Arrestins are cytosolic proteins that regulate G protein-coupled receptor (GPCR) desensitization, internalization, trafficking, and signaling1,2. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets them for internalization via clathrin-coated pits3,4. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G protein effectors into GPCR-based ‘signalsomes’5,6. While the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify divergent arrestin functions is not understood. Using a panel of intramolecular FlAsH-BRET reporters7 to monitor conformational changes in arrestin3, we show here that GPCRs impose distinctive arrestin ‘conformational signatures’ that reflect the stability of the receptor-arrestin complex and role of arrestin3 in activating or dampening downstream signaling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in arrestin3 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average arrestin3 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands8,9 and in identifying factors that dictate arrestin conformation and function.

Purpose: In humans, several distinctive cancers result from mutations that aberrantly activate hedgehog (HH) signal transduction. Here, we investigate the role of HH signaling in ovarian cancer. Experimental Design: We assessed the expression of different components of hedghehog pathway in primary tumor samples and cell lines. We used specific hedghehog pathway blocker to study the effect on clonal growth and proliferation of ovarian cancer cell both in vitro and in vivo. Results: We show that the up-regulation of several HH pathway components is a common feature of primary ovarian tumors and cell lines. However, expression of PATCHED1 (PTCH1), a direct transcriptional target of the HH pathway, is down-regulated in ovarian cancer in direct contrast to the expression observed in other adult solid tumors. Cyclopamine, a specific HH pathway inhibitor, inhibits the proliferation and clonal growth of ovarian tumor cells in vitro and arrests ovarian tumor growth in vivo. Expression of BMI-1, a polycomb gene, is down-regulated in ovarian cancer cells following cyclopamine treatment. Overexpression of PTCH1 phenocopied the effects of cyclopamine; it down-regulated BMI-1and reduced clonal growth in ovarian cancer cell lines. Furthermore, knocking down BMI-1 using small interfering RNA also inhibited the clonal growth of all the ovarian cancer cell lines tested. Conclusions: In brief, the constitutive low-level expression of PTCH1 contributes to proliferation and clonal growth of ovarian cancer cells by an aberrant HH signal. Because the HH pathway can be inhibited by specific inhibitors, these findings point toward possible new treatments to inhibit ovarian cancer growth.

Esophageal squamous cell carcinoma (ESCC) is a common subtype of esophageal cancer that is particularly prevalent in East Asian countries. Our previous expression profile analysis showed that the gene encoding protein tyrosine kinase 7 (PTK7) is upregulated in ESCC tissues. Here, we aimed to validate PTK7 as a prognostic factor and a candidate target for molecular treatment of ESCC. Both RT-PCR and Western blot analysis of tissues from ESCC patients revealed that PTK7 was significantly upregulated in tumor tissue samples of ESCC. Immunohistochemical staining of PTK7 showed that increased expression of PTK7 was inversely correlated with overall survival (P = 0.021). In vitro knockdown of PTK7 inhibited proliferation, survival, wound healing, and invasion of ESCC cells. In addition, PTK7 knockdown decreased phosphorylation of Akt, Erk, and focal adhesion kinase (FAK), important determinants of cell proliferation, survival, and migration. Therefore, our findings suggest that PTK7 has potential as a prognostic marker for ESCC and might also be a candidate for targeted therapy in the treatment of ESCC. (Cancer Sci 2013; 104: 1120-1126 E sophageal cancer is one of the most devastating malignancies. Despite recent developments in its management, advanced esophageal cancer is associated with high rates of local recurrence and distant metastasis.(1,2) Although there has been improvement in clinical outcome through multidisciplinary treatment strategies, (3)(4)(5) such as total mediastinal lymph node dissection, (6) and new diagnostic modalities such as positron emission tomography, (7) these developments have not proven satisfactory in preventing the high recurrence rates. Currently, the prognosis is only promising if the tumor is detected early and resected completely.Esophageal squamous cell carcinoma (ESCC) is the most common histologic subtype of esophageal cancer in East Asian countries.(8) It has a unique profile of clinical and anatomical characteristics that is distinct from those of esophageal adenocarcinoma, which is more prevalent in North America, UK, and Australia.(8) To develop prognostic diagnosis and effective therapeutics for ESCC, the identification of biomarkers is of vital importance. In a gene expression profiling analysis to pursue biomarkers, we identified protein tyrosine kinase 7 (PTK7; also known as colon carcinoma kinase-4 or CCK-4), as a candidate biomarker for ESCC. (9) Protein tyrosine kinase 7 is a receptor tyrosine kinase-like molecule containing an extracellular domain with seven immunoglobulin-like loops, a transmembrane domain, and a defective tyrosine kinase domain that resembles a catalytic domain but lacks catalytic activity.(10-12) Mice expressing a truncated form of PTK7 protein die perinatally, revealing a defect in neural tube closure and stereociliary bundle orientation. These findings implicate PTK7 as a regulator of planar cell polarity (PCP).(13) It has been shown that PTK7 recruits RACK1, which affects Dsh recruitment by interaction with PKCd1. (14) Interaction between PTK7...

LXXLL/leucine zipper-containing alternative reading frame (ARF)-binding protein (LZAP) was recently shown to function as a tumor suppressor through inhibition of the NF-B signaling pathway. LZAP is also known as a negative regulator of cell invasion, and its expression was demonstrated to be reduced in several tumor tissues. However, the molecular mechanism of the negative effect of LZAP on cell invasion is unclear. In this study, we identify NLBP as a novel LZAP-binding protein using tandem affinity purification. We demonstrate the negative effects of NLBP on cell invasion and the NF-B signaling pathway. NLBP expression was not detected in hepatocellular carcinoma cells with strong invasive activity, whereas its expression was detected in a hepatocellular carcinoma cell line with no invasive activity. We also demonstrate that these two proteins mutually affect the stability of each other by inhibiting ubiquitination of the other protein. Based on these results, we suggest that NLBP may act as a novel tumor suppressor by inhibiting cell invasion, blocking NF-B signaling, and increasing stability of the LZAP protein.Multistep progressions are involved in tumor formation. Many proteins, including tumor suppressor genes and oncogenes, affect the tumorigenesis of normal cells (1). One of the most important factors for tumor formation is mutations in tumor suppressors or oncogenes, which affect expression levels and/or protein activity (2). Therefore, maintaining oncogenes and tumor suppressor genes in a wild type state is crucial to blocking tumorigenesis. LZAP 3 (also known as Cdk5rap3 or C53 protein) was originally identified as an ARF-binding protein and was found to have numerous functions as a tumor suppressor. These include activation of p53, induction of apoptosis mediated by genotoxic agents leading to inhibition of tumor cell growth, and negative regulation of the checkpoint response by antagonizing checkpoint kinases to promote cyclin-dependent kinase 1 activation (3-6). Recently, it was shown that upon depletion of LZAP expression, NF-B-dependent MMP-9 expression, and cellular invasion were increased (7). In addition, LZAP protein was reduced in ϳ30% of human head and neck squamous cell carcinomas (7).Although LZAP has been functionally shown to be a tumor suppressor, the molecular mechanism of how LZAP blocks tumorigenesis is not yet clear. To gain further insight into the molecular mechanism of LZAP functions on tumorigenesis, we performed biochemical tandem repeat affinity purification and identified KIAA0776 as a novel LZAP-binding protein (NLBP). NLBP encodes an uncharacterized protein of 794 amino acids and does not contain any conserved domains. In this study, we show that NLBP is a novel LZAP-binding protein that may function as a tumor suppressor by inhibiting cell invasion, blocking NF-B signaling, and increasing the stability of the LZAP protein.

Endothelial cell proliferation and migration is essential to angiogenesis. Typically, proliferation and chemotaxis of endothelial cells is driven by growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF activates phospholipases (PLCs) – specifically PLCγ1 – that are important for tubulogenesis, differentiation and DNA synthesis. However, we show here that VEGF, specifically through VEGFR2, induces phosphorylation of two serine residues on PLCβ3, and this was confirmed in an ex vivo embryoid body model. Knockdown of PLCβ3 in HUVEC cells affects IP3 production, actin reorganization, migration and proliferation; whereas migration is inhibited, proliferation is enhanced. Our data suggest that enhanced proliferation is precipitated by an accelerated cell cycle, and decreased migration by an inability to activate CDC42. Given that PLCβ3 is typically known as an effector of heterotrimeric G-proteins, our data demonstrate a unique crosstalk between the G-protein and receptor tyrosine kinase (RTK) axes and reveal a novel molecular mechanism of VEGF signaling and, thus, angiogenesis.