The atomic-level mechanisms by which G protein-coupled receptors (GPCRs) transmit extracellular ligand binding events through their transmembrane helices to activate intracellular G proteins remain unclear. Using a comprehensive library of mutations covering all 352 residues of the GPCR CXC chemokine receptor 4 (CXCR4), we identified 41 amino acids that are required for signaling induced by the chemokine ligand CXCL12 (stromal cell-derived factor 1). CXCR4 variants with each of these mutations do not signal properly but remain folded, based on receptor surface trafficking, reactivity to conformationally sensitive monoclonal antibodies, and ligand binding. When visualized on the structure of CXCR4, the majority of these residues form a continuous intramolecular signaling chain through the transmembrane helices; this chain connects chemokine binding residues on the extracellular side of CXCR4 to G proteincoupling residues on its intracellular side. Integrated into a cohesive model of signal transmission, these CXCR4 residues cluster into five functional groups that mediate (i) chemokine engagement, (ii) signal initiation, (iii) signal propagation, (iv) microswitch activation, and (v) G protein coupling. Propagation of the signal passes through a "hydrophobic bridge" on helix VI that coordinates with nearly every known GPCR signaling motif. Our results agree with known conserved mechanisms of GPCR activation and significantly expand on understanding the structural principles of CXCR4 signaling.T he CXC chemokine receptor 4 (CXCR4) belongs to the G protein-coupled receptor (GPCR) superfamily of proteins, the largest class of integral membrane proteins encoded in the human genome, comprising greater than 30% of current drug targets. Deregulation of CXCR4 expression in multiple human cancers, its role in hematopoietic stem cell migration, and the utilization of CXCR4 by HIV-1 for T-cell entry, make this receptor an increasingly important therapeutic target (1). One FDA-approved drug against CXCR4 is currently on the market (Mozobil, for hematopoietic stem cell mobilization), and multiple additional drugs against this target are in development for oncology and other indications (2).The crystal structures of class A GPCR superfamily members in their active and inactive conformations (reviewed in refs. 3 and 4) provide unprecedented insight into the structural basis of ligand binding, G protein coupling, and activation of GPCRs via rearrangements of transmembrane (TM) helices. GPCR helices V and VI in particular, and in some cases III and VII, are known to undergo significant conformational changes upon activation (5-7). However, static images alone have not been able to explain the residue-level mechanisms underlying the dynamic helical shifts that mediate GPCR signal transduction. Additionally, only inactive state structures have been solved for CXCR4 and most other GPCRs (8,9). Over the last two decades, extensive mutagenesis studies of GPCRs in general [collectively describing >8,000 mutations (gpcrdb.org)] and of CX...

In mammalian oocytes, meiosis arrests at prophase I. Meiotic resumption requires activation of Maturation-Promoting Factor (MPF), comprised of a catalytic Cyclin-dependent kinase-1 (Cdk1) and a regulatory subunit cyclin B and results in germinal vesicle breakdown (GVBD). Cyclic AMP (cAMP)-mediated Protein Kinase A (PKA) activity sustains prophase arrest by inhibiting Cdk1. However, the link between PKA activity and MPF inhibition remains unclear. Cdc25 phosphatases can activate Cdks by removing inhibitory phosphates from Cdks. Thus one method for sustaining prophase arrest could be inhibition of the activity of the Cdc25 protein required for MPF activation. Indeed, studies in Xenopus identify Cdc25C as a target of PKA activity in meiosis. However, in mice, studies suggest that Cdc25B is the phosphatase essential for GVBD and, therefore, the likely target of PKA activity. To assess these questions, we targeted a potential PKA substrate, a highly conserved serine 321 residue of Cdc25B and evaluated the effect on oocyte maturation. A Cdc25B-Ser321Ala point mutant mRNA induces GVBD when injected into prophase-arrested oocytes more rapidly than wild type mRNA. Using fluorescently-tagged proteins we also determined that the mutant protein enters the nucleus more rapidly than its wildtype counterpart. These data suggest that phosphorylation of the Ser321 residue plays a key role in the negative regulation and localization of Cdc25B during prophase arrest. PKA also phosphorylates a wildtype Cdc25B protein but not a Ser321Ala mutant protein in vitro. Mutation of Ser321 in Cdc25B also affects its association with a sequestering protein, 14-3-3. Our studies suggest that Cdc25B is a direct target of PKA in prophase-arrested oocytes and that Cdc25B phosphorylation results in its inhibition and sequestration by the 14-3-3 protein.

Embryonic development of the pancreas is marked by an early phase of dramatic morphogenesis, in which pluripotent progenitor cells of the developing pancreatic epithelium give rise to the full array of mature exocrine and endocrine cell types. The genetic determinants of acinar and islet cell lineages are somewhat well defined; however, the molecular mechanisms directing ductal formation and differentiation remain to be elucidated. The complex ductal architecture of the pancreas is established by a reiterative program of progenitor cell expansion and migration known as branching morphogenesis, or tubulogenesis, which proceeds in mouse development concomitantly with peak Pdx1 transcription factor expression. We therefore evaluated Pdx1 expression with respect to lineage-specific markers in embryonic sections of the pancreas spanning this critical period of duct formation and discovered an unexpected population of nonislet Pdx1-positive cells displaying physical traits of branching. We then established a 3D cell culture model of branching morphogenesis using primary pancreatic duct cells and identified a transient surge of Pdx1 expression exclusive to branching cells. From these observations we propose that Pdx1 might be involved temporally in a program of gene expression sufficient to facilitate the biochemical and morphological changes necessary for branching morphogenesis. INTRODUCTIONThe primary function of the exocrine pancreas is to produce and secrete digestive enzymes for export to the small intestine. The collection and transport of these enzymes are facilitated by an intricate ductal network of branched epithelial tubules, such that enzymes secreted into smaller peripheral ducts ultimately feed into the larger main pancreatic duct, which in turn flows into the duodenum. This complex structure is established during embryonic development by a coordinated mechanism of progenitor cell proliferation and migration known as branching morphogenesis (Jorgensen et al., 2007). Some of the genetic and biochemical events directing this process are shared among the many organs served by ductal networks-such as the lung, breast, and kidney-and are also somewhat conserved across species (Lu and Werb, 2008). In general, branching morphogenesis is initiated by mesenchymal signaling to epithelial cell growth factor receptors, inducing multiple responses within those epithelial cells to 1) undertake temporary cytoskeletal reorganization that will facilitate cell motility, 2) inhibit proliferation, and 3) suppress the original mesenchymal growth factor signal (Metzger and Krasnow, 1999;Affolter et al., 2003). The specific mesenchymal growth factors and epithelial growth factor receptors, their cognate signaling pathways, and transcription factors involved in such processes contributing to branching morphogenesis in the pancreas are not well understood and remain to be elucidated.Pancreatic organogenesis is dependent on the homeodomain transcription factor Pdx1, as demonstrated by pancreatic agenesis observed in Pdx1-null mic...

Keratin 19 is a member of the cytokeratin family that is critical for maintenance of cellular architecture and organization, especially of epithelia. The pancreas has three distinct cell types, ductal, acinar, and islet, each with different functions. Embryologically, the pancreatic and duodenal homeobox 1 (PDX1) homeodomain protein is critical for the initiation of all pancreatic lineages; however, the later differentiation of the endocrine pancreas is uniquely dependent upon high PDX1 expression, whereas PDX1 is down-regulated in the ductal and acinar cell lineages. We find that this down-regulation may be required for normal ductal expression of cytokeratin K19. The K19 promoter-reporter gene assay demonstrates that ectopic PDX1 inhibits K19 reporter gene activity in primary pancreatic ductal cells. This is reinforced by our findings that retrovirally mediated stable transduction of PDX1 in primary pancreatic ductal cells suppresses K19 expression, and short interfering RNA to PDX1 in Min6 insulinoma cells results in the induction of normally undetectable K19. Complementary functional and biochemical approaches led to the unexpected finding that a multimeric complex of PDX1 and two members of the TALE homeodomain factor family, MEIS1a and PBX1b, regulates K19 gene transcription through a specific cis-regulatory element (؊341 to ؊325) upstream of the K19 transcription start site. These data suggest a unifying mechanism whereby PDX1, myeloid ecotropic viral insertion site (MEIS), and pre-B-cell leukemia transcription factor 1 (PBX) may regulate ductal and acinar lineage specification during pancreatic development. Specifically, concomitant PDX1 suppression and MEIS isoform expression result in proper ductal and acinar lineage specification. Furthermore, PDX1 may inhibit the ductal differentiation program in the pancreatic endocrine compartment, particularly beta cells.Cytokeratins are members of the intermediate filament family that are critical to the maintenance of cell and tissue integrity (1). In addition, they influence membrane and subcellular localization of proteins. The cytokeratin family consists of at least 20 members that are categorized as acidic type I, comprising keratins 9 -20, or basic type II, comprising keratins 1-8. Typically, cytokeratins form heterodimers between one type I member and one type II member. Keratin 19 (K19) 2 is expressed in epithelia and substitutes for keratin 18 in heterodimerization with keratin 8. Among the pancreatic cell types, K19 is specifically expressed in pancreatic ducts in vivo and in primary pancreatic ductal cells in vitro that our laboratory has successfully isolated and characterized (2). We have previously demonstrated that K19 expression is modulated by the KLF4 and Sp1 zinc-finger transcription factors, contributing to its tissue specificity in the pancreas (3). This activity is mediated by a short cis-regulatory region containing an overlapping binding site for KLF4 and Sp1 within the K19 promoter. KLF4 has a higher binding affinity and is the predominan...

BackgroundPancreas organogenesis is the result of well-orchestrated and balanced activities of transcription factors. The homeobox transcription factor PDX-1 plays a crucial role in the development and function of the pancreas, both in the maintenance of progenitor cells and in determination and maintenance of differentiated endocrine cells. However, the activity of homeobox transcription factors requires coordination with co-factors, such as PBX and MEIS proteins. PBX and MEIS proteins belong to the family of three amino acid loop extension (TALE) homeodomain proteins. In a previous study we found that PDX-1 negatively regulates the transcriptional activity of the ductal specific keratin 19 (Krt19). In this study, we investigate the role of different domains of PDX-1 and elucidate the functional interplay of PDX-1 and MEIS1 necessary for Krt19 regulation.Methodology/Principal FindingsHere, we demonstrate that PDX-1 exerts a dual manner of regulation of Krt19 transcriptional activity. Deletion studies highlight that the NH2-terminus of PDX-1 is functionally relevant for the down-regulation of Krt19, as it is required for DNA binding of PDX-1 to the Krt19 promoter. Moreover, this effect occurs independently of PBX. Second, we provide insight on how PDX-1 regulates the Hox co-factor MEIS1 post-transcriptionally. We find specific binding of MEIS1 and MEIS2 to the Krt19 promoter using IP-EMSA, and siRNA mediated silencing of Meis1, but not Meis2, reduces transcriptional activation of Krt19 in primary pancreatic ductal cells. Over-expression of PDX-1 leads to a decreased level of MEIS1 protein, and this decrease is prevented by inhibition of the proteasome.Conclusions/SignificanceTaken together, our data provide evidence for a dual mechanism of how PDX-1 negatively regulates Krt19 ductal specific gene expression. These findings imply that transcription factors may efficiently regulate target gene expression through diverse, non-redundant mechanisms.

Pancreatic ductal adenocarcinoma is the overwhelmingly predominant form of pancreatic cancer and the second most common type of gastrointestinal cancer (behind colorectal cancer) in the United States. Recent exciting advances in two areas of pancreatic ductal adenocarcinoma (i.e., the development and characterization of genetically engineered mouse models and the dissection of the genetic basis of hereditary forms in families) have been illuminating. These preclinical models and clinical syndromes provide the first tangible basis for progress in screening and prevention in high-risk populations and in the development of molecular diagnostics and experimental therapeutics.Gastrointestinal cancers comprise a wide spectrum of tumors. Their diversity is highlighted further by their variable incidences and mortality rates. Historically, colorectal cancer has served as the paradigm for our understanding of the genetics of cancers. This has proven extremely helpful in the development and characterization of mouse models, dissecting hereditary syndromes, identifying novel diagnostic strategies, implementation of chemopreventive strategies, and integrating biologics into conventional therapy. Only in recent years has increased attention been applied to pancreatic cancer, the second most common type of gastrointestinal cancer in the United States. Yet, a typical discussion surrounding pancreatic cancer turns to one of pessimism; however, we would advocate that biomedical research and clinical medicine underlying pancreatic cancer emulate the pathways of historic progress in colorectal cancer. The purpose of this commentary is to provide an overview of the very exciting developments in hereditary syndromes and in preclinical models, and that we are at the cusp of translating this knowledge into better strategies for screening, diagnosis, and therapy of pancreatic cancer. We cannot be exhaustive in each of these strategies but will highlight how knowledge about hereditary pancreatic cancer can be used to screen for this disease in high-risk patients and how preclinical models can be interrogated for the development of new diagnostics and therapeutics. These approaches have the opportunity to improve our understanding and management of patients with sporadic pancreatic cancer.Pancreatic cancers comprise a variety of malignancies, including the preponderant form, pancreatic ductal adenocarcinoma (PDAC), and islet cell tumors (part of a class of neuroendocrine tumors), rare acinar cell tumors, mucinous neoplasms (divided further into mucinous cystic neoplasms and intraductal papillary mucinous neoplasms), lymphoma, and sarcoma. There are approximately 37,000 new cases of PDAC each year in the United States, with an increasing annual incidence in African-Americans. Median survival for PDAC is 6 months, and 5-year survival is <5%. When PDAC is identified in its early stage, 5-year survival approaches 20%. Yet, most PDAC cases present at late stages, when therapies are limited. These staggering data spawned attention on P...

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