Agonist-selektive Phosphorylierung und Dephosphorylierung des Ratten-Somatostatin-Rezeptors 2A

G-Protein-gekoppelte Rezeptoren stellen eine wichtige Proteingruppe pharmakologischer Zielstrukturen dar. Über den G-Protein-gekoppelten Somatostatin-Rezeptor 2A wird die Wirkung von Octreotid vermittelt, das bei Akromegalie und anderen neuroendokrinen Tumoren eingesetzt wird. Gegenwärtig befindet sich die Nachfolgesubstanz Pasireotid (SOM230) in klinischer Evaluation, welches als Pan-Somatostatin-Analogon hohe Affinitäten für vier der fünf Somatostatin-Rezeptoren besitzt und deswegen die klinische Einsetzbarkeit erweitert. G-Protein-gekoppelte Rezeptoren und ihre Signalweiterleitungen unterliegen vielfältigen Regulationsmechanismen wie der Phosphorylierung, Desensitisierung und Internalisierung. Für den in dieser Arbeit genutzten Ratten-Somatostatin-Rezeptor 2A (rsst2A) ist bekannt, dass er nach Agonist-Bindung an einem Cluster von vier Carboxyl-terminalen Threoninen, dem 353TTETQRT359-Motiv, phosphoryliert wird, woraufhin der Rezeptor internalisiert. Die Agonist-induzierte Phosphorylierung wird schnell dephosphoryliert und der Rezeptor kehrt binnen einer Stunde voll funktionsfähig an die Membran zurück. In der vorliegenden Arbeit sollten die molekularen Mechanismen der Regulation des Ratten-Somatostatin-Rezeptors 2A (rsst2A) sowie Agonist-spezifische Effekte, insbesondere im Vergleich von Octreotid und SOM230, untersucht werden

A Transplantable Phosphorylation Probe for Direct Assessment of G Protein-Coupled Receptor Activation

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 sst2 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 sst3 and sst5 receptors but was only a partial agonist at the sst2 receptor. Octreotide exhibited potent agonistic properties at the sst2 receptor but produced very little sst5 receptor activation. Like octreotide, somatoprim was a full agonist at the sst2 receptor. Unlike octreotide, somatoprim was also a potent agonist at the sst5 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

Agonist-selective internalization of human somatostatin receptors.

<p>HEK293 cells stably expressing sst₂, sst₃ or sst₅ receptors were treated with either 1 µM SS-14, octreotide, pasireotide or somatoprim for 0 or 30 min. Cells were fixed, immunoflurescently stained with anti-sst₂ {UMB-1}, anti-sst₃ {UMB-5} or anti-sst₅ {UMB-4} antibodies, and examined by confocal microscopy. Shown are representative images from one of three independent experiments performed in duplicate. <i>Scale bar</i>, 20 µm.</p

Ligand binding properties of sst₅-sst₂CT receptors.

<p>Ligand binding assays were carried out as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039458#s2" target="_blank">Materials and Methods</a>”. The half-maximal inhibitory concentrations (IC₅₀) were analyzed by nonlinear regression curve fitting using the computer program GraphPad Prism. Data are presented as the mean of three independent experiments performed in triplicate.</p

Agonist-stimulated ³⁵S-GTPγS binding.

<p>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-sst_2ACT or the rat sst₃-sst_2ACT receptor. Values represent means of triplicate determinations. SE values were smaller than 15%. Three replicate experiments gave similar results.</p

Agonist-selective phosphorylation of the human sst₂ receptor.

<p>(<i>Top</i>) Schematic representation of the human sst₂ receptor indicating the phosphate acceptor sites S341/343, T353/354 and T356/359 within its carboxyl-terminal tail. (<i>Bottom</i>) 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).</p

Agonist-selective phosphorylation of the sst₃-sst₂CT chimera.

<p>(<i>Top</i>) Schematic representation of the rat sst₃-sst₂CT chimera indicating the phosphate acceptor sites S341/343, T353/354 and T356/359 within the carboxyl-terminal tail. (<i>Bottom</i>) HEK293 cells stably expressing the rat sst₃-sst_2ACT receptor were either not exposed or exposed to concentrations of 10⁻¹² to 10⁻⁵ M SS-14, octreotide, pasireotide or somatoprim for 5 min. The levels of phosphorylated rsst₃-sst_2ACT receptors were then determined using anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} or 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).</p

Agonist-selective phosphorylation of the human sst₅ and sst₅-sst₂CT chimera.

<p>(<i>A</i>,<i>Top panel</i>) Schematic representation of the sst₅-sst₂CT receptor indicating the phosphate acceptor sites S341/343, T353/354 and T356/359 within the carboxyl-terminal tail. (<i>A</i>, <i>Bottom</i>) HEK293 cells stably expressing the sst₅-sst_2ACT 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 sst₅-sst_2ACT receptors were then determined using the phosphosite-specific antibodies anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} and anti-pT356/pT359 {0522}. (<i>B</i>,<i>Top panel</i>) Schematic representation of the human sst₅ receptor indicating the phosphate acceptor site T333 within its carboxyl-terminal tail. (<i>B</i>, <i>Bottom</i>) HEK293 cells stably expressing the human sst₅ receptors 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 sst₅ receptors were then determined using the phosphosite-specific antibodies anti-pT333 {3567}. 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).</p