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Probing the stoichiometry of β2-adrenergic receptor phosphorylation by targeted mass spectrometry.

Gao S, Malbon C, Wang HY - J Mol Signal (2014)

Bottom Line: The results demonstrated that pS356 is the dominant site of protein phosphorylation.Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of β2AR.Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with β2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Health Sciences Center, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA. shujuan.gao@stonybrook.edu.

ABSTRACT

Background: Protein phosphorylation of G-protein-coupled receptors (GPCR) is central to the myriad of functions that these ubiquitous receptors perform in biology. Although readily addressable with the use of phospho-specific antibodies, analysis phosphorylation at the level of stoichiometry requires receptor isolation and advanced proteomics. We chose two key sites of potential phosphorylation of human beta2-adrenergic receptor (β2AR residues S355 and S356) to ascertain the feasibility of applying targeted mass spectrometry to establishing the stoichiometry of the phosphorylation.

Method: We stimulated HEK293 cells stably expressing Flag-tagged β2AR-eGFP with 10 μM beta-adrenergic agonist (isoproterenol) and made use of proteomics and targeted mass spectrometry (MS) to quantify the molar ration of phosphorylation on S355 and S356 versus non-phosphorylated receptor in agonist-treated cells.

Results: Phosphorylation of either S355 or S356 residue occurred only for agonist-occupied β2AR. The results demonstrated that pS356 is the dominant site of protein phosphorylation. The abundance of the p356 was 8.6-fold more than that of pS355. Calculation of the molar ratio of phosphorylated (pS355 plus pS356) versus non-phosphorylated receptor reveals that at high occupancy of the receptor only 12.4% of the β2AR is phosphorylated at these sites.

Conclusions: Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of β2AR. Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with β2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.

No MeSH data available.


β2AR phosphorylation in HEK293 cells challenged with beta-adrenergic agonist. HEK293 clones stably expressing N-terminal Flag-tagged, C-terminal eGFP-tagged β2AR were treated either with 100 nM isoproterenol for the indicated times (A) or with varying concentrations of the beta-adrenergic agonist, isoproterenol for 5 min (B). β2AR was immunoprecipitated with anti-Flag immunoadsorption beads. SDS-PAGE and immunoblotting were performed as indicated in the Materials and methods section. The phosphorylation of β2AR was normalized to total β2AR. The data were quantified and are displayed as percentage of maximum phosphorylation of each site (s). The experimental data shown is of a single analysis performed in triplicate and replicated multiple times with similar results.
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Figure 1: β2AR phosphorylation in HEK293 cells challenged with beta-adrenergic agonist. HEK293 clones stably expressing N-terminal Flag-tagged, C-terminal eGFP-tagged β2AR were treated either with 100 nM isoproterenol for the indicated times (A) or with varying concentrations of the beta-adrenergic agonist, isoproterenol for 5 min (B). β2AR was immunoprecipitated with anti-Flag immunoadsorption beads. SDS-PAGE and immunoblotting were performed as indicated in the Materials and methods section. The phosphorylation of β2AR was normalized to total β2AR. The data were quantified and are displayed as percentage of maximum phosphorylation of each site (s). The experimental data shown is of a single analysis performed in triplicate and replicated multiple times with similar results.

Mentions: β2AR are phosphorylated on distinct sites by a variety of protein kinases in cells stimulated with beta-adrenergic agonist. We initially probed for phosphorylation events on five critical residues, namely S262 (substrate for PKA), S345/S346 (substrate for PKA), and S355/S356 (substrate for GRK). HEK293 clones that were transfected and selected for stable expression of N-terminally Flag-tagged, C-terminally eGFP-tagged β2AR (Flag-β2AR-eGFP) were employed throughout. Clones were treated briefly with beta-adrenergic agonist (10 μM isoproterenol, see Figure 1). The phosphorylation of the sites displayed both a time- and a dose-dependent response to agonist treatment. In unstimulated cells, phosphorylation of S262 was very low. Upon stimulation with isoproterenol, phosphoS262 could be detected readily at 2 min. Phosphorylation of S262 was maximal at ~5 minutes, declining thereafter. Within 30 min of agonist, phosphorylation of S262 is only about 50% of maximal. S345, S346 residues showed higher basal phosphorylation compared to that of S262. Phosphorylation of S345 and S346 was observed to increase within 2 min following isoproterenol treatment, peaking at 5 min. This greater phosphorylation persisted for at least 20 min and slowly declined thereafter. The phosphorylation of GRK-specific sites, S356/S356, showed a very different pattern than those specific for PKA-catalyzed phosphorylation. Whereas basal phosphorylation was readily detected on other sites, phosphorylation of S355/S356 was not detectable in the unstimulated cells. The peak level of phosphorylation was attained within 10 min post stimulation and persisted for 30 min. Importantly, whereas phosphorylation of PKA sites was found to be maximal at 10 nM isoproterenol, phosphorylation of the GRK sites was maximal only at concentrations of isoproterenol 1.0 μM or higher (Figure 1B).


Probing the stoichiometry of β2-adrenergic receptor phosphorylation by targeted mass spectrometry.

Gao S, Malbon C, Wang HY - J Mol Signal (2014)

β2AR phosphorylation in HEK293 cells challenged with beta-adrenergic agonist. HEK293 clones stably expressing N-terminal Flag-tagged, C-terminal eGFP-tagged β2AR were treated either with 100 nM isoproterenol for the indicated times (A) or with varying concentrations of the beta-adrenergic agonist, isoproterenol for 5 min (B). β2AR was immunoprecipitated with anti-Flag immunoadsorption beads. SDS-PAGE and immunoblotting were performed as indicated in the Materials and methods section. The phosphorylation of β2AR was normalized to total β2AR. The data were quantified and are displayed as percentage of maximum phosphorylation of each site (s). The experimental data shown is of a single analysis performed in triplicate and replicated multiple times with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4022239&req=5

Figure 1: β2AR phosphorylation in HEK293 cells challenged with beta-adrenergic agonist. HEK293 clones stably expressing N-terminal Flag-tagged, C-terminal eGFP-tagged β2AR were treated either with 100 nM isoproterenol for the indicated times (A) or with varying concentrations of the beta-adrenergic agonist, isoproterenol for 5 min (B). β2AR was immunoprecipitated with anti-Flag immunoadsorption beads. SDS-PAGE and immunoblotting were performed as indicated in the Materials and methods section. The phosphorylation of β2AR was normalized to total β2AR. The data were quantified and are displayed as percentage of maximum phosphorylation of each site (s). The experimental data shown is of a single analysis performed in triplicate and replicated multiple times with similar results.
Mentions: β2AR are phosphorylated on distinct sites by a variety of protein kinases in cells stimulated with beta-adrenergic agonist. We initially probed for phosphorylation events on five critical residues, namely S262 (substrate for PKA), S345/S346 (substrate for PKA), and S355/S356 (substrate for GRK). HEK293 clones that were transfected and selected for stable expression of N-terminally Flag-tagged, C-terminally eGFP-tagged β2AR (Flag-β2AR-eGFP) were employed throughout. Clones were treated briefly with beta-adrenergic agonist (10 μM isoproterenol, see Figure 1). The phosphorylation of the sites displayed both a time- and a dose-dependent response to agonist treatment. In unstimulated cells, phosphorylation of S262 was very low. Upon stimulation with isoproterenol, phosphoS262 could be detected readily at 2 min. Phosphorylation of S262 was maximal at ~5 minutes, declining thereafter. Within 30 min of agonist, phosphorylation of S262 is only about 50% of maximal. S345, S346 residues showed higher basal phosphorylation compared to that of S262. Phosphorylation of S345 and S346 was observed to increase within 2 min following isoproterenol treatment, peaking at 5 min. This greater phosphorylation persisted for at least 20 min and slowly declined thereafter. The phosphorylation of GRK-specific sites, S356/S356, showed a very different pattern than those specific for PKA-catalyzed phosphorylation. Whereas basal phosphorylation was readily detected on other sites, phosphorylation of S355/S356 was not detectable in the unstimulated cells. The peak level of phosphorylation was attained within 10 min post stimulation and persisted for 30 min. Importantly, whereas phosphorylation of PKA sites was found to be maximal at 10 nM isoproterenol, phosphorylation of the GRK sites was maximal only at concentrations of isoproterenol 1.0 μM or higher (Figure 1B).

Bottom Line: The results demonstrated that pS356 is the dominant site of protein phosphorylation.Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of β2AR.Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with β2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Health Sciences Center, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA. shujuan.gao@stonybrook.edu.

ABSTRACT

Background: Protein phosphorylation of G-protein-coupled receptors (GPCR) is central to the myriad of functions that these ubiquitous receptors perform in biology. Although readily addressable with the use of phospho-specific antibodies, analysis phosphorylation at the level of stoichiometry requires receptor isolation and advanced proteomics. We chose two key sites of potential phosphorylation of human beta2-adrenergic receptor (β2AR residues S355 and S356) to ascertain the feasibility of applying targeted mass spectrometry to establishing the stoichiometry of the phosphorylation.

Method: We stimulated HEK293 cells stably expressing Flag-tagged β2AR-eGFP with 10 μM beta-adrenergic agonist (isoproterenol) and made use of proteomics and targeted mass spectrometry (MS) to quantify the molar ration of phosphorylation on S355 and S356 versus non-phosphorylated receptor in agonist-treated cells.

Results: Phosphorylation of either S355 or S356 residue occurred only for agonist-occupied β2AR. The results demonstrated that pS356 is the dominant site of protein phosphorylation. The abundance of the p356 was 8.6-fold more than that of pS355. Calculation of the molar ratio of phosphorylated (pS355 plus pS356) versus non-phosphorylated receptor reveals that at high occupancy of the receptor only 12.4% of the β2AR is phosphorylated at these sites.

Conclusions: Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of β2AR. Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with β2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.

No MeSH data available.