids, which are required along with Gβγ for GRK2 activity (40). Thus, we turned to a cell-based BRET assay to examine recruitment of GRK2 to an activated GPCR. Although we were able to measure phosphorylation of the β2AR(Y326A) by GRK2 in intact cells (Fig. 6B), we have thus far been unable to detect a significant ISO-stimulated BRET signal between β2AR-GFP, β2AR-Venus, β2AR-GFP10, and luciferase-GRK2 or between β2AR-luciferase and GRK2-GFP10. The β2AR(Y326A) variant also did not yield a measurable ISO-promoted BRET response. However, we were able to detect agonist-dependent recruitment of GRK2 to the α2AAR, as indicated by the NOR-induced BRET signal observed between α2AAR-GFP2 and luciferase-GRK2 in a time course experiment (Fig. 7A). Co-expression of a constant level of RlucII-GRK2 with increasing concentrations of α2AAR-GFP2 led to an increase in basal and agonist-induced BRET signals in a saturable fashion (Fig. 7B), indicating a specific interaction between the two proteins. For all subsequent BRET experiments, acceptor/donor ratios yielding maximal BRET were used, and the readings were collected 6 min following receptor activation. These conditions provided stable signals and a good dynamic range.

Bottom Line: We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment.We suggest that a surface of GRK2, including Leu(4), Val(7), Leu(8), Val(11), and Ser(12), directly interacts with receptors, whereas residues such as Asp(10), Tyr(13), Ala(16), Met(17), Gly(475), Val(477), and Ile(485) are more important for kinase domain closure and activation.Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biochemistry and the Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

Show MeSH