A Screening Strategy Based on Differential Binding of Ligand to Receptor and to Binding Proteins: Screening for Compounds Interacting with Corticotrophin-Releasing Factor-Binding Protein

The ligand-receptor interaction has been commonly used in development of high throughput screening assays for new drugs. In some cases, an endogenous ligand interacts not only with membrane receptors but also with soluble binding proteins. Corticotrophin-releasing factor (CRF) is an important stress neurotransmitter/hormone involved in both the central and peripheral nervous systems. CRF exerts its function by interacting with CRFR1 and CRFR2 receptors. In addition, CRF-binding protein (CRF-BP) binds CRF with high affinity. Accordingly, CRF-BP has been suggested to play an important role in modulating CRF function. Based on the potential involvement of CRF-BP in many neurological disorders, it is desirable to develop a screening assay to look for drugs that either mimic or interfere with CRF binding to CRF-BP. An assay was developed to monitor the interactions of radiolabeled CRF with human/rat CRF-BP and the mouse CRFR1 (mCRFR1) receptor. By carefully examining the binding characteristics of radiolabeled CRF to mCRFR1, the assay was able to identify compounds that bind to CRF-BP with high affinity and have little or no affinity for mCRFR1 receptors. Based on a mathematical model, we have verified the screening system with several well-characterized CRF ligands that all have different affinities for CRF receptors and CRF-BP.     

Structural basis for hormone recognition by the Human CRFR2{alpha} G protein-coupled receptor

The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2α isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2α ECD bound to each of the Ucn peptides. The CRFR2α ECD forms the same fold observed for the CRFR1 and mouse CRFR2β ECDs but contains a unique N-terminal α-helix formed by its pseudo signal peptide. The CRFR2α ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the α-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2α Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.     

The Effects of Corticoptropin-Releasing Factor and the Urocortins on Striatal Dopamine Release Induced by Electrical Stimulation—An in vitro Superfusion Study

The members of the CRF peptide family, corticotropin-releasing factor (CRF), urocortin I (Ucn I), urocortin II (Ucn II) and urocortin III (Ucn III) coordinate endocrine and behavioral responses to stress. CRF has also been demonstrated to stimulate dopamine (DA) synthesis.In our study, a superfusion system was used to investigate the effects of this peptide family on striatal DA release following electrical stimulation. The involvement of the CRF receptors was studied by pretreatment of rat striatal slices with selective CRF antagonists. CRF and Ucn I increased the release of [³H]DA while Ucn II and Ucn III were ineffective. The CRFR1 antagonist antalarmin inhibited the [³H]DA release induced by electrical stimulation and enhanced by CRF and Ucn I. The CRFR2 antagonist astressin-2B was ineffective.These results suggest that CRF and Ucn I mediate DA release through the activation of CRFR1. Ucn II and Ucn III are not involved in this process.Special Issue Dedicated to Miklós Palkovits.     

High-affinity binding of urocortin and astressin but not CRF to G protein-uncoupled CRFR1

The structure-activity relationship (SAR) between the recently identified neuropeptide urocortin (Ucn) and corticotropin-releasing factor (CRF) receptor, type 1 (CRFR1), has been investigated. To this end, rat Ucn (rUcn), ovine CRF (oCRF) and chimeric peptides of rUcn and oCRF were synthesized and tested for their binding affinity and potency to stimulate cAMP production in human embryonic kidney (HEK) 293 cells stably transfected with cDNA encoding rat CRFR1 (rCRFR1). In binding studies with [125I-TyrO]oCRF or [3H-Leu9]rUcn as radioligand, it was observed that rUcn but not oCRF bound in a similar fashion as the CRF antagonist astressin with high affinity to rCRFR1 coupled to G protein or uncoupled from G protein by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Consequently, rUcn was found to exert a significantly lower potency than oCRF to stimulate cAMP accumulation in transfected cells. CD spectroscopic investigations and reverse-phase HPLC (RPHPLC) retention behavior of the peptides suggested a more pronounced amphipatic alpha-helical character of rUcn when compared to oCRF and the chimeric peptides.     

The role of corticotropin-releasing factor receptors in stress and anxiety

Corticotropin releasing factor (CRF) is a critical integratorof the hypothalamic-pituitary-adrenal (HPA) axis in responseto stress. CRF and its related molecule urocortin (UCN) bindCRF receptor 1 (CRFR1) and CRFR2 with distinct affinities. Micedeficient for CRFR1 or CRFR2 were generated in order to determinethe physiological role of these receptors. While CRFR1-mutantmice show a depleted stress response and display anxiolytic-likebehavior, CRFR2-mutant mice are hypersensitive to stress anddisplay anxiogenic-like behavior. Both CRFR1- and CRFR2-mutantmice show normal basal feeding and weight gain, but CRFR2-mutantmice exhibit decreased food intake following a stress of fooddeprivation. While CRFR2-mutant mice display increased levelsof CRF mRNA in the central nucleus of the amygdala (cAmyg) butnot in the paraventricular nucleus of the hypothalamus (PVN),the CRFR1-mutant mice express high levels of CRF in the PVNbut normal levels in the cAmyg. CRFR2-mutant mice also displayincreased levels of Ucn mRNA and protein in the edinger westphalnucleus (EW) as well as an increased number of cells expressingUcn. The levels of these CRF-receptor ligands reflect the stateof the receptor-deficient mice. These results demonstrate apossible modulatory function of CRFR2 in response to CRFR1 stimulationof the HPA axis or anxiety.     

Regulation of corticotropin-releasing factor receptor type 2beta mRNA by mitogen-activated protein kinases in aortic smooth muscle cells

The actions of the corticotropin-releasing factor (CRF) family of peptides are mediated by the seven transmembrane-domain G-protein-coupled receptors, the CRF receptors. CRF receptor type 2beta (CRFR2beta) messenger RNA (mRNA) is expressed primarily in the cardiovascular system, where its levels are decreased by urocortin 1 (Ucn1), a novel peptide in the CRF family. In a previous study, we reported that CRFR2beta mRNA levels were partially down-regulated via the cAMP-protein kinase A pathway. This study focused on the involvement of the intracellular mitogen-activated protein (MAP) kinase pathway in the modulation of CRFR2beta mRNA levels. Ribonuclease protection assays showed that decreases in CRFR2beta mRNA levels induced by Ucn1 and cAMP were attenuated by the p38 MAP kinase inhibitor SB202190 or SB203580. This finding suggested that the p38 MAP kinase pathway was involved in this regulation. Anisomycin, a classic p38 kinase activator, increased CRFR2beta mRNA levels in A7r5 cells. This effect of anisomycin was completely reversed by H7, a serine/threonine kinase inhibitor, while both p38 kinase and MAP kinase kinase inhibitors failed to block the increase in CRFR2beta mRNA levels caused by anisomycin. As anisomycin can activate Jun amino terminal kinases, as well as p38 MAP kinase, it is possible that other MAP kinases, such as Jun amino terminal kinases, also contribute to the increase in gene levels. Alternatively, anisomycin may increase CRFR2beta mRNA levels indirectly as a consequence of blocking protein synthesis.     

Endothelin-converting enzyme-1 actions determine differential trafficking and signaling of corticotropin-releasing factor receptor 1 at high agonist concentrations

CRF receptor 1 (CRF(1)), a key neuroendocrine mediator of the stress response, has two known agonists corticotropin-releasing factor (CRF) and urocortin 1 (Ucn1). Here we report that endothelin-converting enzyme-1 (ECE-1) differentially degrades CRF and Ucn1; ECE-1 cleaves Ucn1, but not CRF, at critical residue Arginine-34/35', which is essential for ligand-receptor binding. At near K(D) agonist concentration (30 nm), both Ucn1- and CRF-mediated Ca(2+) mobilization are ECE-1 dependent. Interestingly, at high agonist concentration (100 nm), Ucn1-mediated Ca(2+) mobilization remains ECE-1 dependent, whereas CRF-mediated mobilization becomes independent of ECE-1 activity. At high agonist concentration, ECE-1 inhibition disrupted Ucn1-, but not CRF-induced CRF(1) recycling and resensitization, but did not prolong the association of CRF(1) with β-arrestins. RNA interference-mediated knockdown of Rab suggests that both Ucn1- and CRF-induced CRF(1) resensitization is dependent on activity of Rab11, but not of Rab4. CRF(1) behaves like a class A G protein-coupled receptor with respect to transient β-arrestins interaction. We propose that differential degradation by ECE-1 is a novel mechanism by which CRF(1) receptor is protected from overactivation by physiologically relevant high concentrations of higher affinity ligand to mediate distinct resensitization and downstream signaling.     

Urocortins and the regulation of gastrointestinal motor function and visceral pain

Urocortin (Ucn) 1, 2 and 3 are corticotropin-releasing factor (CRF)-related peptides recently characterized in mammals. Urocortin 1 binds with high affinity to CRF type 1 (CRF1) and type 2 (CRF2) receptors while Ucn 2 and Ucn 3 are selective CRF2 ligands. They also have a distinct pattern of distribution, both in the brain and the gastrointestinal tract, compatible with a role mediating, with CRF, the response to stress. In rats and mice, Ucn 1 injected centrally or peripherally inhibited gastric emptying and stimulated colonic propulsive motor function, mimicking the effects of stress or exogenous CRF. Centrally administered Ucn 2 inhibited gastric emptying with similar potency as CRF, while Ucn 1 and Ucn 3 were less potent. However, after peripheral administration, Ucn 1 and Ucn 2 were more potent than CRF. In mice, centrally administered Ucn 1 and 2 stimulated colonic motility with lower potency than CRF, and Ucn 3 was inactive. Studies with selective CRF1 and CRF2 antagonists demonstrated that the gastric-inhibitory and colonic-stimulatory effects of exogenously administered Ucns are mediated through CRF2 and CRF1 receptors, respectively. In addition, Ucn 2 showed visceral anti-nociceptive activity associated with the selective activation of CRF2 receptors. These observations suggest that, acting centrally and peripherally, Ucns might play a significant role in the modulation of gastrointestinal motor and pain responses during stress and stress-related pathophysiological conditions.     

The effects of corticotropin-releasing factor and the urocortins on hypothalamic gamma-amino butyric acid release--the impacts on the hypothalamic-pituitary-adrenal axis

Corticotropin-releasing factor (CRF) and the urocortins (UCNs) are structurally and pharmacologically related neuropeptides which regulate the endocrine, autonomic, emotional and behavioral responses to stress. CRF and UCN1 activate both CRF receptors (CRFR1 and CRFR2) with CRF binding preferentially to CRFR1 and UCN1 binding equipotently to both receptors. UCN2 and UCN3 activate selectively CRFR2. Previously an in vitro study demonstrated that superfusion of both CRF and UCN1 elevated the GABA release elicited by electrical stimulation from rat amygdala, through activation of CRF1 receptors. In the present experiments, the same in vitro settings were used to study the actions of CRF and the urocortins on hypothalamic GABA release. CRF and UCN1 administered in equimolar doses increased significantly the GABA release induced by electrical stimulation from rat hypothalamus. The increasing effects of CRF and UCN1 were inhibited considerably by the selective CRFR1 antagonist antalarmin, but were not influenced by the selective CRFR2 antagonist astressin 2B. UCN2 and UCN3 were ineffective. We conclude that CRF1 receptor agonists induce the release of GABA in the hypothalamus as well as previously the amygdala. We speculate that CRF-induced GABA release may act as a double-edged sword: amygdalar GABA may disinhibit the hypothalamic CRF release, leading to activation of the hypothalamic-pituitary-adrenal axis, whereas hypothalamic GABA may inhibit the hypothalamic CRF release, terminating this activation.     

An oxidation resistant radioligand for corticotropin-releasing factor receptors.

The methionine residues in Tyr-corticotropin-releasing factor (CRF) and Tyr-sauvagine radioligands are subject to oxidation, which renders them biologically inactive. Therefore [Tyr(0,) Gln(1,) Leu(17)]sauvagine (YQLS), in which the methionine was replaced with leucine was synthesized and labeled with (125)Iodine using chloramine-T. Mass spectroscopy revealed that chloramine-T-treatment did not oxidize YQLS. (125)I-YQLS bound with high affinity to cells expressing the murine CRF receptor 1 (CRFR1), CRF receptor 2 (CRFR2), and the mouse brain regions known to express both CRF receptors. (125)I-YQLS chemically cross-linked to CRFR1. In conclusion, (125)I-YQLS is oxidation-resistant, high affinity radioligand that can be chemically cross-linked to the CRF receptors.     

Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells

Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo-pituitary-adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF(1) receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF(2) receptor) with little or no binding affinity for the CRF(1) receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF(1) receptor and CRF(2) receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF(1) receptor, while Ucn2 increased the levels via the CRF(2) receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.     

A soluble form of the first extracellular domain of mouse type 2beta corticotropin-releasing factor receptor reveals differential ligand specificity

The heptahelical receptors for corticotropin-releasing factor (CRF), CRFR1 and CRFR2, display different specificities for CRF family ligands: CRF and urocortin I bind to CRFR1 with high affinity, whereas urocortin II and III bind to this receptor with very low affinities. In contrast, all the urocortins bind with high affinities, and CRF binds with lower affinity to CRFR2. The first extracellular domain (ECD1) of CRFR1 is important for ligand recognition. Here, we characterize a bacterially expressed soluble protein, ECD1-CRFR2beta, corresponding to the ECD1 of mouse CRFR2beta. The K(i) values for binding to ECD1-CRFR2beta are: astressin = 10.7 (5.4-21.1) nm, urocortin I = 6.4 (4.7-8.7) nm, urocortin II = 6.9 (5.8-8.3) nm, CRF = 97 (22-430) nm, urocortin III = sauvagine >200 nm. These affinities are similar to those for binding to a chimeric receptor in which the ECD1 of CRFR2beta replaces the ECD of the type 1B activin receptor (ALK4). The ECD1-CRFR2beta possesses a disulfide arrangement identical to that of the ECD1 of CRFR1, namely Cys(45)-Cys(70), Cys(60)-Cys(103), and Cys(84)-Cys(118). As determined by circular dichroism, ECD1-CRFR2beta undergoes conformational changes upon binding astressin. These data reinforce the importance of the ECD1 of CRF receptors for ligand recognition and raise the interesting possibility that different ligands having similar affinity for the full-length receptor may, nevertheless, have different affinities for microdomains of the receptor.     

Juxtamembrane region of the amino terminus of the corticotropin releasing factor receptor type 1 is important for ligand interaction

The functional properties of the amino terminus (NT) of the corticotropin releasing factor (CRF) receptor type 1 (R1) were studied by use of murine (m) CRFR1 and rat (r) parathyroid hormone (PTH)/parathyroid hormone-related peptide receptor (PTH1R) chimeras. The chimeric receptor CXP, in which the NT of mCRFR1 was annealed to the TMs of PTH1R, and the reciprocal hybrid, PXC, bound radiolabeled analogues of sauvagine and PTH(3--34), respectively. Neither hybrid bound radiolabeled CRF or PTH(1--34). CRF and PTH(1--34) weakly stimulated intracellular cAMP accumulation in COS-7 cells transfected with PXC and CXP, respectively. Thus the NT is required for ligand binding and the TMs are required for agonist-stimulated cAMP accumulation. Replacing individual intercysteine segments of PXC with their mCRFR1 counterparts did not rescue CRF or sauvagine radioligand binding or stimulation of cAMP accumulation. Replacement of residues 1--31 of mCRFR1 with their PTH1R counterparts resulted in a chimeric receptor, PEC, which had normal CRFR1 functional properties. In addition, a series of chimeras (F1PEC--F6PEC) were generated by replacement of the NT intercysteine residues of PEC with their PTH1R counterparts. Only F1PEC, F2PEC, and F3PEC showed detectable CRF and sauvagine radioligand binding. All of the PEC chimeras except F5PEC increased cAMP accumulation. These data indicate that the Cys(68)(-)Glu(109) domain is important for binding and that the Cys(87)(-)Cys(102) region plays an important role in CRFR1 activation.     

Regulation of corticotropin-releasing factor receptor type 2beta mRNA via cyclic AMP pathway in A7r5 aortic smooth muscle cells

Corticotropin-releasing factor receptor type 2beta (CRF R2beta) is a member of the Class B heptahelical G protein-coupled receptors. This receptor is positively coupled to adenylate cyclase and is bound preferentially by the CRF-related peptides, urocortin (Ucn), Ucn II and Ucn III. In the rodent, CRF R2beta messenger RNA (mRNA) is expressed in the cardiovascular system, where its levels can be modulated by Ucn. In the present study, we investigated regulation of CRF R2beta levels by Ucn in A7r5 aortic smooth muscle cells. Ribonuclease protection assays show that A7r5 cells expressed the CRF R2beta subtype, which had two isoforms differing in one codon at the junction of exons 3 and 4. Ucn induced accumulation of intracellular cAMP via CRF R2beta in this cell line. In addition to the treatment with Ucn, cAMP agonists or analogues themselves caused a significant decrease in CRF R2beta mRNA levels. Blockade of Ucn- or cAMP-induced decreases in CRF R2beta mRNA levels by H7, a broad protein kinase inhibitor, suggested that a protein kinase pathway might be involved in this regulation. H89, a protein kinase A inhibitor, partially blocked Ucn- or cAMP-induced decreases in CRF R2beta mRNA levels. Thus, Ucn induces intracellular cAMP to downregulate CRF R2beta mRNA expression in A7r5 cells.     

Dissection of corticotropin‐releasing factor system involvement in locomotor sensitivity to methamphetamine

Sensitivity to the euphoric and locomotor‐activating effects of drugs of abuse may contribute to risk for excessive use and addiction. Repeated administration of psychostimulants such as methamphetamine (MA) can result in neuroadaptive consequences that manifest behaviorally as a progressive escalation of locomotor activation, termed psychomotor sensitization. The present studies addressed the involvement of specific components of the corticotropin‐releasing factor (CRF) system in locomotor activation and psychomotor sensitization induced by MA (1, 2 mg/kg) by utilizing pharmacological approaches, as well as a series of genetic knockout (KO) mice, each deficient for a single component of the CRF system: CRF‐R1, CRF‐R2, CRF, or the CRF‐related peptide Urocortin 1 (Ucn1). CRF‐R1 KO mice did not differ from wild‐type mice in sensitization to MA, and pharmacological blockade of CRF‐R1 with CP‐154,526 (15, 30 mg/kg) in DBA/2J mice did not selectively attenuate either the acquisition or expression of MA‐induced sensitization. Deletion of either of the endogenous ligands of CRF‐R1 (CRF, Ucn1) either enhanced or had no effect on MA‐induced sensitization, providing further evidence against a role for CRF‐R1 signaling. Interestingly, deletion of CRF‐R2 attenuated MA‐induced locomotor activation, elucidating a novel contribution of the CRF system to MA sensitivity, and suggesting the participation of the endogenous urocortin peptides Ucn2 and Ucn3. Immunohistochemistry for Fos was used to visualize neural activation underlying CRF‐R2‐dependent sensitivity to MA, identifying the basolateral and central nuclei of the amygdala as neural substrates involved in this response. Our results support further examination of CRF‐R2 involvement in neural processes associated with MA addiction.     

Corticotropin-releasing factor receptor subtypes mediating nutritional suppression of estrous behavior in Syrian hamsters

Caloric deprivation inhibits reproduction, including copulatory behaviors, in female mammals. Decreases in metabolic fuel availability are detected in the hindbrain, and this information is relayed to the forebrain circuits controlling estrous behavior by neuropeptide Y (NPY) projections. In the forebrain, the nutritional inhibition of estrous behavior appears to be mediated by corticotropin-releasing factor (CRF) or urocortin-signaling systems. Intracerebroventricular (ICV) infusion of the CRF antagonist, astressin, prevents the suppression of lordosis by food deprivation and by NPY treatment in Syrian hamsters. These experiments sought to determine which CRF receptor type(s) is involved. ICV infusion of the CRF receptor subtype CRFR2-selective agonists urocortin 2 and 3 (UCN2, UCN3) inhibited sexual receptivity in hormone-primed, ovariectomized hamsters. Furthermore, the CRFR2-selective antagonist, astressin 2B, prevented the inhibition of estrous behavior by UCN2 and by NPY, consistent with a role for CRFR2. On the other hand, astressin 2B did not prevent the inhibition of behavior induced by 48-h food deprivation or ICV administration of CRF, a mixed CRFR1 and CRFR2 agonist, suggesting that activation of CRFR1 signaling is sufficient to inhibit sexual receptivity in hamsters. Although administration of CRFR1-selective antagonists (NBI-27914 and CP-154,526) failed to reverse the inhibition of receptivity by CRF treatment, we could not confirm their biological effectiveness in hamsters. The most parsimonious interpretation of these findings is that, although NPY inhibits estrous behavior via downstream CRFR2 signaling, food deprivation may exert its inhibition via both CRFR1 and CRFR2 and that redundant neuropeptide systems may be involved.     

Molecular recognition of corticotropin-releasing factor by its G-protein-coupled receptor CRFR1

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.     

Corticotropin-releasing factor requires CRF binding protein to potentiate NMDA receptors via CRF receptor 2 in dopamine neurons

Stress increases addictive behaviors and is a common cause of relapse. Corticotropin-releasing factor (CRF) plays a key role in the modulation of drug taking by stress. However, the mechanism by which CRF modulates neuronal activity in circuits involved in drug addiction is poorly understood. Here we show that CRF induces a potentiation of NMDAR (N-methyl-D-aspartate receptor)-mediated synaptic transmission in dopamine neurons of the ventral tegmental area (VTA). This effect involves CRF receptor 2 (CRF-R2) and activation of the phospholipase C (PLC)-protein kinase C (PKC) pathway. We also find that this potentiation requires CRF binding protein (CRF-BP). Accordingly, CRF-like peptides, which do not bind the CRF-BP with high affinity, do not potentiate NMDARs. These results provide evidence of the first specific roles for CRF-R2 and CRF-BP in the modulation of neuronal activity and suggest that NMDARs in the VTA may be a target for both drugs of abuse and stress.