A hormone-dependent module regulating energy balance.

Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. Our results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance.

The CD100 receptor interacts with its plexin B2 ligand to regulate epidermal γδ T cell function.

γδ T cells respond rapidly to keratinocyte damage, providing essential contributions to the skin wound healing process. The molecular interactions regulating their response are unknown. Here, we identify a role for interaction of plexin B2 with the CD100 receptor in epithelial repair. In vitro blocking of plexin B2 or CD100 inhibited γδ T cell activation. Furthermore, CD100 deficiency in vivo resulted in delayed repair of cutaneous wounds due to a disrupted γδ T cell response to keratinocyte damage. Ligation of CD100 in γδ T cells induced cellular rounding via signals through ERK kinase and cofilin. Defects in this rounding process were evident in the absence of CD100-mediated signals, thereby providing a mechanistic explanation for the defective wound healing in CD100-deficient animals. The discovery of immune functions for plexin B2 and CD100 provides insight into the complex cell-cell interactions between epithelial resident γδ T cells and the neighboring cells they support.

Inositol-1,4,5-trisphosphate receptor regulates hepatic gluconeogenesis in fasting and diabetes.

In the fasted state, increases in circulating glucagon promote hepatic glucose production through induction of the gluconeogenic program. Triggering of the cyclic AMP pathway increases gluconeogenic gene expression via the de-phosphorylation of the CREB co-activator CRTC2 (ref. 1). Glucagon promotes CRTC2 dephosphorylation in part through the protein kinase A (PKA)-mediated inhibition of the CRTC2 kinase SIK2. A number of Ser/Thr phosphatases seem to be capable of dephosphorylating CRTC2 (refs 2, 3), but the mechanisms by which hormonal cues regulate these enzymes remain unclear. Here we show in mice that glucagon stimulates CRTC2 dephosphorylation in hepatocytes by mobilizing intracellular calcium stores and activating the calcium/calmodulin-dependent Ser/Thr-phosphatase calcineurin (also known as PP3CA). Glucagon increased cytosolic calcium concentration through the PKA-mediated phosphorylation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs), which associate with CRTC2. After their activation, InsP(3)Rs enhanced gluconeogenic gene expression by promoting the calcineurin-mediated dephosphorylation of CRTC2. During feeding, increases in insulin signalling reduced CRTC2 activity via the AKT-mediated inactivation of InsP(3)Rs. InsP(3)R activity was increased in diabetes, leading to upregulation of the gluconeogenic program. As hepatic downregulation of InsP(3)Rs and calcineurin improved circulating glucose levels in insulin resistance, these results demonstrate how interactions between cAMP and calcium pathways at the level of the InsP(3)R modulate hepatic glucose production under fasting conditions and in diabetes.

The CREB coactivator CRTC2 links hepatic ER stress and fasting gluconeogenesis.

In fasted mammals, circulating pancreatic glucagon stimulates hepatic gluconeogenesis in part through the CREB regulated transcription coactivator 2 (CRTC2, also referred to as TORC2). Hepatic glucose production is increased in obesity, reflecting chronic increases in endoplasmic reticulum (ER) stress that promote insulin resistance. Whether ER stress also modulates the gluconeogenic program directly, however, is unclear. Here we show that CRTC2 functions as a dual sensor for ER stress and fasting signals. Acute increases in ER stress triggered the dephosphorylation and nuclear entry of CRTC2, which in turn promoted the expression of ER quality control genes through an association with activating transcription factor 6 alpha (ATF6alpha, also known as ATF6)--an integral branch of the unfolded protein response. In addition to mediating CRTC2 recruitment to ER stress inducible promoters, ATF6alpha also reduced hepatic glucose output by disrupting the CREB-CRTC2 interaction and thereby inhibiting CRTC2 occupancy over gluconeogenic genes. Conversely, hepatic glucose output was upregulated when hepatic ATF6alpha protein amounts were reduced, either by RNA interference (RNAi)-mediated knockdown or as a result of persistent stress in obesity. Because ATF6alpha overexpression in the livers of obese mice reversed CRTC2 effects on the gluconeogenic program and lowered hepatic glucose output, our results demonstrate how cross-talk between ER stress and fasting pathways at the level of a transcriptional coactivator contributes to glucose homeostasis.

Neural cells secrete a unique repertoire of proteins.

Proteins that are released from cells consist of those in the extracellular matrix, as well as extracellular signaling and adhesion molecules. The majority of these extracellular proteins are, however, unknown. To determine their identity, we have used a proteomics approach to define proteins released from neurons, astrocytes and neural precursor cells. Using two-dimensional gels and liquid chromatography/mass spectrometry technology, it is shown that while astrocytes release a relatively small number of proteins, neurons and neuronal precursor cells release a larger number of proteins with more functional diversity. Although there is overlap between the different cell types, the exact composition of the extracellular protein pool is unique for each cell population. The various subsets of extracellular neural proteins include those involved in cellular Redox regulation and chaperones. In addition, many proteolytic enzymes are found outside of the cell. These data show that the extracellular space within the nervous system has a more diverse protein composition than previously thought.

Expression of functional kappa-opioid receptors on murine dendritic cells.

Endogenous and exogenous opioids are known to exert direct effects on the immune system and the expression of functional opioid receptors has been reported for several immune cell types. Since dendritic cells are important inducers and regulators of immune responses, we investigated whether murine dendritic cells express functional kappa-opioid receptors. FACScan analysis and radioligand binding studies revealed the expression of kappa-opioid receptors by murine dendritic cells, which by RT-PCR were also shown to express kappa-opioid mRNA. In a primary allogenic mixed-lymphocyte reaction the kappa-agonists dynorphin A and U50488H suppressed the capacity of dendritic cells to induce T-cell proliferation in a concentration-dependent manner. Preincubation with the kappa-specific antagonist nor-binaltrophimine abolished the observed effect, indicating specificity. In contrast, antigen uptake by dendritic cells as well as phenotypic maturation of dendritic cells were not influenced by the kappa-agonists dynorphin A and U50488H. In summary our data demonstrate that dendritic cells express functional kappa-opioid receptors and that specific agonists exert a direct effect on these cells. Therefore, dendritic cells might be involved in the interaction of the neuroendocrine hormones and the immune system.

MALDI-MS analysis of peptides modified with photolabile arylazido groups.

The ability of MALDI-MS to analyze photolabile arylazido peptide derivatives was investigated. Peptides containing UV-labile p-azidobenzoyl groups were subjected to MALDI-MS analysis in a variety of matrices. As standard MALDI-MS employs a UV laser (337 nm), we investigated conditions that would allow detection of the intact molecule ions for these light-sensitive peptides. When using alpha-cyano-4-hydroxycinnamic acid (ACHC) or 2,5 dihydroxybenzoic acid (DHB) as the matrix, photoinduced degradation products were prevalent. In contrast, when employing the matrix sinapinic acid, the intact molecule ion corresponding with the azido peptide was the predominant signal. The protection of photolabile azido derivatives correlates with the UV absorbance properties of the matrix employed, i.e., sinapinic acid, which exhibits a strong absorbance near 337 nm, most efficiently protects the azido derivative from photodegradation.

Autocrine/paracrine regulation of pituitary function by activin, inhibin and follistatin.

The precise regulation of the anterior pituitary is achieved by the cell-specific and combined actions of central, peripheral and local factors. Activins, inhibins, and follistatins were first discovered as gonadal factors with actions on FSH production from pituitary gonadotropes. With the realization that these factors are expressed in a wide array of tissues, including the pituitary, it became apparent that the functional importance of activins, inhibins, and follistatins extends beyond the reproductive axis and that they often exert their effects by autocrine/paracrine mechanisms. As members of the TGF-beta superfamily, activins and inhibins control and orchestrate many physiological processes and are vital for the development, the growth, and the functional integrity of most tissues, including the pituitary. Activins exert effects on multiple pituitary cell types but the best-characterized pituitary targets of the autocrine/paracrine function of activins are the gonadotropes. The autocrine/paracrine function of the activin-binding proteins, follistatins, constitutes an important local mechanism to modulate activin bioactivity while the restricted actions of gonadal inhibins to betaglycan-expressing gonadotropes provides a secondary mode of regulation of cell-specific actions of activins. The aim of this review is to highlight and evaluate experimental evidence that supports the roles of activins, inhibins, and follistatins as autocrine, paracrine, and/or endocrine modulators of the pituitary.

Sequence of horse (Equus caballus) apoA-II. Another example of a dimer forming apolipoprotein.

Apolipoprotein A-II, the second major apolipoprotein of human HDL, also has been observed in a variety of mammals; however, it is either present in trace amounts or absent in other mammals. In humans and chimpanzee, and probably in other great apes, apoA-II with a cysteine at residue 6 is able to form a homodimer. In other primates as well as other mammals, apoA-II, lacking a cysteine residue, is monomeric. However, horse HDL has been reported to contain dimeric apoA-II that following reduction forms monomers. In this report, we extend these observations by reporting on the first complete sequence for a horse apolipoprotein and by demonstrating that horse apoA-II also contains a cysteine residue at position 6. Both the intact protein and its enzymatic fragments were analyzed by chemical sequence analysis and time-of-flight MALDI-MS (matrix assisted laser desorption ionization mass spectrometry). We also obtained molecular mass data on dimeric and monomeric apoA-II using electrospray-ionization mass spectrometry (ESI-MS). The data are compared with other mammalian sequences of apoA-II and are discussed in terms of resulting similarities and variations in the primary sequences.

Posttranslational processing of human and mouse urocortin 2: characterization and bioactivity of gene products.

Mouse (m) and human (h) urocortin 2 (Ucn 2) were identified by molecular cloning strategies and the primary sequence of their mature forms postulated by analogy to closely related members of the corticotropin-releasing factor (CRF) neuropeptide family. Because of the paucity of Ucn 2 proteins in native tissues, skin, muscle, and pancreatic cell lines were transduced with lentiviral constructs and secretion media were used to isolate and characterize Ucn 2 products and study processing. Primary structures were assigned using a combination of Edman degradation sequencing and mass spectrometry. For mUcn 2, transduced cells secreted a 39 amino acid peptide and the glycosylated prohormone lacking signal peptide; both forms were C-terminally amidated and highly potent to activate the type 2 CRF receptor. Chromatographic profiles of murine tissue extracts were consistent with cleavage of mUcn 2 prohormone to a peptidic form. By contrast to mUcn 2, mammalian cell lines transduced with hUcn 2 constructs secreted significant amounts of an 88 amino acid glycosylated hUcn 2 prohormone but were unable to further process this molecule. Similarly, WM-266-4 melanoma cells that express endogenous hUcn 2 secreted only the glycosylated prohormone lacking the signal peptide and unmodified at the C terminus. Although not amidated, hUcn 2 prohormone purified from overexpressing lines activated CRF receptor 2. Hypoxia and glycosylation, paradigms that might influence secretion or processing of gene products, did not significantly impact hUcn 2 prohormone cleavage. Our findings identify probable Ucn 2 processing products and should expedite the characterization of these proteins in mammalian tissues.

Antagonism of activin by activin chimeras.

Activins are pluripotent hormones/growth factors that belong to the TGF-ß superfamily of growth and differentiation factors (GDFs). They play a role in cell growth, differentiation and apoptosis, endocrine function, metabolism, wound repair, immune responses, homeostasis, mesoderm induction, bone growth, and many other biological processes. Activins and the related bone morphogenic proteins (BMPs) transduce their signal through two classes of single transmembrane receptors. The receptors possess intracellular serine/threonine kinase domains. Signaling occurs when the constitutively active type II kinase domain phosphorylates the type I receptor, which upon activation, phosphorylates intracellular signaling molecules. To generate antagonistic ligands, we generated chimeric molecules that disrupt the receptor interactions and thereby the phosphorylation events. The chimeras were designed based on available structural data to maintain high-affinity binding to type II receptors. The predicted type I receptor interaction region was replaced by residues present in inactive homologs or in related ligands with different type I receptor affinities.

Proteomic analysis yields an unexpected trans-acting point in control of the human sympathochromaffin phenotype.

BACKGROUND: The secretory protein chromogranin A (CHGA) plays a necessary role in formation of catecholamine storage vesicles and gives rise to a catecholamine release-inhibitory fragment. Because genetic variation in the proximal human CHGA promoter predicts autonomic function and blood pressure, we explored how a common genetic variant alters transcription of the gene. METHODS AND RESULTS: Bioinformatic analysis suggested that the common G-462A promoter variant (rs9658634) may disrupt as many as 3 transcriptional control motifs: LEF1, COUP-TF, and PPARγ-RXRα. During electrophoretic mobility shifts, chromaffin cell nuclear proteins bound specifically to the A (though not G) allele of CHGA promoter G-462A. On oligonucleotide affinity chromatography followed by electrospray ionization followed by 2-dimensional (tandem) mass spectrometry analysis of A allele eluates, the transcription factor LEF1 (lymphoid enhancer-binding factor-1) was identified. Interaction of LEF1 with the A allele at G-462A was confirmed by supershift. On cotransfection, LEF1 discriminated between the allelic variants, especially in chromaffin cells. Allele specificity of trans-activation by LEF1 was transferable to an isolated G-462A element fused to a heterologous (SV40) promoter. Because ß-catenin (CTNNB1) can heterodimerize with LEF1, we tested the effect of cotransfection of this factor and again found A allele-specific perturbation of CHGA transcription. CONCLUSIONS: Common genetic variation within the human CHGA promoter alters the interaction of specific factors in trans with the promoter, with LEF1 identified by proteomic analysis and confirmed by supershift. Coexpression experiments show functional effects of LEF1 and CTNNB1 on CHGA promoter. The findings document a novel role for components of the immune and WNT pathways in control of human sympathochromaffin phenotypes.

The junctional adhesion molecule JAML is a costimulatory receptor for epithelial gammadelta T cell activation.

Gammadelta T cells present in epithelial tissues provide a crucial first line of defense against environmental insults, including infection, trauma, and malignancy, yet the molecular events surrounding their activation remain poorly defined. Here we identify an epithelial gammadelta T cell-specific costimulatory molecule, junctional adhesion molecule-like protein (JAML). Binding of JAML to its ligand Coxsackie and adenovirus receptor (CAR) provides costimulation leading to cellular proliferation and cytokine and growth factor production. Inhibition of JAML costimulation leads to diminished gammadelta T cell activation and delayed wound closure akin to that seen in the absence of gammadelta T cells. Our results identify JAML as a crucial component of epithelial gammadelta T cell biology and have broader implications for CAR and JAML in tissue homeostasis and repair.

FoxL2 and Smad3 coordinately regulate follistatin gene transcription.

Follistatin is a transcriptional target and a modulator of activin action. Through an autocrine/paracrine loop, activin controls follistatin levels and thus regulates its own bioavailability. In gonadotropic alphaT3-1 cells, activin induces follistatin transcription primarily through the action of Smad3 at an intronic Smad-binding element (SBE1). Using a proteomics approach, we searched for endogenous alphaT3-1 proteins that participate in SBE1-mediated transcription. We identified FoxL2, a member of the forkhead family, as a candidate modulator of SBE1 function. Mutations of FoxL2 are associated with the blepharophimosis/ptosis/epicanthus inversus syndrome characterized with craniofacial defects and premature ovarian failure. FoxL2 localizes to alpha-glycoprotein subunit- and follicle-stimulating hormone beta-positive cells of the adult mouse pituitary and is present in alphaT3-1 and LbetaT2 cells, but its pituitary actions remain largely unknown. We have determined that FoxL2 binds to a forkhead-binding element (FKHB) located just downstream of the SBE1 site of the follistatin gene and functions as a Smad3 partner to drive SBE1-mediated transcription in alphaT3-1 cells treated with activin. Chromatin immunoprecipitation assays confirm that endogenous FoxL2 and Smad3 are recruited to the intronic enhancer of the follistatin gene where the SBE1 and FKHB sites are located. Exogenous FoxL2 enhances SBE1-mediated transcription, and short hairpin RNA-mediated knockdown of endogenous FoxL2 protein compromises this effect in alphaT3-1 cells. FoxL2 directly associates with Smad3 but not Smad2 or Smad4. This association between Smad3 and FoxL2 is mediated by the MH2 domain of Smad3 and is dependent on an intact forkhead domain in FoxL2. Altogether, these observations highlight a novel role for FoxL2 and suggest that it may function as a transcriptional regulator and a coordinator of Smad3 targets.

GRP78 and Cripto form a complex at the cell surface and collaborate to inhibit transforming growth factor beta signaling and enhance cell growth.

Cripto is a multifunctional cell surface protein with important roles in vertebrate embryogenesis and the progression of human tumors. While Cripto has been shown to modulate multiple signaling pathways, its binding partners do not appear to fully explain its molecular actions. Therefore, we conducted a screen aimed at identifying novel Cripto-interacting proteins. This screen led to our identification of glucose-regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone that is also expressed at the surfaces of tumor cells. Here we demonstrate that Cripto and GRP78 interact at the cell surfaces of multiple cell lines and that their interaction is independent of prior association within the ER. Interestingly, short hairpin RNA knockdown of endogenous GRP78 resulted in enhanced transforming growth factor beta (TGF-beta) signaling, indicating that like Cripto, GRP78 inhibits this pathway. We further show that when coexpressed, GRP78 and Cripto collaborate to antagonize TGF-beta responses, including Smad phosphorylation and growth inhibition of prostate cancer cells grown under anchorage-dependent or -independent conditions. Finally, we provide evidence that cells coexpressing GRP78 and Cripto grow much more rapidly in soft agar than do cells expressing either protein individually. Together, our results indicate that these proteins bind at the cell surface to enhance tumor growth via the inhibition of TGF-beta signaling.

Activin A/bone morphogenetic protein (BMP) chimeras exhibit BMP-like activity and antagonize activin and myostatin.

Activins and bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family of growth and differentiation factors that induce signaling in target cells by assembling type II and type I receptors at the cell surface. Ligand residues involved in type II binding are located predominantly in the C-terminal region that forms an extended beta-sheet, whereas residues involved in type I binding are located in the alpha-helical and preceding loop central portion of the molecule. To test whether the central residues are sufficient to determine specificity toward type I receptors, activin A/BMP chimeras were constructed in which the central residues (45-79) of activin A were replaced with corresponding residues of BMP2 and BMP7. The chimeras were assessed for activin type II receptor (Act RII) binding, activin-like bioactivity, and BMP-like activity as well as antagonistic properties toward activin A and myostatin. ActA/BMP7 chimera retained Act RII binding affinity comparable with wild type activin A, whereas ActA/BMP2 chimera showed a slightly reduced affinity toward Act RII. Both the chimeras were devoid of significant activin bioactivity in 293T cells in the A3 Lux reporter assay up to concentrations 10-fold higher than the minimal effective activin A concentration (approximately 4 nM). In contrast, these chimeras showed BMP-like activity in a BRE-Luc assay in HepG2 cells as well as induced osteoblast-like phenotype in C2C12 cells expressing alkaline phosphatase. Furthermore, both the chimeras activated Smad1 but not Smad2 in C2C12 cells. Also, both the chimeras antagonized ligands that signal via activin type II receptor, such as activin A and myostatin. These data indicate that activin residues in the central region determine its specificity toward type I receptors. ActA/BMP chimeras can be useful in the study of receptor specificities and modulation of transforming growth factor-beta members, activins, and BMPs.

Increase in expression levels and resistance to sulfhydryl oxidation of peroxiredoxin isoforms in amyloid beta-resistant nerve cells.

Peroxiredoxins (Prxs) are a ubiquitously expressed family of thiol peroxidases that reduce hydrogen peroxide, peroxynitrite, and hydroperoxides using a highly conserved cysteine. There is substantial evidence that oxidative stress elicited by amyloid beta (Abeta) accumulation is a causative factor in the pathogenesis of Alzheimer disease (AD). Here we show that Abeta-resistant PC12 cell lines exhibit increased expression of multiple Prx isoforms with reduced cysteine oxidation. Abeta-resistant PC12 cells also display higher levels of thioredoxin and thioredoxin reductase, two enzymes critical for maintaining Prx activity. PC12 cells and rat primary hippocampal neurons transfected with wild type Prx1 exhibit increased Abeta resistance, whereas mutant Prx1, lacking a catalytic cysteine, confers no protection. Using an antibody that specifically recognizes sulfinylated and sulfonylated Prxs, it is demonstrated that primary rat cortical nerve cells exposed to Abeta display a time-dependent increase in cysteine oxidation of the catalytic site of Prxs that can be blocked by the addition of the thiol-antioxidant N-acetylcysteine. In support of previous findings, expression of Prx1 is higher in post-mortem human AD cortex tissues than in age-matched controls. In addition, two-dimensional gel electrophoresis and mass spectrometry analysis revealed that Prx2 exists in a more oxidized state in AD brains than in control brains. These findings suggest that increased Prx expression and resistance to sulfhydryl oxidation in Abeta-resistant nerve cells is a compensatory response to the oxidative stress initiated by chronic pro-oxidant Abeta exposure.

Proteins that bind to the RERMS region of beta amyloid precursor protein.

The main objective of this study was to investigate the biological function of beta amyloid precursor protein (APP), in particular its nerve growth factor-like activity. We hypothesize that the extracellular domain containing the sequence RERMS, amino acids 328-332 of APP(695), represents the active site for this function. Binding assays using peptide fragments of this domain have demonstrated specific and saturable binding to the cell surface with affinity in the low nanomolar range. This induced our quest for an APP-specific receptor. We chose different peptide fragments of the RERMS domain as ligands and displacing agents on affinity columns to purify APP-binding molecules. Amino acid microsequencing yielded partial sequences of serum albumin, actin, two novel proteins of 41 and 63kDa, and human Collapsin Response Mediator Protein-2 (hCRMP-2). Because both APP and hCRMP-2 promote neuronal outgrowth and use a common signaling pathway, APP could be acting through a semaphorin receptor as well.

Distinct structural and functional roles of conserved residues in the first extracellular domain of receptors for corticotropin-releasing factor and related G-protein-coupled receptors.

The G-protein-coupled receptor B1 family includes corticotropin-releasing factor (CRF), growth hormone-releasing hormone, incretin, and pituitary adenylate cyclase-activating polypeptide receptors. The three-dimensional NMR structure of the first extracellular domain (ECD1) of CRF receptor 2beta (CRF-R2beta), free and complexed with astressin, comprises a Sushi domain. This domain is stabilized in part by a salt bridge between Asp(65) and Arg(101). Analogous residues are conserved in other members of the B1 family. To address the importance of the salt bridge residues within this receptor family, we studied the effects of mutating the residues in full-length CRF-R2beta and isolated ECD1. Mutation D65A or D65R/R101D resulted in loss of the canonical disulfide arrangement, whereas R101A retained the Cys(4)-Cys(6) disulfide bond. The mutations resulted in misfolding within the ECD1 as determined by NMR and 1-anilino-8-naphthalenesulfonate binding but did not prevent cell surface expression. The D65A mutation in CRF-R2beta greatly reduced binding and activation, but the R101A substitution had only a small effect. Similar effects were seen on astressin binding to the ECD1. The different interactions of Asp(65) and Arg(101), deduced from the three-dimensional structure of the complex, are consistent with the differential effects seen in the mutants. The reduction in binding of Asp(65) mutants is a consequence of a distinct Asp(65)-Trp(71) interaction, which stabilizes the ligand-binding loop. Hence, loss of the salt bridge leads to disruption of the overall fold but does not abolish function. Because homologous mutations in other B1 receptors produce similar effects, these conserved residues may play similar roles in the entire receptor family.

F-spondin promotes nerve precursor differentiation.

Cells in the developing nervous system secrete a large number of proteins that regulate the migration and differentiation of their neighbors. It is shown here that a clonal central nervous system cell line secretes a protein that causes both a rat hippocampal progenitor cell line and primary cortical neural cells to differentiate into cells with the morphological and biochemical features of neurons. This protein was identified as F-spondin. Analysis of F-spondin isoforms secreted from transfected cells shows that the core protein without the thrombospondin type 1 repeats is sufficient to promote neuronal differentiation when adsorbed to a surface. F-spondin can also inhibit neurite outgrowth while allowing the expression of nerve-specific proteins when present in a soluble form at high concentrations. Therefore, F-spondin can alter cell differentiation in multiple ways, depending upon its concentration and distribution between substrate-attached and soluble forms.