Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant unusually preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employ static and dynamic structural methods and observe that, compared to R132H, the R132Q active site adopts a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling reveals a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Capturing Differences in the Regulation of LRRK2 Dynamics and Conformational States by Small Molecule Kinase Inhibitors.

Mutations in the human leucine rich repeat protein kinase-2 (LRRK2) create risk factors for Parkinson's disease, and pathological functions of LRRK2 are often correlated with aberrant kinase activity. Past research has focused on developing selective LRRK2 kinase inhibitors. In this study, we combined enhanced sampling simulations with HDX-MS to characterize the inhibitor-induced dynamic changes and the allosteric communications within the C-terminal domains of LRRK2, LRRK2RCKW. We find that the binding of MLi-2 (a type I kinase inhibitor) stabilizes a closed kinase conformation and reduces the global dynamics of LRRK2RCKW, leading to a more compact LRRK2RCKW structure. In contrast, the binding of Rebastinib (a type II kinase inhibitor) stabilizes an open kinase conformation, which promotes a more extended LRRK2RCKW structure. By probing the distinct effects of the type I and type II inhibitors, key interdomain interactions are found to regulate the communication between the kinase domain and the GTPase domain. The intermediate states revealed in our simulations facilitate the efforts toward in silico design of allosteric modulators that control LRRK2 conformations and potentially mediate the oligomeric states of LRRK2 and its interactions with other proteins.

Capturing the Dynamic Conformational Changes of Human Isocitrate Dehydrogenase 1 (IDH1) upon Ligand and Metal Binding Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Human isocitrate dehydrogenase 1 (IDH1) is a highly conserved metabolic enzyme that catalyzes the interconversion of isocitrate and α-ketoglutarate. Kinetic and structural studies with IDH1 have revealed evidence of striking conformational changes that occur upon binding of its substrates, isocitrate and NADP+, and its catalytic metal cation. Here, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to build a comprehensive map of the dynamic conformational changes experienced by IDH1 upon ligand binding. IDH1 proved well-suited for HDX-MS analysis, allowing us to capture profound changes in solvent accessibility at substrate binding sites and at a known regulatory region, as well as at more distant local subdomains that appear to support closure of this protein into its active conformation. HDX-MS analysis suggested that IDH1 is primarily purified with NADP(H) bound in the absence of its metal cation. Subsequent metal cation binding, even in the absence of isocitrate, was critical for driving large conformational changes. WT IDH1 folded into its fully closed conformation only when the full complement of substrates and metal was present. Finally, we show evidence supporting a previously hypothesized partially open conformation that forms prior to the catalytically active state, and we propose this conformation is driven by isocitrate binding in the absence of metal.

Target binding triggers hierarchical phosphorylation of human Argonaute-2 to promote target release.

Argonaute (Ago) proteins play a central role in post-transcriptional gene regulation through RNA interference (RNAi). Agos bind small RNAs (sRNAs) including small interfering RNAs (siRNAs) and microRNAs (miRNAs) to form the functional core of the RNA-induced silencing complex (RISC). The sRNA is used as a guide to target mRNAs containing either partially or fully complementary sequences, ultimately leading to downregulation of the corresponding proteins. It was previously shown that the kinase CK1α phosphorylates a cluster of residues in the eukaryotic insertion (EI) of Ago, leading to the alleviation of miRNA-mediated repression through an undetermined mechanism. We show that binding of miRNA-loaded human Ago2 to target RNA with complementarity to the seed and 3' supplementary regions of the miRNA primes the EI for hierarchical phosphorylation by CK1α. The added negative charges electrostatically promote target release, freeing Ago to seek out additional targets once it is dephosphorylated. The high conservation of potential phosphosites in the EI suggests that such a regulatory strategy may be a shared mechanism for regulating miRNA-mediated repression.

Proteins are the chemical 'workhorses' of the cell: some help maintain a cell's shape or structure, while others carry out the chemical reactions necessary for life. Organisms therefore need to keep tight control over the production of proteins in their cells, so that the right amount of each protein is made at the right time, in the right place. Instructions for making new proteins are encoded in a type of molecule called messenger RNA. Each messenger RNA contains the instructions for one protein, which are then 'read' and carried out by special cellular machinery called ribosomes. The cell can control how much protein it produces by regulating both the levels of different messenger RNA and the amount of protein ribosomes are allowed to make from those instructions. The main way to regulate the levels of messenger RNA is through their transcription from the genome. However, this needs fine tuning. Cells can do this in a highly specific way using molecules called microRNAs. A microRNA works by directing a protein called Argonaute to the messenger RNA that it targets. Once Argonaute arrives, it can call in additional 'helper proteins' to shut down, or reduce, protein production from that messenger RNA, or alternatively to break down the messenger RNA altogether. Cells can use an enzyme called CK1α to attach bulky chemical groups onto a specific part of the Argonaute protein, in a reaction termed phosphorylation. The ability to carry out this reaction (and to reverse it) also seems to be important for microRNAs to do their job properly, but why has remained unknown. Bibel et al. wanted to determine what triggers CK1α to phosphorylate Argonaute, and how this affects interactions between microRNAs, Argonaute and their target messenger RNAs. A series of 'test tube' experiments looked at the interaction between purified CK1α and Argonaute under different conditions. These demonstrated that CK1α could only carry out its phosphorylation reaction when Argonaute was already interacting with a microRNA and its corresponding messenger RNA. Further measurements revealed that phosphorylation of Argonaute made it detach from the messenger RNA more quickly. This suggests that phosphorylation might be a way to let Argonaute seek out new messenger RNAs after blocking protein production at its first 'target'. These results shed new light on a fundamental mechanism that cells use to control protein production. Bibel et al. propose that this mechanism may be shared across many different species and could one day help guide the development of new medical therapies based on microRNAs.

LRRK2 dynamics analysis identifies allosteric control of the crosstalk between its catalytic domains.

The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease-related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a "cap" that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2.

To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.

Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein.

The COVID-2019 pandemic is the most severe acute public health threat of the twenty-first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS-CoV-2 coronavirus. Here, we examine the architecture and self-assembly properties of the SARS-CoV-2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS-CoV. While the N2b domain forms a dimer in solution, addition of the C-terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen-deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α-helix that self-associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS-CoV-2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self-assembly properties of a key protein in the SARS-CoV-2 life cycle, with implications for both drug design and antibody-based testing.

Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein.

The COVID-2019 pandemic is the most severe acute public health threat of the twenty-first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS-CoV-2 coronavirus. Here, we examine the architecture and self-assembly properties of the SARS-CoV-2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS-CoV. While the N2b domain forms a dimer in solution, addition of the C-terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen-deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α-helix that self-associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS-CoV-2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self-assembly properties of a key protein in the SARS-CoV-2 life cycle, with implications for both drug design and antibody-based testing.

Differential Local Stability Governs the Metamorphic Fold Switch of Bacterial Virulence Factor RfaH.

RfaH, a two-domain protein from a universally conserved NusG/Spt5 family of regulators, is required for the transcription and translation of long virulence and conjugation operons in many Gram-negative bacterial pathogens. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA element. Upon recruitment, the C-terminal domain of RfaH refolds from an α-hairpin, which is bound to RNA polymerase binding site within the N-terminal domain, into an unbound ß-barrel that interacts with the ribosome. Although structures of the autoinhibited (α-hairpin) and active (ß-barrel) states and plausible refolding pathways have been reported, how this reversible switch is encoded within RfaH sequence and structure is poorly understood. Here, we combined hydrogen-deuterium exchange measurements by mass spectrometry and nuclear magnetic resonance with molecular dynamics to evaluate the differential local stability between both RfaH folds. Deuteron incorporation reveals that the tip of the C-terminal hairpin (residues 125-145) is stably folded in the autoinhibited state (∼20% deuteron incorporation), whereas the rest of this domain is highly flexible (>40% deuteron incorporation), and its flexibility only decreases in the ß-folded state. Computationally predicted ΔG agree with these results by displaying similar anisotropic stability within the tip of the α-hairpin and on neighboring N-terminal domain residues. Remarkably, the ß-folded state shows comparable structural flexibility than nonmetamorphic homologs. Our findings provide information critical for understanding the metamorphic behavior of RfaH and other chameleon proteins and for devising targeted strategies to combat bacterial infections.

α2 Integrin-Dependent Suppression of Pancreatic Adenocarcinoma Cell Invasion Involves Ectodomain Regulation of Kallikrein-Related Peptidase-5.

Previous reports demonstrate that the α2-integrin (α2) mediates pancreatic ductal adenocarcinoma (PDAC) cell interactions with collagens. We found that while well-differentiated cells use α2 exclusively to adhere and migrate on collagenI, poorly differentiated PDAC cells demonstrate reduced reliance on, or complete loss of, α2. Since well-differentiated PDAC lines exhibit reduced in vitro invasion and α2-blockade suppressed invasion of well-differentiated lines exclusively, we hypothesized that α2 may suppress the malignant phenotype in PDAC. Accordingly, ectopic expression of α2 retarded in vitro invasion and maintenance on collagenI exacerbated this effect. Affymetrix profiling revealed that kallikrein-related peptidase-5 (KLK5) was specifically upregulated by α2, and reduced α2 and KLK5 expression was observed in poorly differentiated PDAC cells in situ. Accordingly, well-differentiated PDAC lines express KLK5, and KLK5 blockade increased the invasion of KLK5-positive lines. The α2-cytoplasmic domain was dispensable for these effects, demonstrating that the α2-ectodomain and KLK5 coordinately regulate a less invasive phenotype in PDAC.

Modification of the L1-CAM carboxy-terminus in pancreatic adenocarcinoma cells.

The neural cell adhesion molecule L1 has recently been shown to be expressed in pancreatic adenocarcinoma (PDAC) cells. In this report, we demonstrate that L1 is expressed by moderately- to poorly-differentiated PDAC cells in situ, and that L1 expression is a predictor of poor patient survival. In vitro, reduced reactivity of an anti-L1 carboxy-terminus-specific antibody was observed in the more poorly differentiated fast-growing (FG) variant of the COLO357 population, versus its well-differentiated slow-growing (SG) counterpart, even though they express equivalent total L1. The carboxy-terminus of L1 mediates binding to the MAP kinase-regulating protein RanBPM and mutation of T1247/S1248 within this region attenuates the expression of malignancy associated proteins and L1-induced tumorigenicity in mice. Therefore, we reasoned that the differential epitope exposure observed might be indicative of modifications responsible for regulating these events. However, epitope mapping demonstrated that the major determinant of binding was actually N1251; mutation of T1247 and S1248, alone or together, had little effect on C20 binding. Moreover, cluster assays using CD25 ectodomain/L1 cytoplasmic domain chimeras demonstrated the N1251-dependent, RanBPM-independent stimulation of erk phosphorylation in these cells. Reactivity of this antibody also reflects the differential exposure of extracellular epitopes in these COLO357 sublines, consistent with the previous demonstration of L1 ectodomain conformation modulation by intracellular modifications. These data further support a central role for L1 in PDAC, and define a specific role for carboxy-terminal residues including N1251 in the regulation of L1 activity in PDAC cells.

Inside-out regulation of L1 conformation, integrin binding, proteolysis, and concomitant cell migration.

Previous reports on the expression of the cell adhesion molecule L1 in pancreatic ductal adenocarcinoma (PDAC) cells range from absent to high. Our data demonstrate that L1 is expressed in poorly differentiated PDAC cells in situ and that threonine-1172 (T1172) in the L1 cytoplasmic domain exhibits steady-state saturated phosphorylation in PDAC cells in vitro and in situ. In vitro studies support roles for casein kinase II and PKC in this modification, consistent with our prior studies using recombinant proteins. Importantly, T1172 phosphorylation drives, or is associated with, a change in the extracellular structure of L1, consistent with a potential role in regulating the shift between the closed conformation and the open, multimerized conformation of L1. We further demonstrate that these distinct conformations exhibit differential binding to integrins alphavbeta3 and alphavbeta5 and that T1172 regulates cell migration in a matrix-specific manner and is required for a disintegrin and metalloproteinase-mediated shedding of the L1 ectodomain that has been shown to regulate cell migration. These data define a specific role for T1172 of L1 in regulating aspects of pancreatic adenocarcinoma cell phenotype and suggest the need for further studies to elucidate the specific ramifications of L1 expression and T1172 phosphorylation in the pathobiology of pancreatic cancer.

Syk tyrosine kinase acts as a pancreatic adenocarcinoma tumor suppressor by regulating cellular growth and invasion.

We have identified the nonreceptor tyrosine kinase syk as a marker of differentiation/tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). Syk expression is lost in poorly differentiated PDAC cells in vitro and in situ, and stable reexpression of syk in endogenously syk-negative Panc1 (Panc1/syk) cells retarded their growth in vitro and in vivo and reduced anchorage-independent growth in vitro. Panc1/syk cells exhibited a more differentiated morphology and down-regulated cyclin D1, akt, and CD171, which are overexpressed by Panc1 cells. Loss of PDAC syk expression in culture is due to promoter methylation, and reversal of promoter methylation caused reexpression of syk and concomitant down-regulation of CD171. Moreover, suppression of syk expression in BxPC3 cells caused de novo CD171 expression, consistent with the reciprocal expression of syk and CD171 we observe in situ. Importantly, Panc1/syk cells demonstrated dramatically reduced invasion in vitro. Affymetrix analysis identified statistically significant regulation of >2000 gene products by syk in Panc1 cells. Of these, matrix metalloproteinase-2 (MMP2) and tissue inhibitor of metalloproteinase-2 were down-regulated, suggesting that the MMP2 axis might mediate Panc1/mock invasion. Accordingly, MMP2 inhibition suppressed the in vitro invasion of Panc1/mock cells without effect on Panc1/syk cells. This study demonstrates a prominent role for syk in regulating the differentiation state and invasive phenotype of PDAC cells.

Tyrosine and serine phosphorylation regulate the conformation and subsequent threonine phosphorylation of the L1 cytoplasmic domain.

Previously we identified threonine-1172 (T1172) in the cytoplasmic domain of the cell adhesion molecule L1 as phosphorylated in pancreatic cancer cells. Although both CKII- and PKC-blockade suppressed this modification, only CKII was capable of phosphorylating T1172 of a recombinant L1 cytoplasmic domain, suggesting the requirement for additional events to facilitate availability of T1172 to PKC. In this study, we demonstrate that the region around T1172 exists in distinct conformations based on both T1172 phosphorylation and the integrity of surrounding residues. We further demonstrate the role of membrane-proximal and membrane-distal residues in regulating cytoplasmic domain conformation, and that modification of 3 of the 4 tyrosines in the L1 cytoplasmic domain promote conformational changes that facilitate other events. In particular, phenylalanine-substitution of tyrosine-1151 or tyrosine-1229 promote opening up of the cytoplasmic domain in a manner that facilitates phosphorylation of the other 3 tyrosines, as well as phosphorylation of T1172 by PKCalpha. Importantly, we show that phosphorylation of serine-1181 is required for T1172 phosphorylation by CKII. These data define a specific role for secondary structure in regulating the availability of T1172 that facilitates phosphorylation by PKC.

The extracellular matrix differentially regulates the expression of PTHrP and the PTH/PTHrP receptor in FG pancreatic cancer cells.

OBJECTIVES: Previous studies by our laboratory have demonstrated that parathyroid hormone-related protein (PTHrP) and its receptor (PTH/PTHrP receptor) are commonly expressed in pancreatic cancer and suggest their participation in the progression of this devastating disease. It has also been demonstrated that one of the major hallmarks of pancreatic adenocarcinoma is an increased production of the extracellular matrix (ECM), a critical regulator of diverse cellular processes such as differentiation, proliferation, and angiogenesis. The present study focused on the relationship between the PTHrP and ECM axes in the pathobiology of pancreatic cancer. METHOD AND RESULTS: Using the FG pancreatic adenocarcinoma cell line, we demonstrate a significant inverse correlation between FG cell proliferation and PTHrP expression that depended on the ECM protein on which the cells were cultured (P < 0.05). Generally, ECM proteins that promoted the strongest proliferation, including type I collagen, type IV collagen, and laminin, resulted in decreased expression of PTHrP. Conversely, ECM proteins that promoted the weakest proliferation, including fibronectin, vitronectin, and BSA, resulted in increased expression of PTHrP. A similar trend was found between FG cell proliferation and the PTH/PTHrP receptor expression, with Pearson correlation coefficients of 0.480 (mRNA) and -0.591 (protein). CONCLUSION: These observations demonstrate a unique functional relationship between the ECM and PTHrP axes and have important implications for our understanding of the complex mechanisms responsible for the progression of pancreatic cancer and its metastases.

Extracellular signal-regulated kinase (ERK)-dependent gene expression contributes to L1 cell adhesion molecule-dependent motility and invasion.

The cell adhesion molecule L1 has been implicated in a variety of motile processes, including neurite extension, cerebellar cell migration, extravasation, and metastasis. Homophilic or heterophilic L1 binding and concomitant signaling have been shown to promote cell motility in the short term. In this report, L1 is also shown to induce and maintain a motile and invasive phenotype by promoting gene transcription. In the presence of serum or platelet-derived growth factor, L1 promotes heightened and sustained activation of the extracellular signal-regulated kinase pathway. Activation of this pathway then induces the expression of motility- and invasion-associated gene products, including the beta(3)-integrin subunit, small GTPases, and the cysteine proteases cathepsin-L and -B. Induction of integrin alpha(v)beta(3) and rac-1 is shown to contribute directly to L1-dependent haptotaxis, whereas induction of cathepsins-L and -B promotes matrix invasion. This study provides a novel translational mechanism to account for the association between L1 expression and motile processes involved in metastasis and development.

Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration.

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, we show that Netrin-1 is expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. We demonstrate that alpha6beta4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of alpha6beta4 and alpha3beta1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells.