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Several lines of evidence over the last decade have established that G protein-coupled receptors (GPCRs) can signal in the absence of their natural ligand which results in ligand-independent or constitutive activity. Natural genetic mutation, overexpression and site-directed mutagenesis all result in constitutive activation of GPCRs. Of the 100 leading pharmaceutical products in 2000, 39, wholly or in part, acted through a GPCR-mediated mechanism, a fact that underlines the extreme importance of GPCRs as pharmaceutical drug targets. In addition, the sequencing of the human genome and database mining has revealed that there are hundreds of putative orphan GPCRs for which the natural ligands have not been identified. These orphan GPCRs have largely been inaccessible to drug discovery because traditional methods have mainly relied on ligand-dependent binding assays to discover and pharmacologically characterize potential drug candidates from this receptor class. In the absence of ligand identification, constitutively active receptors allow for a logical and direct way forward through the drug discovery pathway by providing the tool necessary to find modulators of this receptor class in a ligand-independent fashion.

Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role in normal growth and brain development and protect brain cells from several forms of injury. The effects of IGFs are mediated by type-I and type-II receptors and modulated by potentially six specific binding proteins that form high-affinity complexes with IGFs in blood and cerebrospinal fluid (CSF) and under most circumstances inactivate them. Because brain injury is commonly associated with increases in IGFs and their associated binding proteins, we hypothesized that displacement of this large "pool" of endogenous IGF from the binding proteins would elevate "free" IGF levels to elicit neuroprotective effects comparable to those produced by administration of exogenous IGF. A human IGF-I analog [(Leu24, 59, 60, Ala31)hIGF-I] with high affinity to IGF-binding proteins (Ki = 0.3-3.9 nM) and no biological activity at the IGF receptors (Ki = >10,000 nM) increased the levels of "free, bioavailable" IGF-I in the CSF. Intracerebroventricular administration of this analog up to 1h after an ischemic insult to the rat brain had a potent neuroprotective action comparable to IGF-I. This novel strategy for increasing "free" IGF levels in the brain may be useful for the treatment of stroke and other neurodegenerative diseases.

In Alzheimer's disease (AD) there are dramatic reductions in human corticotropin-releasing factor (hCRF) concentration and reciprocal increases in CRF receptor density in the cortex. hCRF-binding protein (hCRF-BP), hCRF/hCRF-BP complex, and "free" hCRF were measured in 10 brain regions from control and AD postmortem human tissue. In the control brains hCRF-BP was heterogenously distributed and levels were at least 10-fold higher on a molar basis than total hCRF levels, suggesting that one major role of the binding protein is to limit the actions of hCRF at the hCRF receptors. Concordant with this hypothesis, the percentage of total hCRF that was in the bound inactive form ranged from 65 to 90% in most areas examined, with the exception of the caudate and globus pallidus where only 15 and 40% were complexed, respectively. hCRF-BP concentrations were similar in the control and AD groups except for Brodmann area (BA) 39 where there was a small but significant decrease in the AD group. Complexed hCRF levels were significantly decreased in BA 8/BA 9, BA 22, BA 39, nucleus basalis, and globus pallidus in the Alzheimer's group and free hCRF levels were significantly decreased only in three brain areas, BA 4, BA 39, and caudate; substantial (40%) but nonsignificant decreases were also noted in BA 8/BA 9 and BA 22. These data demonstrate that (1) a large proportion of the total hCRF in human brain is complexed to hCRF-BP and thus unavailable for hCRF receptor activation, (2) reductions in total hCRF alone do not necessarily predict reductions in bioactive free hCRF, and (3) total hCRF levels and hCRF-BP levels appear to be the main factors determining the quantity of bound and free hCRF in human brain.

Evidence favors a role for corticotropin-releasing factor (CRF) in learning and memory processes. A binding protein (CRF-BP) with the ability to inactivate CRF provides a novel target to modulate endogenous levels of CRF. The present studies employed three measures of information processing in rats in order to examine the impact of CRF system activation resulting from administration of CRF-BP ligand inhibitors, which increase levels of "free CRF." Acquisition of a visual discrimination paradigm and retention of a inhibitory avoidance task were dose dependently facilitated by central administration of a CRF-BP ligand inhibitor. CRF-BP ligand inhibitor treatment also improved performance in an active avoidance paradigm in aged animals. No nonspecific anorexic effects of the active dose of CRF-BP ligand inhibitor were detected in a food intake test. Moreover, the magnitude of in vivo efficacy of the CRF-BP ligand inhibitor peptide in producing a mild increase in motor activity was dissociated from that of a postsynaptic CRF receptor agonist that exerted robust and long-lasting activity increases. Thus, CRF-BP ligand inhibitors appear to elicit generalized learning enhancement effects without mimicking the robust nonspecific behavioral actions of a CRF receptor agonist.

Elevation of the neuropeptide corticotropin-releasing factor (CRF) in the brain is associated with a reduction of food intake and body weight gain in normal and obese animals. A protein that binds CRF and the related peptide, urocortin, with high affinity, CRF-binding protein (CRF-BP), may play a role in energy homeostasis by inactivating members of this peptide family in ingestive and metabolic regulatory brain regions. Intracerebroventricular administration in rats of the high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which dissociates CRF or urocortin from CRF-BP and increases endogenous brain levels of "free" CRF or urocortin significantly blunted exaggerated weight gain in Zucker obese subjects and in animals withdrawn from chronic nicotine. Chronic administration of CRF suppressed weight gain nonselectively by 60% in both Zucker obese and lean control rats, whereas CRF-BP ligand inhibitor treatment significantly reduced weight gain in obese subjects, without altering weight gain in lean control subjects. Nicotine abstinent subjects, but not nicotine-naive controls, experienced a 35% appetite suppression and a 25% weight gain reduction following acute and chronic administration, respectively, of CRF-BP ligand inhibitor. In marked contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand inhibitor did not stimulate adrenocorticotropic hormone secretion or elevate heart rate and blood pressure. These results provide support for the hypothesis that the CRF-BP may function within the brain to limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP may represent a novel and functionally selective target for the symptomatic treatment of excessive weight gain associated with obesity of multiple etiology.

The actions of CRF in the brain and in the periphery are mediated through multiple binding sites. There are three receptors, CRF1, CRF2 alpha and CRF2 beta, which encode 411, 415 and 431 amino acid proteins and transduce signals via the stimulation of intracellular cAMP production. The recent identification of high-affinity non-peptide CRF receptor antagonists should allow for rapid progress in drug development of CRF receptor antagonists. In addition to the receptors, the actions of CRF in brain and in the periphery can also be modulated by a binding protein of 322 amino acids. Ligands of CRF-BP, such as CRF (6-33) can elevate brain levels of 'free' CRF and improve learning and memory without stress-like side effects of CRF receptor agonists. Urocortin, a mammalian CRF-related peptide with close sequence homology to fish urotensin, interacts with CRF1, CRF2 receptors and with CRF-BP. These data indicate that CRF receptor antagonists may be useful for the treatment of the disease states where CRF is elevated such as anxiety and depression, anorexia nervosa and stroke and that ligand inhibitors of CRF-BP may be used to elevate brain levels of 'free' urocortin and other CRF-related peptides.

Here we demonstrate that urocortin, a new mammalian member of the corticotropin-releasing factor (CRF) neuropeptide family has high affinity for both the recombinant human CRF binding protein (CRF-BP) and for a membrane-associated form of the protein solubilized from postmortem human cerebrocortical brain tissue. The rank order of binding potency for both the human recombinant CRF-BP and for the solubilized human brain CRF-BP is: urotensin > hCRF > urocortin > sauvagine. The bound hCRF/hCRF-BP complex was detected in the postmortem human brain tissue using an ELISA assay specific for the hCRF/hCRF-BP complex. A large proportion (65%) of the endogenous hCRF was found to be complexed to the CRF-BP and thus unavailable for CRF receptor activation. Incubation of human brain postmortem tissue extracts with urocortin and urotensin resulted in a dramatic decrease in hCRF/hCRF-BP levels and a concomitant increase in "free' hCRF levels. Thus, urocortin and other putative CRF-related peptides may elevate endogenous levels of "free' hCRF in brain by displacing hCRF from the binding protein. These data define an indirect endogenous mechanism for activation of CRF receptors by new mammalian members of the CRF family of neuropeptides.

Corticotrophin-releasing factor (CRF) acts within both the brain and the periphery to coordinate the overall response of the body to stress. The involvement of the CRF systems in a variety of both CNS and peripheral disease states has stimulated great interest in this peptide as a potential site of therapeutic intervention. The recent cloning of multiple CRF receptor subtypes has precipitated a new era in CRF research that has allowed precise molecular, pharmacological and anatomical examination of mammalian CRF receptors. In this article, Derek Chalmers and colleagues highlight the major differences between the two classes of CRF receptors, CRF1 and CRF2, and a functionally related CRF-binding protein, and discuss the relevance of these sites to the ongoing development of CRF-based therapeutics.

In Alzheimer's disease (AD) there are dramatic reductions in the content of corticotropin releasing factor (CRF), reciprocal increases in CRF receptors, and morphological abnormalities in CRF neurons in affected brain areas. Cognitive impairment in AD patients is associated with a lower cerebrospinal fluid concentration of CRF, which is known to induce increases in learning and memory in rodents. This suggests that CRF deficits contribute to cognitive impairment. The identification in post-mortem brain of CRF-binding protein (CRF-BP), a high-affinity binding protein that inactivates CRF, and the differential distribution of CRF-BP and CRF receptors, provides the potential for improving learning and memory without stress effects of CRF receptor agonists. Here we show that ligands that dissociate CRF from CRF-BP increase brain levels of 'free CRF' in AD to control levels and show cognition-enhancing properties in models of learning and memory in animals without the characteristic stress effects of CRF receptor agonists.

Corticotropin releasing factor (CRF) binding protein (CRF-BP) was measured in media and cell lysates of primary rat astrocytes, microglia and neurons with the use of a ligand immunoradiometric assay (LIRMA). A low basal level of CRF-BP was detected in the media and cell lysates from primary neuronal and astrocyte cells after 48 h in culture. No basal expression of CRF-BP was detected in cell lysates or media from primary microglial cultures. The CRF-BP expressed in cultured astrocytes and neurons had the same pharmacological characteristics as the human recombinant molecule. After forskolin, IBMX or forskolin/IBMX treatment, a robust increase in secreted CRF-BP levels in the media from astrocytes and neurons, but not microglia, was observed. An increase in CRF-BP-like immunoreactivity in cell lysates was also observed after IBMX/forskolin treatment. In situ hybridization analysis revealed that CRF-BP mRNA was increased in primary cultured astrocytes after IBMX/forskolin stimulation suggesting that regulation was at the level of gene transcription. 'Axon sparing' lesions produced with 0.12 M quinolinic acid in PBS injected intracerebrally (unilaterally into dorsal hippocampus) resulted in loss of CRF-BP expression in neurons. These data provide evidence for the differential localization and regulation of CRF-BP in different cell types in brain and suggest that CRF-BP expression may be locally increased in disease states associated with astrocytosis and gliosis.

A 37-kDa corticotropin releasing factor (CRF) binding protein (CRF-BP) was purified from human plasma by repeated affinity purification and subsequently sequenced and cloned. The human and rat CRF-BP cDNAs encode proteins of 322 amino acids with one putative signal sequence, one N-glycosylation site, and 10 conserved cysteines. Human CRF-BP binds human CRF with high affinity but has low affinity for the ovine peptide. In contrast, sheep CRF-BP binds human and ovine CRF with high affinity. The CRF-BP gene consists of seven exons and six introns and is located on chromosome 13 and loci 5q of the mouse and human genomes, respectively. CRF-BP inhibits the adrenocorticotrophic hormone (ACTH) releasing properties of CRF in vitro. CRF-BP dimerizes after binding CRF and clears the peptide from blood. This clearance mechanism protects the maternal pituitary gland from elevated plasma CRF levels found during the third trimester of human pregnancy. CRF-BP is expressed in the brains of all species so far tested but is uniquely expressed in human liver and placenta. In brain, CRF-BP is membrane associated and is predominantly expressed in the cerebral cortex and subcortical limbic structures. In some brain areas CRF-BP colocalizes with CRF and CRF receptors. The protein is also present in pituitary corticotropes, where it is under positive glucocorticoid control, and is likely to locally modulate CRF-induced ACTH secretion. The ligand requirements of the CRF receptor and the CRF-BP can be distinguished in that central human CRF fragments, such as CRF (6-33) and CRF (9-33), have high affinity for CRF-BP but low affinity for the CRF receptor. The binding protein's ability to inhibit CRF-induced ACTH secretion can be reversed by CRF (6-33) and CRF (9-33), suggesting that ligand inhibitors may have utility in elevating free CRF levels in disease states associated with decreased CRF. Thus, by controlling the amount of free CRF which activates CRF receptors, it is likely that the CRF-BP is an important modulator of CRF both in the CNS and in the periphery.

We have previously isolated, cloned, and characterized a protein that specifically binds and inactivates the peptide corticotropin-releasing factor. The integrity of the disulfide bonds in the binding protein is essential for this activity as reduction abolishes the protein's ability to bind corticotropin-releasing factor. The disulfide arrangement of the 10 cysteines present in the mature protein was established by analysis of proteolytically cleaved protein and sequence analysis of cystine containing fragments. A pattern is observed where each cysteine is connected to the next one in a sequential manner. Inspection of the genomic DNA encoding for this protein reveals that four of the domains defined by disulfide linkage coincide with four different exons.

The human CRF-binding protein gene has been cloned and mapped to the distal region of chromosome 13 and loci 5q in the mouse and human genomes, respectively. The gene consists of 7 exons and 6 introns. The mature protein has 10 cysteines and 5 tandem disulfide bridges 4 of which are contained within exons 3, 5, 6, and 7. One bridge is shared by exons 3 and 4. The signal peptide and the first 3 amino acids of the mature protein were coded for by an extreme 5' exon. Primer extension analyses revealed the transcriptional initiation site to be located 32 bp downstream from a consensus TATA box. The promoter sequence contained a number of putative promoter elements including an AP-1 site, three ER-half sites, the immunoglobulin enhancer elements NF-kappa B and INF-1, and the liver-specific enhancers LFA1 and LFB1.

This study is the first to use CRH-binding protein (CRH-BP) purified from human plasma to investigate how the CRH-BP affects the physiological activity of CRH. After incubation at 37 C for 15 min, purified CRH-BP reduced the ACTH-releasing activity of synthetic human (h) CRH in a dose-dependent fashion; using hCRH and CRH-BP at concentrations commonly found in late gestational maternal plasma, (1.5 and 100 ng/mL, respectively) an average 76% reduction in ACTH release was obtained. No effect was observed under the same conditions on ACTH release induced by ovine (o) CRH, which does not bind to CRH-BP. Kinetic estimations are presented to show that the extent of binding of CRH to its BP (and, hence, the reduction in its bioactivity) depends on the time available for binding and the concentration of reactants. Equilibrium between CRH and its BP at the concentrations used in the bioactivity studies take place within 400 s. Since long term secretion of placental CRH into the peripheral circulation occurs during the third trimester of pregnancy, we suggest that the presence of circulating CRH BP may partly explain how markedly elevated plasma levels of CRH coexist with normal ACTH levels at this time. We also propose that stress-induced CRH release will not be similarly quenched by the CRH-BP in the hypothalamic portal system, as the concentration of CRH released will be high, and the exposure time before reaching pituitary corticotropes will be low. Using pituitary cells constantly bathed in BP premixed with hCRH or BP alone (to mimic the situations in pregnancy and nonpregnancy, respectively), we show that short concentrated pulses of synthetic CRH (10 ng in 5 s) or rat stalk median eminence extract (one tenth stalk median eminence in 5 s) retain their ability to induce ACTH secretion despite the presence of CRH-BP. It is, thus, possible that CRH can still exert its role as a stress hormone in late gestation.