Falko Nagel

Reassessment of sst3 Somatostatin Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-5

Objective: The frequent overexpression of somatostatin receptors (sst) in neuroendocrine tumors provides the molecular basis for the diagnostic and therapeutic application of stable somatostatin analogs. Whereas octreotide acts mainly via the sst2 receptor, the novel pan-somatostatin analog pasireotide exhibits particular high affinity for the sst5 receptor. To determine whether a patient is a candidate for octreotide or pasireotide therapy, it is important to evaluate the somatostatin receptor status. However, so far highly specific rabbit monoclonal antibodies have been developed for the sst2 receptor only (clone UMB-1). Methods: Here, we have extensively characterized a novel rabbit monoclonal antibody for the human sst5 receptor (clone UMB-4). In a comparative immunohistochemical study, the expression of sst5 and sst2 receptors was assessed using UMB-4 and UMB-1, respectively. Results: Western blot experiments unequivocally demonstrated that UMB-4 selectively detected its cognate sst5 receptor and did not cross-react with other proteins present in crude tissue homogenates. UMB-4 yielded a highly effective immunostaining of distinct cell populations in formalin-fixed, paraffin-embedded human tissues with a predominance of plasma membrane staining. In the pituitary, sst5 was present on all growth hormone (GH)- and adrenocorticotropin hormone (ACTH)-producing cells whereas sst2 was only observed on a subpopulation of GH-positive cells. Consequently, sst5 was detectable on the majority of GH and ACTH adenomas. In contrast, sst2 was only seen on GH but not on ACTH adenomas. Conclusions:The rabbit monoclonal antibodies UMB-4 and UMB-1 will facilitate the assessment of the somatostatin receptor status of human tumors during routine histopathological examinations.

Reassessment of CXCR4 Chemokine Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-2

Background The CXCR4 chemokine receptor regulates migration and homing of cancer cells to specific metastatic sites. Determination of the CXCR4 receptor status will provide predictive information for disease prognosis and possible therapeutic intervention. However, previous attempts to localize CXCR4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have produced predominant nuclear and occasional cytoplasmic staining but did not result in the identification of bona fide cell surface receptors. Methodology/Principal Findings In the present study, we extensively characterized the novel rabbit monoclonal anti-CXCR4 antibody (clone UMB-2) using transfected cells and tissues from CXCR4-deficient mice. Specificity of UMB-2 was demonstrated by cell surface staining of CXCR4-transfected cells; translocation of CXCR4 immunostaining after agonist exposure; detection of a broad band migrating at M r 38,000–43,000 in Western blots of homogenates from CXCR4-expressing cells; selective detection of the receptor in tissues from CXCR4+/+ but not from CXCR4−/− mice; and abolition of tissue immunostaining by preadsorption of UMB-2 with its immunizing peptide. In formalin-fixed, paraffin-embedded human tumor tissues, UMB-2 yielded highly effective plasma membrane staining of a subpopulation of tumor cells, which were often heterogeneously distributed throughout the tumor. A comparative analysis of the mouse monoclonal antibody 12G5 and other frequently used commercially available antibodies revealed that none of these was able to detect CXCR4 under otherwise identical conditions. Conclusions/Significance Thus, the rabbit monoclonal antibody UMB-2 may prove of great value in the assessment of the CXCR4 receptor status in a variety of human tumors during routine histopathological examination.

Hierarchical organization of multi-site phosphorylation at the CXCR4 C terminus.

The chemokine receptor CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. Upon stimulation by the endogenous ligand CXCL12, CXCR4 becomes phosphorylated at multiple sites in its C-terminal domain. Mutations in the CXCR4 gene affecting C-terminal phosphorylation sites are a hallmark of WHIM syndrome, a genetic disorder characterized by a gain-of-CXCR4-function. To better understand how multi-site phosphorylation of CXCR4 is organized and how perturbed phosphorylation might affect CXCR4 function, we developed novel phosphosite-specific CXCR4 antibodies and studied the differential regulation and interaction of three C-terminal phosphorylation sites in human embryonic kidney cells (HEK293). CXCL12 promoted a robust phosphorylation at S346/347 which preceded phosphorylation at S324/325 and S338/339. After CXCL12 washout, the phosphosites S338/339 and S324/325 were rapidly dephosphorylated whereas phosphorylation at S346/347 was long-lasting. CXCL12-induced phosphorylation at S346/347 was staurosporine-insensitive and mediated by GRK2/3. WHIM syndrome-associated CXCR4 truncation mutants lacking the S346/347 phosphosite and the recently identified E343K WHIM mutant displayed strongly impaired phosphorylation at S324/325 and S338/339 as well as reduced CXCL12-induced receptor internalization. Relevance of the S346-S348 site was confirmed by a S346-348A mutant showing strongly impaired CXCL12-promoted phosphorylation at S324/325 and S338/339, defective internalization, gain of calcium mobilization, and reduced desensitization. Thus, the triple serine motif S346-S348 contains a major initial CXCR4 phosphorylation site and is required for efficient subsequent multi-site phosphorylation and receptor regulation. Hierarchical organization of CXCR4 phosphorylation explains why small deletions at the extreme CXCR4 C terminus typically associated with WHIM syndrome severely alter CXCR4 function.

Structural Determinants of Agonist-Selective Signaling at the sst2A Somatostatin Receptor

The clinically used somatostatin (SS-14) analogs octreotide and pasireotide (SOM230) stimulate distinct species-specific patterns of sst(2A) somatostatin receptor phosphorylation and internalization. Like SS-14, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues, namely S341, S343, T353, T354, T356, and T359, which in turn leads to a robust endocytosis of both rat and human sst(2A) receptors. Unlike SS-14, pasireotide fails to induce any substantial phosphorylation or internalization of the rat sst(2A) receptor. Nevertheless, pasireotide is able to stimulate a selective phosphorylation of S341 and S343 of the human sst(2A) receptor followed by a clearly detectable receptor sequestration. Here, we show that transplantation of amino acids 1-180 of the human sst(2A) receptor to the rat sst(2A) receptor facilitates pasireotide-induced internalization. Conversely, construction of a rat-human sst(2A) chimera conferred resistance to pasireotide-induced internalization. We then created a series of site-directed mutants leading to the identification of amino acids 27, 30, 163, and 164 that when exchanged to their human counterparts facilitated pasireotide-driven S341/S343 phosphorylation and internalization of the rat sst(2A) receptor. Exchange of these amino acids to their rat counterparts completely blocked the pasireotide-mediated internalization of the human sst(2A) receptor. Notably, octreotide and SS-14 stimulated a full phosphorylation and internalization of all mutant sst(2A) receptors tested. Together, these findings suggest that pasireotide activates the sst(2A) receptor via a molecular switch that is structurally and functionally distinct from that turned on during octreotide-driven sst(2A) activation.

Phosphorylation of Threonine 333 Regulates Trafficking of the Human sst5 Somatostatin Receptor

The frequent overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of novel multireceptor somatostatin analogs. Although the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (T333) and 347 (T347), which enabled us to selectively detect either the T333-phosphorylated or the T347-phosphorylated form of sst5. We show that agonist-mediated phosphorylation occurs at T333, whereas T347 is constitutively phosphorylated in the absence of agonist. We further demonstrate that the multireceptor somatostatin analog pasireotide and the sst5-selective ligand L-817,818 but not octreotide or KE108 were able to promote a detectable T333 phosphorylation. Interestingly, BIM-23268 was the only sst5 agonist that was able to stimulate T333 phosphorylation to the same extent as natural somatostatin. Agonist-induced T333 phosphorylation was dose-dependent and selectively mediated by G protein-coupled receptor kinase 2. Similar to that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. However, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were rapidly completed within minutes. We also identify protein phosphatase 1γ as G protein-coupled receptor phosphatase for the sst5 receptor. Together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal T333. In addition, we identify G protein-coupled receptor kinase 2-mediated phosphorylation and protein phosphatase 1γ-mediated dephosphorylation of T333 as key regulators of rapid internalization and recycling of the human sst5 receptor.

Reevaluation of sst1 somatostatin receptor expression in human normal and neoplastic tissues using the novel rabbit monoclonal antibody UMB-7

BACKGROUND The somatostatin receptor 1 (sst1) is widely distributed throughout the body and is also present in neoplastic tissues. However, little is known about its precise tissue distribution, regulation and function, which may in part be due to the lack of specific monoclonal anti-sst1 antibodies. METHODS We have characterized the novel rabbit monoclonal anti-human sst1 antibody UMB-7 using sst1-expressing cells and human pituitary samples. The antibody was then used for immunohistochemical staining of a large panel of formalin-fixed, paraffin-embedded human tissues. RESULTS Western blot analyses of BON-1 cells and human pituitary revealed a broad band migrating at a molecular weight of 45,000-60,000. After enzymatic deglycosylation the size of this band decreased to a molecular weight of 45,000. UMB-7 yielded an efficient immunostaining of distinct cell populations in the human tissue samples with a predominance of plasma membrane staining, which was completely abolished by preadsorption of UMB-7 with its immunizing peptide. The sst1 receptor was detected in anterior pituitary, pancreatic islets, distal tubules, enteric ganglion cells and nerve fibers, chief cells of the gastric mucosa, macrophages and mast cells. In addition, sst1 was observed in pituitary adenomas, gastrointestinal neuroendocrine tumors and pheochromocytoma as well as in pancreatic adenocarcinomas, gastric carcinomas, urinary bladder carcinomas and sarcomas. CONCLUSIONS UMB-7 may prove of great value in the identification of sst1-expressing tumors during routine histopathological examinations. This may open up new routes for diagnostic and therapeutic intervention.

UMB-3, a novel rabbit monoclonal antibody, for assessing μ-opioid receptor expression in mouse, rat and human formalin-fixed and paraffin-embedded tissues.

BACKGROUND The immunohistochemical localization of the μ-opioid receptor (MOR, MOP) has been studied in detail in mouse and rat brain using a variety of polyclonal antibodies. However, biochemical analysis of the MOR signaling complex in vivo has been hampered by the lack of suitable monoclonal antibodies for efficient immunoprecipitation of the receptor protein from native sources. Moreover, previous immunohistochemical investigations were restricted to frozen sections from perfusion-fixed rodent brain, largely due to the limited availability of MOR antibodies that effectively stain paraffin-embedded tissues. METHODS Here, we extensively characterized the novel rabbit monoclonal anti-MOR antibody UMB-3 using transfected cells and MOR-deficient mice. UMB-3 was also subjected to a comparative immunohistochemical study of formalin-fixed, paraffin-embedded mouse and rat organ samples as well as human normal and neoplastic tissues. RESULTS Specificity of UMB-3 was demonstrated by detection of a broad band migrating at M(r) 70,000-80,000 in immunoprecipitates from crude brain homogenates of MOR+/+ mice but not of MOR⁻/⁻ mice; cell surface staining of MOR-transfected cells; translocation of MOR receptor immunostaining after agonist exposure; distinct immunostaining of neuronal cell bodies and fibers in MOR-expressing brain regions; absence of staining in MOR-deficient mice; and abolition of tissue immunostaining by preadsorption of UMB-3 with its immunizing peptide. CONCLUSIONS The rabbit monoclonal antibody UMB-3 is an excellent tool for immunoprecipitation of MOR from native sources as well as for immunohistochemical staining of MOR in paraffin-embedded tissue samples of rodent and human origin.

Carboxyl-terminal multi-site phosphorylation regulates internalization and desensitization of the human sst2 somatostatin receptor ☆

The somatostatin receptor 2 (sst2) is the pharmacological target of somatostatin analogs that are widely used in the diagnosis and treatment of human neuroendocrine tumors. We have recently shown that the stable somatostatin analogs octreotide and pasireotide (SOM230) stimulate distinct patterns of sst2 receptor phosphorylation and internalization. Like somatostatin, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust receptor endocytosis. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor followed by a partial receptor internalization. Here, we show that exchange of S341 and S343 by alanine is sufficient to block pasireotide-driven internalization, whereas mutation of T353, T354, T356 and T359 to alanine is required to strongly inhibited both octreotide- and somatostatin-induced internalization. Yet, combined mutation of T353, T354, T356 and T359 is not sufficient to prevent somatostatin-driven β-arrestin mobilization and receptor desensitization. Replacement of all fourteen carboxyl-terminal serine and threonine residues by alanine completely abrogates sst2 receptor internalization and β-arrestin mobilization in HEK293 cells. Together, our findings demonstrate for the first time that agonist-selective sst2 receptor internalization is regulated by multi-site phosphorylation of its carboxyl-terminal tail.

Identification of Phosphorylation Sites Regulating sst3 Somatostatin Receptor Trafficking

The human somatostatin receptor 3 (sst3) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst3 receptor is unique among ssts in that it exhibits a very long intracellular C-terminal tail containing a huge number of potential phosphate acceptor sites. Consequently, our knowledge about the functional role of the C-terminal tail in sst3 receptor regulation is very limited. Here, we have generated a series of phosphorylation-deficient mutants that enabled us to determine crucial sites for its agonist-induced β-arrestin mobilization, internalization, and down-regulation. Based on this information, we generated phosphosite-specific antibodies for C-terminal Ser(337)/Thr(341), Thr(348), and Ser(361) that enabled us to investigate the temporal patterns of sst3 phosphorylation and dephosphorylation. We found that the endogenous ligand somatostatin induced a rapid and robust phosphorylation that was completely blocked by the sst3 antagonist NVP-ACQ090. The stable somatostatin analogs pasireotide and octreotide promoted clearly less phosphorylation compared with somatostatin. We also show that sst3 phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst3 receptor occurred at a considerable slower rate. In addition, we also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1α and 1β as key regulators of sst3 phosphorylation and dephosphorylation, respectively. Thus, we here define the C-terminal phosphorylation motif of the human sst3 receptor that regulates its agonist-promoted phosphorylation, β-arrestin recruitment, and internalization of this clinically relevant receptor.

Carboxyl-Terminal Receptor Domains Control the Differential Dephosphorylation of Somatostatin Receptors by Protein Phosphatase 1 Isoforms

We have recently identified protein phosphatase 1β (PP1β) as G protein-coupled receptor (GPCR) phosphatase for the sst2 somatostatin receptor using siRNA knockdown screening. By contrast, for the sst5 somatostatin receptor we identified protein phosphatase 1γ (PP1γ) as GPCR phosphatase using the same approach. We have also shown that sst2 and sst5 receptors differ substantially in the temporal dynamics of their dephosphorylation and trafficking patterns. Whereas dephosphorylation and recycling of the sst2 receptor requires extended time periods of ∼30 min, dephosphorylation and recycling of the sst5 receptor is completed in less than 10 min. Here, we examined which receptor domains determine the selection of phosphatases for receptor dephosphorylation. We found that generation of tail-swap mutants between sst2 and sst5 was required and sufficient to reverse the patterns of dephosphorylation and trafficking of these two receptors. In fact, siRNA knockdown confirmed that the sst5 receptor carrying the sst2 tail is predominantly dephosphorylated by PP1β, whereas the sst2 receptor carrying the sst5 tail is predominantly dephosphorylated by PP1γ. Thus, the GPCR phosphatase responsible for dephosphorylation of individual somatostatin receptor subtypes is primarily determined by their different carboxyl-terminal receptor domains. This phosphatase specificity has in turn profound consequences for the dephosphorylation dynamics and trafficking patterns of GPCRs.