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

Background: Among the five somatostatin receptors (sst1-sst5), the sst3 receptor displays a distinct pharmacological profile. Like sst2, the sst3 receptor efficiently internalizes radiolabeled somatostatin analogs. Unlike sst2, however, internalized sst3 receptors are rapidly transferred to lysosomes for degradation. Apart from this, very little is known about the clinical relevance of the sst3 receptor, which may in part be due to the lack of specific monoclonal sst3 antibodies. Methods: Here, we have extensively characterized the novel rabbit monoclonal anti-human sst3 antibody UMB-5 using transfected cells and receptor-expressing tissues. UMB-5 was then subjected to immunohistochemical staining of a series of 190 formalin-fixed, paraffin-embedded normal and neoplastic human tissues. Results: Specificity of UMB-5 was demonstrated by detection of a broad band migrating at a molecular weight of 70,000–85,000 in immunoblots from human pituitary. After enzymatic deglycosylation, the size of this band decreased to a molecular weight of 45,000. Tissue immunostaining was completely abolished by pre-adsorption of UMB-5 with its immunizing peptide. In addition, UMB-5 detected distinct cell populations in human tissues like pancreatic islands, anterior pituitary, adrenal cortex, adrenal medulla, and enteric ganglia, similar to that seen with a rabbit polyclonal antibody generated against a different carboxyl-terminal epitope of the sst3 receptor. In a comparative immunohistochemical study, UMB-5 yielded predominant plasma membrane staining in the majority of pituitary adenomas, pheochromocytomas, and a subset of neuroendocrine tumors. The sst3 receptor was also present in many glioblastomas, pancreatic, breast, cervix, and ovarian carcinomas. Conclusion: The rabbit monoclonal antibody UMB-5 may prove of great value in the identification of sst3-expressing tumors during routine histopathological examinations. Given its unique trafficking properties, these tumors may be potential candidates for sst3-directed receptor radiotherapy

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

Strukturelle Hirnveränderungen bei Erkrankungen des olfaktorischen Systems

Der Somatostatin-Rezeptor 2A, welcher bei der Behandlung von neuroendokrinen Tumoren mit Octreotid, eine wesentliche Rolle spielt gehört zur Gruppe der G-Protein-gekoppelten Rezeptoren. In der vorliegenden Arbeit wurden die strukturellen Ursachen der Unterschiede in der Rezeptorlokalisation von humanem und Ratten-sst2A nach der Behandlung mit dem neuen Pan-Somatostatin-Analogon Pasireotid (SOM230) untersucht. Es konnte gezeigt werden, dass der Austausch von einer von 4 Aminosäuren im rsst2A gegen sein humanes Gegenstück ausreichte, um eine Internalisierung des Rezeptors nach Stimulation mit SOM230 zu induzieren. Es ist wahrscheinlich, dass diese 4 Aminosäuren zu einer Stabilisierung einer Rezeptorkonformation beitragen, die für die Internalisierung des Rezeptors von entscheidender Bedeutung ist. In einem Radioligand-Bindungsassay ergaben sich keine Unterschiede hinsichtlich der Bindungsaffinität für SS-14, Octreotid und SOM230 zwischen den untersuchten Rezeptoren. Für den hsst2A konnte des weiteren ein schnelleres Rezeptor-Recycling nach SOM230 gegenüber der Behandlung mit Octreotid oder SS-14 beobachtet werden. Außerdem rekrutierte der Rezeptor mit SOM230 im Gegensatz zu SS-14 kein -Arrestin2

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

Hierarchical Organization of Multi-Site Phosphorylation at the CXCR4 C Terminus

<div><p>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.</p></div

Long-lasting phosphorylation at S346/347.

<p><b>A–E</b>, Stably CXCR4-transfected HEK293 cells were stimulated with CXCL12 for 30 min, washed with acidic buffer, and incubated with AMD3100-supplemented medium for the indicated intervals. Aliquots of the lysates were detected in five immunoblots using the indicated antibodies. The HA-tag and the endogenous transferrin receptor (TFR) were detected as loading controls. <b>A</b>,<b>B</b>, Slow recovery of the anti-S341-S352 signal corresponds to long-lasting phosphorylation at S346/347. <b>C</b>,<b>D</b>, Phosphorylation at S338/339 and S324/325 is rapidly reversed after CXCL12 washout. <b>E</b>, Detection with anti-HA demonstrates similar CXCR4 levels in the different samples. <b>F–I</b>, Using densitometry, the signal ratio versus anti-HA was determined for the four phospho-sensitive antibodies. Non-stimulated samples were set as 100% in <i>F</i> and samples undergoing 30 min CXCL12 stimulation were set as 100% in <i>G–I</i>. While there is little dephosphorylation at S346/347 (F,G<b>)</b>, major dephosphorylation occurs already 15 min after CXCL12 washout at S338/339 and S324/324 (H,I). Data represent mean+SEM from 4–5 independent experiments. *: p<0.05; 1way ANOVA followed by Dunnett´s post-test vs. the 30 min CXCL12/0 min washout group.</p

Hierarchical phosphorylation of CXCR4 C-terminal sites.

<p>HEK293 cells were transiently transfected with N-terminal T7 epitope-tagged wildtype CXCR4 (T7-CXCR4) and C-terminal deletion mutants lacking the last 10 and 17 residues (T7-CXCR4-Δ10, T7-CXCR4-Δ17). <b>A–F</b>, Surface receptors were pulse-labeled with anti-T7 antibody in live cells before cells received vehicle (A–C) or CXCL12 (D–F) for 30 min. Cells were fixed and permeabilized before immunofluorescent detection of anti-T7. In the absence of CXCL12 all three receptors are targeted to the plasma membrane showing little internalization (A–C). CXCL12 causes strong internalization of T7-CXCR4 (D) but only weak internalization of the C-terminal truncated receptors (E,F). <b>G</b>, Western blot analyses of CXCL12-induced phosphorylation of T7-CXCR4, T7-CXCR4-Δ10, and T7-CXCR4-Δ17 (CXCR4, Δ10, Δ17). HEK293 cells were transiently transfected with the indicated constructs and harvested before or 15 min after CXCL12 stimulation. Aliquots of the lysates were dephosphorylated using Lambda-Protein Phosphatase (λ-PP). Samples were loaded in four SDS gels, blotted, and immunodetected using anti-S341-S352, anti-pS346/347, anti-pS338/339, and anti-pS324/325. <b>T7-CXCR4:</b> In samples from non-stimulated transfectants λ-PP-treatment slightly enhances the signal of anti-S341-S352 and almost eliminates signals of the phospho-selective antibodies, indicating some constitutive CXCR4 phosphorylation. CXCL12 treatment causes a strong signal reduction of anti-S341-S352 and a strong signal increase of anti-pS346/347, anti-pS338/339, and anti-pS324/325. λ-PP-treatment of stimulated lysates fully restores the anti-S341-S352 signal and eliminates the signal of the phospho-selective antibodies. <b>T7-CXCR4-</b>Δ<b>10:</b> Deletion of the epitope eliminates specific signals of anti-S341-S352 and anti-pS346/347. There is virtually no constitutive phosphorylation at S338/339 and S324/325 in non-stimulated lysates. CXCL12-induced phosphorylation is not detected at S338/339 and strongly reduced at pS324/325 as compared with T7-CXCR4. <b>T7-CXCR4-</b>Δ<b>17:</b> Deletion of CXCR4 residues 336–352 eliminates specific signals of anti-S341-S352, anti-pS346/347, and anti-pS338/339. The mutant shows virtually no constitutive and no CXCL12-induced phosphorylation at S324/325. <b>Anti-T7-tag:</b> Stripping and detection with the anti-tag antibody reveals similar CXCR4 expression levels in all samples. <b>Anti-TFR:</b> Detection of endogenous transferrin receptor (TFR) in a stripped blot demonstrates equal loading. <b>G</b>, Results are representative for three independent experiments with similar results.</p

Specificity of phospho-sensitive anti-CXCR4 antibodies.

<p><b>A</b>, Schematic representation of the anti-pS324/325, anti-pS338/339, anti-pS346/347, and anti-S341-S352 (UMB-2) antibodies with their epitopes in the human CXCR4 receptor. <b>B–G</b>, HEK293 cells stably transfected with hemagglutinin (HA) epitope-tagged CXCR4 received CXCL12 (20 nM) or vehicle for 15 min and were lysed. An aliquot of the lysate from stimulated cells was dephosphorylated using Lambda-Protein Phosphatase (λ-PP). Samples were separated on 10% SDS polyacrylamide gels, blotted, and detected with anti-S341-S352 (B), anti-pS346/347 (C), anti-pS338/339 (D), anti-pS324/325 (E), anti-HA-tag (F), and anti-transferrin receptor (TFR) (G). <b>B</b>, CXCL12 stimulation causes a strong loss of anti-S341-S352 binding; dephosphorylation restores antibody binding. <b>C–E</b>, The phospho-selective antibodies recognize CXCR4 only in the non-dephosphorylated sample from stimulated cells. Anti-pS324/325 produces a non-specific band at approximately 70 kDa (arrowhead in E). <b>H</b>, HEK293 cells transiently transfected with a HA-CXCR4-ST/A mutant (all serines and threonines in the C-terminal domain were converted into alanines) were stimulated with CXCL12. Four aliquots were loaded in a Western blot and separately detected with the indicated antibodies. The phospho-selective antibodies do not detect the CXCL12-stimulated phosphorylation-deficient CXCR4 mutant (the arrow points to 47 kDa which corresponds to the size of CXCR4). <b>F</b>,<b>G</b>,<b>H</b>, Equal loading was controlled in stripped blots by detecting the HA-tag of recombinant expressed CXCR4 and by detecting endogenous TFR.</p

Figure 2

<p><b>Sequential phosphorylation at CXCR4 C-terminal sites. </b><b>A–F</b>, Stably CXCR4-transfected HEK293 cells were stimulated with 20 nM CXCL12 for 0, 2, 5, and 15 min. Aliquots of the lysates were detected in four immunoblots using the indicated antibodies. Blots were stripped and reprobed with anti-HA and anti-transferrin receptor (TFR) to control for equal loading. <b>A</b>,<b>B</b>, CXCL12 induces a rapid loss of the anti-S341-S352 signal and a concomitant immediate increase of the anti-pS346/347 signal. <b>C</b>, Maximal S338/339 phosphorylation requires 15 min CXCL12 stimulation. <b>D</b>, Demonstration of rapid CXCL12-promoted S324/325 phosphorylation. <b>E</b>–<b>I</b>, The signal ratio versus anti-HA was determined for the four anti-CXCR4 antibodies using densitometry. The ratio was set as 100% at 0 min for anti-S341-S352 (G) and at 15 min for the phospho-selective antibodies (G–I). Data represent mean+SEM from 4–5 independent experiments. <b>G</b>, Note complementarity of the anti-S341-S352 and anti-pS346/347 time courses. <b>H</b>,<b>I</b>, Phosphorylation occurs faster at S346/347 than at S338/339 and S324/325. *: p<0.05; 1way ANOVA and Bonferronís multiple comparison post-test for selected groups.</p