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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-stimulated 35S-GTPγS binding.Stimulation of [35S]GTPγS binding by SS-14, Octreotide, Pasireotide and Somatoprim in the concentration range of 10−12 to 10−6 M. Membranes wer prepared from HEK293 cells stably expressing either the human sst2, sst5 and sst5-sst2ACT or the rat sst3-sst2ACT receptor. Values represent means of triplicate determinations. SE values were smaller than 15%. Three replicate experiments gave similar results.
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pone-0039458-g004: Agonist-stimulated 35S-GTPγS binding.Stimulation of [35S]GTPγS binding by SS-14, Octreotide, Pasireotide and Somatoprim in the concentration range of 10−12 to 10−6 M. Membranes wer prepared from HEK293 cells stably expressing either the human sst2, sst5 and sst5-sst2ACT or the rat sst3-sst2ACT receptor. Values represent means of triplicate determinations. SE values were smaller than 15%. Three replicate experiments gave similar results.

Mentions: We then examined the capacity of these compounds to stimulate GTPγS binding in membrane preparations from the same cells (Figure 4). Unlike that seen in sst2 receptor phosphorylation assays, pasireotide was able stimulate GTPγS binding to a similar degree as octreotide or somatoprim suggesting that pasireotide is a G protein-biased ligand. In contrast, octreotide stimulated GTPγS binding in both sst5- and sst5-sst2CT-expressing cells to a much lesser extend than pasireotide or somatoprim suggesting it is indeed a weak partial agonist at the sst5 receptor. Again similar results were obtained with the wild-type sst5 and the sst5-sst2CT 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-stimulated 35S-GTPγS binding.Stimulation of [35S]GTPγS binding by SS-14, Octreotide, Pasireotide and Somatoprim in the concentration range of 10−12 to 10−6 M. Membranes wer prepared from HEK293 cells stably expressing either the human sst2, sst5 and sst5-sst2ACT or the rat sst3-sst2ACT receptor. Values represent means of triplicate determinations. SE values were smaller than 15%. Three replicate experiments gave similar results.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3383726&req=5

pone-0039458-g004: Agonist-stimulated 35S-GTPγS binding.Stimulation of [35S]GTPγS binding by SS-14, Octreotide, Pasireotide and Somatoprim in the concentration range of 10−12 to 10−6 M. Membranes wer prepared from HEK293 cells stably expressing either the human sst2, sst5 and sst5-sst2ACT or the rat sst3-sst2ACT receptor. Values represent means of triplicate determinations. SE values were smaller than 15%. Three replicate experiments gave similar results.
Mentions: We then examined the capacity of these compounds to stimulate GTPγS binding in membrane preparations from the same cells (Figure 4). Unlike that seen in sst2 receptor phosphorylation assays, pasireotide was able stimulate GTPγS binding to a similar degree as octreotide or somatoprim suggesting that pasireotide is a G protein-biased ligand. In contrast, octreotide stimulated GTPγS binding in both sst5- and sst5-sst2CT-expressing cells to a much lesser extend than pasireotide or somatoprim suggesting it is indeed a weak partial agonist at the sst5 receptor. Again similar results were obtained with the wild-type sst5 and the sst5-sst2CT 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