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A transplantable phosphorylation probe for direct assessment of G protein-coupled receptor activation.

Kliewer A, Mann A, Petrich A, Pöll F, Schulz S - PLoS ONE (2012)

Bottom Line: Like octreotide, somatoprim was a full agonist at the sst(2) receptor.Unlike octreotide, somatoprim was also a potent agonist at the sst(5) receptor.Together, we propose the application of a phosphorylation probe for direct assessment of G protein-coupled receptor activation and demonstrate its utility in the pharmacological characterization of novel somatostatin analogs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Jena University Hospital-Friedrich Schiller University Jena, Jena, Germany.

ABSTRACT
The newly developed multireceptor somatostatin analogs pasireotide (SOM230), octreotide and somatoprim (DG3173) have primarily been characterized according to their binding profiles. However, their ability to activate individual somatostatin receptor subtypes (sst) has not been directly assessed so far. Here, we transplanted the carboxyl-terminal phosphorylation motif of the sst(2) receptor to other somatostatin receptors and assessed receptor activation using a set of three phosphosite-specific antibodies. Our comparative analysis revealed unexpected efficacy profiles for pasireotide, octreotide and somatoprim. Pasireotide was able to activate sst(3) and sst(5) receptors but was only a partial agonist at the sst(2) receptor. Octreotide exhibited potent agonistic properties at the sst(2) receptor but produced very little sst(5) receptor activation. Like octreotide, somatoprim was a full agonist at the sst(2) receptor. Unlike octreotide, somatoprim was also a potent agonist at the sst(5) receptor. Together, we propose the application of a phosphorylation probe for direct assessment of G protein-coupled receptor activation and demonstrate its utility in the pharmacological characterization of novel somatostatin analogs.

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Agonist-selective phosphorylation of the human sst2 receptor.(Top) Schematic representation of the human sst2 receptor indicating the phosphate acceptor sites S341/343, T353/354 and T356/359 within its carboxyl-terminal tail. (Bottom) HEK293 cells stably expressing the sst2 receptor were either not exposed or exposed for 5 min to SS-14, octreotide, pasireotide or somatoprim in concentrations ranging from 10−12 to 10−5 M. The levels of phosphorylated sst2 receptors were then determined using the phosphosite-specific antibodies anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} and anti-pT356/pT359 {0522}. Western blots shown are representative of three to five independent experiments for each condition. The positions of the molecular mass markers are indicated on the left (in kDa).
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pone-0039458-g002: Agonist-selective phosphorylation of the human sst2 receptor.(Top) Schematic representation of the human sst2 receptor indicating the phosphate acceptor sites S341/343, T353/354 and T356/359 within its carboxyl-terminal tail. (Bottom) HEK293 cells stably expressing the sst2 receptor were either not exposed or exposed for 5 min to SS-14, octreotide, pasireotide or somatoprim in concentrations ranging from 10−12 to 10−5 M. The levels of phosphorylated sst2 receptors were then determined using the phosphosite-specific antibodies anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} and anti-pT356/pT359 {0522}. Western blots shown are representative of three to five independent experiments for each condition. The positions of the molecular mass markers are indicated on the left (in kDa).

Mentions: Recently, we have generated a set of three phosphosite-specific antibodies, which allowed us to detect selectively the S341/S343-, the T353/T354- and the T356/T359-phosphorylated forms of the sst2 receptor [16], . When HEK293 cells stably expressing the human sst2 receptor were exposed for 5 min to SS-14, octreotide, pasireotide or somatoprim in concentrations ranging from 10−12 to 10−5 M, SS-14, octreotide and somatoprim were able to promote a robust dose-dependent phosphorylation of all three sites (Figure 2). In contrast, pasireotide stimulated only at saturating concentration a detectable phosphorylation of S341/S343 and T356/T359 but not of T353/T354. Considering the high binding affinity of pasireotide this result was unexpected and indicates that, in contrast to octreotide and somatoprim, pasireotide is a partial agonist at the human sst2 receptor. It also shows that the sst2 receptor can exist in distinct active conformations, which favor different patterns of GRK-mediated phosphorylation. Thus, considerable differences may exist between the binding and efficacy profiles of pan-somatostatin analogs. It would therefore be desirable to know the patterns of phosphorylation induced by multireceptor ligands at the level of individual somatostatin receptors. However, at present phosphosite-specific antibodies are only available for the sst2 receptor.


A transplantable phosphorylation probe for direct assessment of G protein-coupled receptor activation.

Kliewer A, Mann A, Petrich A, Pöll F, Schulz S - PLoS ONE (2012)

Agonist-selective phosphorylation of the human sst2 receptor.(Top) Schematic representation of the human sst2 receptor indicating the phosphate acceptor sites S341/343, T353/354 and T356/359 within its carboxyl-terminal tail. (Bottom) HEK293 cells stably expressing the sst2 receptor were either not exposed or exposed for 5 min to SS-14, octreotide, pasireotide or somatoprim in concentrations ranging from 10−12 to 10−5 M. The levels of phosphorylated sst2 receptors were then determined using the phosphosite-specific antibodies anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} and anti-pT356/pT359 {0522}. Western blots shown are representative of three to five independent experiments for each condition. The positions of the molecular mass markers are indicated on the left (in kDa).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383726&req=5

pone-0039458-g002: Agonist-selective phosphorylation of the human sst2 receptor.(Top) Schematic representation of the human sst2 receptor indicating the phosphate acceptor sites S341/343, T353/354 and T356/359 within its carboxyl-terminal tail. (Bottom) HEK293 cells stably expressing the sst2 receptor were either not exposed or exposed for 5 min to SS-14, octreotide, pasireotide or somatoprim in concentrations ranging from 10−12 to 10−5 M. The levels of phosphorylated sst2 receptors were then determined using the phosphosite-specific antibodies anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} and anti-pT356/pT359 {0522}. Western blots shown are representative of three to five independent experiments for each condition. The positions of the molecular mass markers are indicated on the left (in kDa).
Mentions: Recently, we have generated a set of three phosphosite-specific antibodies, which allowed us to detect selectively the S341/S343-, the T353/T354- and the T356/T359-phosphorylated forms of the sst2 receptor [16], . When HEK293 cells stably expressing the human sst2 receptor were exposed for 5 min to SS-14, octreotide, pasireotide or somatoprim in concentrations ranging from 10−12 to 10−5 M, SS-14, octreotide and somatoprim were able to promote a robust dose-dependent phosphorylation of all three sites (Figure 2). In contrast, pasireotide stimulated only at saturating concentration a detectable phosphorylation of S341/S343 and T356/T359 but not of T353/T354. Considering the high binding affinity of pasireotide this result was unexpected and indicates that, in contrast to octreotide and somatoprim, pasireotide is a partial agonist at the human sst2 receptor. It also shows that the sst2 receptor can exist in distinct active conformations, which favor different patterns of GRK-mediated phosphorylation. Thus, considerable differences may exist between the binding and efficacy profiles of pan-somatostatin analogs. It would therefore be desirable to know the patterns of phosphorylation induced by multireceptor ligands at the level of individual somatostatin receptors. However, at present phosphosite-specific antibodies are only available for the sst2 receptor.

Bottom Line: Like octreotide, somatoprim was a full agonist at the sst(2) receptor.Unlike octreotide, somatoprim was also a potent agonist at the sst(5) receptor.Together, we propose the application of a phosphorylation probe for direct assessment of G protein-coupled receptor activation and demonstrate its utility in the pharmacological characterization of novel somatostatin analogs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Jena University Hospital-Friedrich Schiller University Jena, Jena, Germany.

ABSTRACT
The newly developed multireceptor somatostatin analogs pasireotide (SOM230), octreotide and somatoprim (DG3173) have primarily been characterized according to their binding profiles. However, their ability to activate individual somatostatin receptor subtypes (sst) has not been directly assessed so far. Here, we transplanted the carboxyl-terminal phosphorylation motif of the sst(2) receptor to other somatostatin receptors and assessed receptor activation using a set of three phosphosite-specific antibodies. Our comparative analysis revealed unexpected efficacy profiles for pasireotide, octreotide and somatoprim. Pasireotide was able to activate sst(3) and sst(5) receptors but was only a partial agonist at the sst(2) receptor. Octreotide exhibited potent agonistic properties at the sst(2) receptor but produced very little sst(5) receptor activation. Like octreotide, somatoprim was a full agonist at the sst(2) receptor. Unlike octreotide, somatoprim was also a potent agonist at the sst(5) receptor. Together, we propose the application of a phosphorylation probe for direct assessment of G protein-coupled receptor activation and demonstrate its utility in the pharmacological characterization of novel somatostatin analogs.

Show MeSH