VGF function in depression and antidepressant efficacy.

Brain-derived neurotrophic factor (BDNF) is a critical effector of depression-like behaviors and antidepressant responses. Here, we show that VGF (non-acronymic), which is robustly regulated by BDNF/TrkB signaling, is downregulated in hippocampus (male/female) and upregulated in nucleus accumbens (NAc) (male) in depressed human subjects and in mice subjected to chronic social defeat stress (CSDS). Adeno-associated virus (AAV)-Cre-mediated Vgf ablation in floxed VGF mice, in dorsal hippocampus (dHc) or NAc, led to pro-depressant or antidepressant behaviors, respectively, while dHc- or NAc-AAV-VGF overexpression induced opposite outcomes. Mice with reduced VGF levels in the germ line (Vgf+/-) or in dHc (AAV-Cre-injected floxed mice) showed increased susceptibility to CSDS and impaired responses to ketamine treatment in the forced swim test. Floxed mice with conditional pan-neuronal (Synapsin-Cre) but not those with forebrain (αCaMKII-Cre) Vgf ablation displayed increased susceptibility to subthreshold social defeat stress, suggesting that neuronal VGF, expressed in part in inhibitory interneurons, regulates depression-like behavior. Acute antibody-mediated sequestration of VGF-derived C-terminal peptides AQEE-30 and TLQP-62 in dHc induced pro-depressant effects. Conversely, dHc TLQP-62 infusion had rapid antidepressant efficacy, which was reduced in BDNF floxed mice injected in dHc with AAV-Cre, and in NBQX- and rapamycin-pretreated wild-type mice, these compounds blocking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and mammalian target of rapamycin (mTOR) signaling, respectively. VGF is therefore a critical modulator of depression-like behaviors in dHc and NAc. In hippocampus, the antidepressant response to ketamine is associated with rapid VGF translation, is impaired by reduced VGF expression, and as previously reported, requires coincident, rapid BDNF translation and release.

Models and mechanisms of metabolic regulation: genes, stress, and the HPA and HPG axes.

A variety of models have been developed to better understand the mechanisms underlying individual variation in susceptibility to obesity. This review discusses several of these models and explores their role in understanding individual vulnerability to metabolic disease and the environmental factors around which metabolic perturbations occur. Recently, the focus of models has shifted towards heterogeneous populations, in which individuals characterized by a high vulnerability and individuals that are seemingly resistant can be identified. The use of these heterogeneous studies has lead to the identification of several novel biomarkers predicting obesity. This review therefore focuses on nontraditional factors, which are not directly implicated in metabolic regulation. First, the evidence from rodent knockout models for genetic factors involved in obesity is discussed. Second, the role of a stressful environment, particularly the early life environment is investigated along with a discussion of circadian disruption and metabolic disorders. Finally, the impact of sex-steroids, as exemplified by polycystic ovarian syndrome, is discussed. Overall, the data presented in our review demonstrate that in most cases interplay between genetic and environmental factors best predicts disease development. Our review shows that susceptibility to obesity may be explained by complex interactions between traditional homeostatic mechanisms, such as the hypothalamic peptide, and less studied mechanisms, like steroids and neurotrophic factors.

L1 cell adhesion molecule promotes resistance to alcohol-induced silencing of growth cone responses to guidance cues.

Alcohol exposure in utero is a common cause of mental retardation, but the targets and mechanisms of action are poorly understood. Several lines of data point toward alterations in cortical connectivity, suggesting that axon guidance may be vulnerable to alcohol exposure. To test this, we asked whether ethanol directly affects cortical axonal growth cone responses to guidance cues. We find that even low concentrations of ethanol (12.5 mM; 57.2 mg/dl) commonly observed in social drinking prevent growth cone responses to three mechanistically independent guidance cues, Semaphorin3A, Lysophosphatidic Acid, and Netrin-1. However, this effect is highly dependent on substrate; axonal growth cones extending on an L1 cell adhesion molecule (L1CAM) substrate retain responsiveness to cues following exposure to ethanol, while those growing on poly-L-lysine or N-cadherin do not. The effects of ethanol on axon extension are, by contrast, quite modest. Quantitative assessments of the effects of ethanol on the surface distribution of L1CAM in growth cones suggest that L1CAM homophilic interactions may be particularly relevant for retaining growth cone responsiveness following ethanol exposure. Together, our findings indicate that ethanol can directly and generally alter growth cone responses to guidance cues, that a substrate of L1CAM effectively antagonizes this effect, and that cortical axonal growth cone vulnerability to ethanol may be predicted in part based on the environment through which they are extending.

Granins as disease-biomarkers: translational potential for psychiatric and neurological disorders.

The identification of biomarkers represents a fundamental medical advance that can lead to an improved understanding of disease pathogenesis, and holds the potential to define surrogate diagnostic and prognostic endpoints. Because of the inherent difficulties in assessing brain function in patients and objectively identifying neurological and cognitive/emotional symptoms, future application of biomarkers to neurological and psychiatric disorders is extremely desirable. This article discusses the biomarker potential of the granin family, a group of acidic proteins present in the secretory granules of a wide variety of endocrine, neuronal and neuroendocrine cells: chromogranin A (CgA), CgB, Secretogranin II (SgII), SgIII, HISL-19 antigen, 7B2, NESP55, VGF and ProSAAS. Their relative abundance, functional significance, and secretion into the cerebrospinal fluid (CSF), saliva, and the general circulation have made granins tractable targets as biomarkers for many diseases of neuronal and endocrine origin, recently impacting diagnosis of a number of neurological and psychiatric disorders including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia, and schizophrenia. Although research has not yet validated the clinical utility of granins as surrogate endpoints for the progression or treatment of neurological or psychiatric disease, a growing body of experimental evidence indicates that the use of granins as biomarkers might be of great potential clinical interest. Advances that further elucidate the mechanism(s) of action of granins, coupled with improvements in biomarker technology and direct clinical application, should increase the translational effectiveness of this family of proteins in disease diagnosis and drug discovery.

Effects of ethanol on axon outgrowth and branching in developing rat cortical neurons.

Humans exposed prenatally to ethanol can exhibit brain abnormalities and cognitive impairment similar to those seen in patients expressing mutant forms of the L1 cell adhesion molecule (L1CAM). The resemblance suggests that L1CAM may be a target for ethanol, and consistent with this idea, ethanol can inhibit L1CAM adhesion in cell lines and L1CAM-mediated outgrowth and signaling in cerebellar granule neurons. However, it is not known whether ethanol inhibits L1CAM function in other neuron types known to require L1CAM for appropriate development. Here we asked whether ethanol alters L1CAM function in neurons of the rat cerebral cortex. We find that ethanol does not alter axonal polarization, L1CAM-dependent axon outgrowth or branching, or L1CAM recycling in axonal growth cones. Thus, ethanol inhibition of L1CAM is highly dependent on neuronal context.

Expression of VGF mRNA in developing neuroendocrine and endocrine tissues.

Analysis of knockout mice suggests that the neurotropin-inducible secreted polypeptide VGF (non-acronymic) plays an important role in the regulation of energy balance. VGF is synthesized by neurons in the central and peripheral nervous systems (CNS, PNS), as well as in the adult pituitary, adrenal medulla, endocrine cells of the stomach and pancreatic beta cells. Thus VGF, like cholecystokinin, leptin, ghrelin and other peptide hormones that have been shown to regulate feeding and energy expenditure, is synthesized in both the gut and the brain. Although detailed developmental studies of VGF localization in the CNS and PNS have been completed, little is known about the ontogeny of VGF expression in endocrine and neuroendocrine tIssues. Here, we report that VGF mRNA is detectable as early as embryonic day 15.5 in the developing rat gastrointestinal and esophageal lumen, pancreas, adrenal, and pituitary, and we further demonstrate that VGF mRNA is synthesized in the gravid rat uterus, together supporting possible functional roles for this polypeptide outside the nervous system and in the enteric plexus.

Regional differences in neurotrophin availability regulate selective expression of VGF in the developing limbic cortex.

Gene and protein expression patterns in the cerebral cortex are complex and often change spatially and temporally through development. The signals that regulate these patterns are primarily unknown. In the present study, we focus on the regulation of VGF expression, which is limited to limbic cortical areas early in development but later expands into sensory and motor areas. We isolated neurons from embryonic day 17 rat cortex and demonstrate that the profile of VGF expression in perirhinal (expressing) and occipital (nonexpressing) populations in vitro is similar to that in the perinatal cortex in vivo. The addition of neutralizing neurotrophin antibodies indicates that endogenous brain-derived neurotrophic factor (BDNF) is necessary for the normal complement of VGF-expressing neurons in the perirhinal cortex, although endogenous neurotrophin-3 (NT-3) regulates the expression of VGF in a subpopulation of cells. ELISA analysis demonstrates that there is significantly more BDNF present in the perirhinal cortex compared with the occipital cortex in the perinatal period. However, the total amount of NT-3 is similar between the two regions and, moreover, there is considerably more NT-3 than BDNF in both areas, a finding seemingly in conflict with regional VGF expression. Quantification of the extracellular levels of neurotrophins in perirhinal and occipital cultures using ELISA in situ analysis indicates that perirhinal neurons release significantly more BDNF than the occipital population. Furthermore, the amount of NT-3 released by the perirhinal neurons is significantly less than the amount of BDNF. Local injection of BDNF in vivo into a normally negative VGF region results in robust ectopic expression of VGF. These data suggest that the local availability of specific neurotrophins for receptor occupation, rather than the total amount of neurotrophin, is a critical parameter in determining the selective expression of VGF in the developing limbic cortex.

VGF: a novel role for this neuronal and neuroendocrine polypeptide in the regulation of energy balance.

Insight into the mechanisms of action of neurotrophic growth factors has been obtained through the identification and characterization of gene products that are regulated or modified at the transcriptional, translational, and/or posttranslational level in response to neurotrophin treatment. VGF (non-acronymic) was identified approximately 15 years ago as a nerve growth factor (NGF)-regulated transcript in rat PC12 pheochromocytoma cells. Subsequent studies have demonstrated that neurotrophins such as NGF and brain-derived neurotrophic factor induce vgf gene expression relatively rapidly in PC12 cells and cultured cortical neurons, respectively, in comparison to less robust regulation by epidermal growth factor (EGF) and insulin, growth factors which do not trigger the neuronal differentiation of PC12 cells. vgf gene expression is stimulated in vitro by NGF and the ras/map kinase signaling cascade through a CREB-dependent mechanism, while in vivo, VGF mRNA levels are regulated by neuronal activity, including long-term potentiation, seizure, and injury. Both the mRNA and encoded approximately 68-kDa protein (VGF) are selectively synthesized in neuroendocrine and neuronal cells. The predicted VGF sequence is rich in paired basic amino acid residues that are potential sites for proteolytic processing, and VGF undergoes regulated release from dense core secretory vesicles. Although VGF mRNA is synthesized widely, by neurons in the brain, spinal cord, and peripheral nervous system, its expression is particularly abundant in the hypothalamus. In addition, VGF peptides are found in hypophysial, adrenal medullary, gastrointestinal, and pancreatic endocrine cells, suggesting important neuroendocrine functions. Recent analysis of VGF knockout mice indeed demonstrates that VGF plays a critical role in the control of energy homeostasis. VGF knockout mice are thin, small, hypermetabolic, hyperactive, and relatively infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic pro-opiomelanocortin, neuropeptide Y, and agouti-related peptide expression. Coupled with the demonstration that VGF mRNA levels are induced in the normal mouse hypothalamic arcuate nuclei in response to fasting, important central and peripheral roles for VGF in the regulation of metabolism are suggested. Here we review previous studies of VGF in the broader context of its newly recognized role in the control of energy balance and propose several models and experimental approaches that may better define the mechanisms of action of VGF.

Targeted deletion of the Vgf gene indicates that the encoded secretory peptide precursor plays a novel role in the regulation of energy balance.

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Regulated expression during development and following sciatic nerve injury of mRNAs encoding the receptor tyrosine phosphatase HPTPzeta/RPTPbeta.

Three major isoforms of the receptor protein tyrosine phosphatase HPTPzeta/RPTPbeta (RPTPzeta/beta) have been previously identified, two with identical transmembrane and intracellular catalytic domains that differ by virtue of a long cysteine-free extracellular region, and a soluble proteoglycan called phosphacan that lacks the transmembrane and carboxy-terminal catalytic domains. To determine whether these RPTPzeta/beta variants are produced by alternative mRNA splicing of a common primary transcript, we performed genomic Southern analysis and characterized several rat cDNA and genomic RPTPzeta/beta clones. These studies indicated that the three major transcripts which encode phosphacan and the two RPTPzeta/beta phosphatase variants are encoded by a single gene, and further that additional alternative mRNA splicing is likely to result in the deletion of a 7 amino acid insert from the intracellular juxtamembrane region of both long and short phosphatase isoforms. Simultaneous quantitation of the three major isoforms by RNase protection analysis indicated that the mRNA encoding phosphacan had the highest relative abundance in the CNS while that encoding the short phosphatase isoform was most abundant relative to the other RPTPzeta/beta variants in the PNS. Following peripheral nerve crush, all RPTPzeta/beta mRNAs, including phosphacan and the phosphatase variants with and without the 21 base insert, were significantly induced in the distal segments of the sciatic nerve with a time course that correlated well with the response of Schwann cells to this injury.

Developmental expression of VGF mRNA in the prenatal and postnatal rat.

VGF is a developmentally regulated, secretory peptide precursor that is expressed by neurons and neuroendocrine cells and that has its transcription and secretion induced rapidly by neurotrophins and by depolarization. To gain insight into the possible functions and regulation of VGF in vivo, we have characterized the distribution of VGF mRNA in the developing rat nervous system. VGF expression was first detectable at embryonic day 11.5 in the primordia of cranial, sympathetic, and dorsal root ganglia, and its distribution expanded throughout development to include significant expression throughout the brain, spinal cord, and retina of the adult rat. The earliest expression of VGF, therefore, appeared in the peripheral nervous system as developing neurons settled in their designated ganglia. In many regions of the brain, VGF mRNA levels were found to be highest during periods when axonal outgrowth and synaptogenesis predominate. Areas of the central nervous system that contain predominantly dividing cells never displayed any VGF mRNA expression, nor did the vast majority of nonneural tissues.

Expression of VGF mRNA in the adult rat central nervous system.

VGF is a secretory peptide precursor that is expressed and processed by neuronal cells in a cell type-specific fashion. In addition, VGF transcription and secretion are rapidly and relatively selectively induced by neurotrophins and depolarization in vitro. To gain insight into the possible function(s) of VGF in the nervous system, we have carried out a detailed examination of the distribution of VGF mRNA in the adult rat central nervous system by using in situ hybridization. Robust expression was detected in many neurons throughout the brain and spinal cord, in several types of neurons in the retina, and in presumptive chromaffin cells of the adrenal medulla. In the brain, prominent expression of VGF mRNA was observed in neurons of the main and accessory olfactory bulbs; in the anterior olfactory nucleus; in the induseum griseum and taenia tecta; in the olfactory tubercle; in CA1-CA3, the hilus of the dentate gyrus, and the subicular complex of the hippocampal formation; in the piriform, periamygdaloid, transitional, and lateral entorhinal cortices; in the endopiriform nucleus; in the hypothalamus, particularly the preoptic, periventricular, supraoptic, suprachiasmatic, and arcuate nuclei; and in a number of septal, thalamic, amygdaloid, and brainstem nuclei. Labeling was also seen in neurons of the neocortex and transitional cortical areas, particularly in layer V, and in basal ganglia and cerebellum. These data demonstrate that VGF mRNA is expressed much more extensively in the brain than has been described in previous RNA or immunohistochemical studies, and, furthermore, that VGF is widely expressed in the spinal cord and retina.

The messenger RNA encoding VGF, a neuronal peptide precursor, is rapidly regulated in the rat central nervous system by neuronal activity, seizure and lesion.

The VGF gene encodes a neuronal secretory-peptide precursor that is rapidly induced by neurotrophic growth factors and by depolarization in vitro. VGF expression in the animal peaks during critical periods in the developing peripheral and central nervous systems. To gain insight into the possible functions and regulation of VGF in vivo, we have used in situ hybridization to examine the regulation of VGF messenger RNA by experimental manipulations, and have found it to be regulated in the CNS by paradigms that affect electrical activity and by lesion. Inhibition of retinal electrical activity during the critical period of visual development rapidly repressed VGF messenger RNA in the dorsal lateral geniculate nucleus of the thalamus. In the adult, kainate-induced seizures transiently induced VGF messenger RNA in neurons of the dentate gyrus, hippocampus, and cerebral cortex within hours. Cortical lesion strongly induced VGF messenger RNA in ipsilateral cortex within hours, and strongly repressed expression in ipsilateral striatum. Ten days postlesion there was a delayed induction of VGF messenger RNA in a portion of deafferented striatum where compensatory cortical sprouting has been detected. Expression of the neuronal secretory-peptide precursor VGF is therefore modulated in vivo by monocular deprivation, seizure, and cortical lesion, paradigms which lead to neurotrophin induction, synaptic remodeling and axonal sprouting.

Comparison of VGF and trk mRNA distributions in the developing and adult rat nervous systems.

The vgf gene encodes a neuronal secretory-peptide precursor that is rapidly induced by neurotrophic growth factors and by depolarization in vitro. To gain insight into the possible functions and regulation of VGF in vivo, we characterized the expression of VGF messenger RNA (mRNA) by in situ hybridization, during development and in adult brain, and directly compared it to the distributions of mRNAs encoding neurotrophin receptors TrkA, TrkB, and TrkC. Overlap in VGF and trk mRNA expression exists, and though no trk mRNA was exclusively colocalized with VGF message, the best correlation was found between the distributions of the VGF and trkB kinase mRNAs.

Expression and polarization of VGF in developing hippocampal neurons.

VGF is an NGF-inducible protein whose expression closely correlates with PC12 cell neurite outgrowth. Immunolocalization of VGF in PC12 cells by electron microscopy revealed labeling of a subpopulation of large dense core vesicles. To determine whether VGF expression may be involved in neural and axonal differentiation, we examined VGF immunoreactivity in developing cultured hippocampal neurons. Most VGF appeared to be in vesicles which were present in neurons from the earliest developmental stages and were particularly abundant in a small number of neurons, most of which were GABAergic. VGF became restricted to axons after dendrites had begun to mature, a stage more closely associated with synaptogenesis than axonal polarization. Colocalization studies suggest that VGF-containing and synaptophysin-containing vesicles are distinct and differentially distributed.

Activation of codependent transcription factors is required for transcriptional induction of the vgf gene by nerve growth factor and Ras.

Nerve growth factor (NGF) treatment of PC12 cells leads to the elaboration of a neuronal phenotype, including the induction of neuronally expressed genes such as vgf. To study vgf transcription, we have created chimeric vgf/beta-globin genes in which vgf promoter sequences drive the expression of the beta-globin reporter gene or of a chimeric beta-globin gene fused to 3' untranslated vgf gene sequences. We have found that the level of inducibility of the latter construct by NGF resembles that of the endogenous vgf gene. Using transient transfection of the chimeric reporter genes into PC12 cells, into PC12 subclones expressing activated or dominantly interfering mutant Ras proteins, and into PC12 variants expressing specific NGF receptor/Trk mutants, we show that transcriptional regulation of the vgf promoter by NGF is mediated through a Ras-dependent signaling pathway. By mutational analysis of the vgf promoter, we have identified three promoter elements involved in mediating transcriptional induction by NGF and Ras. In addition to the cyclic AMP-responsive element (CRE), which binds to ATF-1, ATF-2, and CRE-binding protein in PC12 nuclear extracts, a novel CCAAT element and its binding proteins were identified, which, like the CRE, is necessary but not sufficient for the Ras-dependent induction of the vgf gene by NGF. We also identify a G(S)G element unusually located between the TATA box and transcriptional start site, which binds the NGF- and Ras-induced transcription factor, NGFI-A, and amplifies the transcriptional response. Integrating data from studies of vgf promoter regulation and NGF signal transduction, we present a model for vgf gene induction in which transcriptional activation is achieved through the persistent, direct activation of multiple interacting transcription factors binding to CRE and CCAAT elements, coordinated with the delayed transcription factor action at a G(S)G element resulting from the induced expression of NGFI-A.

Comparison of RPTP zeta/beta, phosphacan, and trkB mRNA expression in the developing and adult rat nervous system and induction of RPTP zeta/beta and phosphacan mRNA following brain injury.

The receptor protein tyrosine phosphatase (RPTP) zeta/beta and a major isoform, phosphacan, a chondroitin sulfate proteoglycan that contains the RPTP zeta/beta extracellular domain but not the transmembrane and intracellular phosphatase domains, are expressed abundantly in the nervous system, primarily by astroglia. Because of similarities in the expression patterns of RPTP zeta/beta and the receptor tyrosine kinase TrkB, we investigated whether RNAs encoding these proteins were co-localized during development, which would suggest that these molecules might functionally interact in vivo. By in-situ hybridization, we noted extensive areas of overlap in the expression of trkB and RPTP zeta/beta mRNAs in the developing peripheral and central nervous systems. Analysis with a probe specific for the catalytic TrkB isoform suggested that RPTP zeta/beta and non-catalytic trkB mRNAs were co-expressed in particular regions of the nervous system while the catalytic trkB and RPTP zeta/beta transcripts were also, but to a lesser extent. RPTP zeta/beta and phosphacan expression were extremely similar, differing particularly in the level of expression in the ventricular and subventricular zones, hippocampus, and ependyma. Furthermore, both RPTP zeta/beta and phosphacan mRNAs were found in several subsets of neurons as well as astrocytes. Following CNS injury, we observed robust induction of RPTP zeta/beta mRNA in areas of axonal sprouting, and of both RPTP zeta/beta and phosphacan mRNAs in areas of glial scarring, implying that the encoded proteins and the cell adhesion molecules and extracellular matrix proteins to which they bind may contribute to recovery from injury and perhaps regulation of axonal regrowth in the nervous system.

Stimulation of vgf gene expression by NGF is mediated through multiple signal transduction pathways involving protein phosphorylation.

The vgf gene encodes one of the most rapidly induced neuronal mRNAs identified in NGF-treated PC12 cells. Maximal inhibition of VGF mRNA induction was achieved using K-252a, an inhibitor of the NGF-receptor Trk tyrosine kinase, and by mutating both Y490 (SHC association site) and Y785 (PLC-gamma 1 association site) of Trk. Inhibitors of the NGF-activated protein kinase N (PKN) were found to partially and in some cases transiently block VGF induction by NGF while in PKA-deficient PC12 cells, VGF induction by NGF was comparable to that observed in parental PC12 cells. The binding of NGF to Trk therefore activates redundant signal transduction pathways which converge to regulate vgf gene expression.

Characterization of a corticotropin-releasing hormone-responsive element in the rat proopiomelanocortin gene promoter and molecular cloning of its binding protein.

A corticotropin-releasing hormone (CRH) and cAMP-responsive region (-236/-133) in the rat POMC gene promoter previously reported to confer CRH/cAMP responsiveness to heterologous reporter constructs has been characterized. DNAse footprint analysis revealed that multiple elements in this region were bound by nuclear proteins from the POMC expressing AtT20 cells. When these individual DNA elements were separately tested in heterologous reporter constructs for CRH induction, only one element, designated PCRH-RE (POMC CRH responsive element, -171/-160) was found to give strong CRH stimulation (5- to 7-fold). This element appears novel as to the possible binding factors, although it has homology to the mouse metallothionein metal regulatory element. Gel shift analyses of the PCRH-RE with AtT20 cell nuclear extracts showed marked stimulation of retarded nucleoproteins following CRH stimulation, suggesting that the possible binding factor(s) may mediate transcriptional regulation at this site. The activity of PCRH-RE binding protein was inhibited by divalent cations, with Cu2+ and Cd2+ being most effective; Zn2+ had no effect, indicating that this binding factor(s) is functionally distinct from the metallothionein metal regulatory element binding protein. A 2.6 kilobase cDNA clone encoding a protein (PCRH-REB-1) binding to this element was isolated by Southwestern screening of an AtT20 expression library with radiolabeled PCRH-RE oligonucleotides. This clone was used to isolate several other cDNA clones to determine the sequence corresponding to the entire coding region of the protein (PCRH-REB), which proved to be identical to a recently described DNA binding protein of the replication factor C complex, mRFC140/Mouse Southwestern. Primer extension and Northern blot analysis revealed that the size of the full length mRNA is about 4.9 kilobases. PCRH-REB mRNA expression is not restricted to corticotrophs but is present in a broad tissue distribution as evaluated by reverse transcription polymerase chain reaction analysis. A bacterially expressed beta-galactosidase-PCRH-REB-1 fusion protein was shown to bind PCRH-RE efficiently. Furthermore, binding of the PCRH-REB-1 fusion protein to the POMC CRH-responsive element was inhibited by divalent cations with similar sensitivities to those observed using AtT20 nuclear extracts. The predicted PCHR-REB protein sequence presents several interesting motifs: one p-Loop motif (ATP binding site), nine protein kinase A phosphorylation sites (implying a possible role in responding to the CRH-induced cAMP signal), and regions of homology to proteins involved in DNA replication and repair. PCRH-REB is, therefore, a potential transacting factor binding to a major CRH-responsive element in the POMC promoter.