Novel high-affinity photoactivatable antagonists of corticotropin-releasing factor (CRF) photoaffinity labeling studies on CRF receptor, type 1 (CRFR1).

Novel photoactivatable antagonists of human/rat corticotropin-releasing factor (h/rCRF) have been synthesized and characterized. The N-terminal amino acid D-phenylalanine in astressin ?cyclo(30-33) [D-Phe12, Nle21,38, Glu30, Lys33]h/rCRF-(12-41)?, a potent CRF peptide antagonist, was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. Additionally, His32 of astressin was substituted by either alanine or tyrosine for specific radioactive labeling with 125I at either His13 or Tyr32, respectively. The photoactivatable CRF antagonists were tested for their ability to displace 125I-labeled Tyr0 ovine CRF ([125I-labeled Tyr0]oCRF) in binding experiments and to inhibit oCRF-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1) or human Y-79 retinoblastoma cells known to carry endogenous functional human CRFR1 (hCRFR1). ATB-cyclo(30-33)[Nle21,38, Glu30, Ala32, Lys33]h/rCRF-(13-41) (compound 1) was found to bind with higher affinity to rat or human CRFR1 when compared with ATB-cyclo(30-33)[Nle21,38, Glu30, Tyr32, Lys33]h/rCRF-(13-41) (compound 2) and exhibited higher inhibition of oCRF-stimulated cAMP accumulation in HEK 293 cells stably transfected with cDNA coding for rCRFR1 (HEK-rCRFR1 cells) or Y-79 cells. A highly glycosylated, 66-kDa protein was identified with SDS/PAGE, when the radioactively iodinated compounds 1 or 2 were covalently linked to rCRFR1. The specificity of the photoactivatable 125I-labeled CRF antagonists was demonstrated with SDS/PAGE by the finding that these analogs could be displaced from the receptor by their corresponding nonlabeled form, but not other unrelated peptides such as vasoactive intestinal peptide. The observed molecular size of the receptor was in agreement with the size of CRFR1 found in rat pituitary (66 kDa), but was significantly larger than the size of CRFR1 found in rat cerebellum and olfactory bulb (53 kDa).     

Functional and protein chemical characterization of the N-terminal domain of the rat corticotropin-releasing factor receptor 1

Rat corticotropin-releasing factor receptor 1 (rCRFR1) was produced either in transfected HEK 293 cells as a complex glycosylated protein or in the presence of the mannosidase I inhibitor kifunensine as a high mannose glycosylated protein. The altered glycosylation did not influence the biological function of rCRFR1 as demonstrated by competitive binding of rat urocortin (rUcn) or human/rat corticotropin-releasing factor (h/rCRF) and agonist-induced cAMP accumulation. The low production rate of the N-terminal domain of rCRFR1 (rCRFR1-NT) by transfected HEK 293 cells, was increased by a factor of 100 in the presence of kifunensine. The product, rCRFR1-NT-Kif, bound rUcn specifically (K(D) = 27 nM) and astressin (K(I) = 60 nM). This affinity was 10-fold lower than the affinity of full length rCRFR1. However, it was sufficiently high for rCRFR1-NT-Kif to serve as a model for the N-terminal domain of rCRFR1. With protein fragmentation, Edman degradation, and mass spectrometric analysis, evidence was found for the signal peptide cleavage site C-terminally to Thr(23) and three disulfide bridges between precursor residues 30 and 54, 44 and 87, and 68 and 102. Of all putative N-glycosylation sites in positions 32, 38, 45, 78, 90, and 98, all Asn residues except for Asn(32) were glycosylated to a significant extent. No O-glycosylation was observed.     

The ligand-selective Domains of Corticotropin-Releasing Factor Type 1 and Type 2 Receptor Reside in Different Extracellular Domains

The nonselective human corticotropin-releasing factor (hCRF) receptor 1 (hCRFR1) and the ligand-selective Xenopus CRFR1 (xCRFR1), xCRFR2, and hCRFR2alpha were compared. To understand the interactions of hCRF, ovine CRF (oCRF), rat urocortin (rUcn), and sauvagine, ligands with different affinities for type 1 and type 2 CRFRs, chimeric and mutant receptors of hCRFR1, xCRFR1, hCRFR2alpha, and xCRFR2 were constructed. In cyclic AMP stimulation and CRF-binding assays, it was established that different extracellular regions of CRFR1 and CRFR2 conferred their ligand selectivities. The ligand selectivity of xCRFR1 resided in five N-terminal amino acids, whereas the N-terminus of both CRFR2 proteins did not contribute to their ligand selectivities. Chimeric receptors in which the first extracellular domain of hCRFR1 replaced that of hCRFR2alpha or xCRFR2 showed a similar pharmacological profile to the two parental CRFR2 molecules. Chimeric receptors carrying the N-terminal domain of xCRFR1 linked to hCRFR2alpha or xCRFR2 displayed a novel pharmacological profile. hCRF, rUcn, and sauvagine were bound with high affinity, whereas oCRF was bound with low affinity. Furthermore, when three or five residues of xCRFR1 (Gln76, Gly81, Val83, His88, Leu89; or Gln76, Gly81, Val83) were introduced into receptor chimeras carrying the N-terminus of hCRFR1 linked to xCRFR2, the same novel pharmacology was observed. These data indicate a compensation mechanism of two differentially selecting regions located in different domains of both xCRFR1 and CRFR2.     

Residues of corticotropin releasing factor-binding protein (CRF-BP) that selectively abrogate binding to CRF but not to urocortin 1

Corticotropin releasing factor-binding protein (CRF-BP) binds CRF and urocortin 1 (Ucn 1) with high affinity, thus preventing CRF receptor (CRFR) activation. Despite recent progress on the molecular details that govern interactions between CRF family neuropeptides and their cognate receptors, little is known concerning the mechanisms that allow CRF-BP to bind CRF and Ucn 1 with picomolar affinity. We conducted a comprehensive alanine scan of 76 evolutionarily conserved residues of CRF-BP and identified several residues that differentially affected the affinity for CRF over Ucn 1. We determined that both neuropeptides derive their similarly high affinity from distinct binding surfaces on CRF-BP. Alanine substitutions of arginine 56 (R56A) and aspartic acid 62 (D62A) reduce the affinity for CRF by approximately 100-fold, while only marginally affecting the affinity for Ucn 1. The selective reduction in affinity for CRF depends on glutamic acid 25 in the CRF peptide, as substitution of Glu(25) reduces the affinity for CRF-BP by approximately 2 orders of magnitude, but only in the presence of both Arg(56) and Asp(62) in human CRF-BP. We show that CRF-BP(R56A) and CRF-BP(D62A) have lost the ability to inhibit CRFR1-mediated responses to CRF that activate luciferase induction in HEK293T cells and ACTH release from cultured rat anterior pituitary cells. In contrast, both CRF-BP mutants retain the ability to inhibit Ucn 1-induced CRFR1 activation. Collectively our findings demonstrate that CRF-BP has distinct and separable binding surfaces for CRF and Ucn 1, opening new avenues for the design of ligand-specific antagonists based on 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.     

Regulation of the coupling to different G proteins of rat corticotropin-releasing factor receptor type 1 in human embryonic kidney 293 cells

The regulation of G protein activation by the rat corticotropin-releasing factor receptor type 1 (rCRFR1) in human embryonic kidney (HEK)293 (HEK-rCRFR1) cell membranes was studied. Corresponding to a high and low affinity ligand binding site, sauvagine and other peptidic CRFR1 ligands evoked high and low potency responses of G protein activation, differing by 64-fold in their EC(50) values as measured by stimulation of [(35)S]GTPgammaS binding. Contrary to the low potency response, the high potency response was of lower GTPgammaS affinity, pertussis toxin (PTX)-insensitive, and homologously desensitized. Distinct desensitization was also observed in the adenylate cyclase activity, when its high potency stimulation was abolished and the activity became low potently inhibited by sauvagine. From these results and immunoprecipitation of [(35)S]GTPgammaS-bound Galpha(s) and Galpha(i) subunits it is concluded that the high and low potency [(35)S]GTPgammaS binding stimulation reflected coupling to G(s) and G(i) proteins, respectively, only G(s) coupling being homologously desensitized. Immunoprecipitation of [(35)S]GTPgammaS-bound Galpha(q/11) revealed additional coupling to G(q/11), which also was homologously desensitized. Although Galpha(q/11) coupling was PTX-insensitive, half of the sauvagine-stimulated accumulation of inositol phosphates in the cells was PTX-sensitive, suggesting involvement of G(i) in addition to G(q/11)in the stimulation of inositol metabolism. It is concluded that CRFR1 signals through at least two different ways, one leading to G(s)- and G(q/11)-mediated signaling steps and desensitization and another leading to G(i) -mediated signals without being desensitized. Furthermore, the concentrations of the stimulating ligand and GTP and desensitization may be part of a regulatory mechanism determining the actual ratio of the coupling of CRFR1 to different G proteins.     

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2).

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid d-Phe in aSvg-30 [d-Phe11,His12]Svg(11-40) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[His12]Svg was tested for its ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRF1) or mouse CRF receptor, type 2beta (mCRF2beta). Furthermore, the ability of ATB-[His12]Svg(12-40) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF1 (HEK-rCRF1 cells) or mCRF2beta (HEK-mCRF2beta cells) was determined. Unlike astressin and photo astressin, ATB-[His12]Svg(12-40) showed high selective binding to mCRF2beta (Ki = 3.1 +/- 0.2 nm) but not the rCRF1 receptor (Ki = 142.5 +/- 22.3 nm) and decreased Svg-stimulated cAMP activity in mCRF2beta-expressing cells in a similar fashion as aSvg-30. A 66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB-[His12]Svg(12-40) was covalently linked to mCRF2beta receptor. The specificity of the photoactivatable 125I-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).     

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.     

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.     

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.     

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.     

Differential responsiveness of CRF receptor subtypes to N-terminal truncation of peptidic ligands

The role of the N-terminal domains of corticotropin-releasing factor (CRF) and CRF-like peptides in receptor subtype selectivity, ligand affinity and biological potency was investigated. Therefore, human CRF(12-41), human URP(12-38) and antisauvagine-30 (aSvg) were N-terminally prolonged by consecutive addition of one or two amino acids. The peptides obtained were tested for their binding affinities to rat CRF1 and murine CRF(2beta) receptor, and their capability to stimulate cAMP-release by HEK cells producing either receptor.It was observed that human CRF N-terminally truncated by eight residues was bound with high affinity to CRF2 receptor (Ki=5.4nM), whereas affinity for CRF1 receptor was decreased (Ki=250 nM). A similar shift of affinity was found with sauvagine (Svg) analogs. Truncation of human URP analogs did not affect their preference for CRF(2beta) receptor, but reduced their affinity. Changes in affinity were positively correlated with changes in potency. These results indicated that CRF1 receptor was more stringent in its structural requirements for ligands to exhibit high affinity binding than CRF(2beta) receptor.     

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.     

The brain corticotropin-releasing factor (CRF) receptor is of lower apparent molecular weight than the CRF receptor in anterior pituitary. Evidence from chemical cross-linking studies

The ligand binding subunits of the corticotropin-releasing factor (CRF) receptors in brain and anterior pituitary of a number of species have been identified by chemical affinity cross-linking using the homobifunctional cross-linking agent disuccinimidyl suberate and 125I-Tyr0-oCRF (ovine CRF). In homogenates of rat, monkey, and human cerebral cortex, 125I-Tyr0-oCRF was covalently incorporated into a protein of Mr = 58,000. Under identical conditions in the anterior pituitary of rat, monkey, cow, and pig, 125I-Tyr0-oCRF was incorporated into a protein of apparent Mr = 75,000. The specificity of the labeling was typical of the CRF binding site since both the cerebral cortex- and pituitary-labeled proteins exhibited the appropriate pharmacological rank order profile characteristic of the CRF receptor (Nle21,Tyr32-oCRF approximately equal to rat/human CRF approximately equal to ovine CRF approximately equal to alpha-helical CRF(6-41) greater than alpha-helical oCRF(9-41) greater than or equal to oCRF(7-41) greater than rat/human CRF(1-20) approximately equal to vasoactive intestinal peptide). In addition to the major labeled proteins, 125I-Tyr0-oCRF was incorporated into higher molecular weight peptides which may represent precursors and into lower molecular weight components which may represent fragments of the major labeled proteins or altered forms of the CRF binding subunit. In summary, these data indicate a heterogeneity between brain and pituitary CRF receptors with the ligand binding subunit of the brain CRF receptor residing on a Mr = 58,000 protein, while in the anterior pituitary, the identical binding subunit resides on a protein of apparent Mr = 75,000.     

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.     

Functional corticotropin-releasing factor (CRF) receptors in mouse spleen: evidence from adenylate cyclase studies

Radioligand binding studies have previously identified a high affinity, magnesium-dependent, guanine nucleotide-sensitive binding site for corticotropin-releasing factor (CRF) in mouse spleen. In order to determine the functional nature of these CRF binding sites, we examined the effects of CRF on adenylate cyclase activity in mouse spleen homogenates. The stimulation of adenylate cyclase activity was dependent on time, tissue protein concentration, and guanine nucleotides. CRF-stimulated adenylate cyclase activity was evident in the presence of guanosine-5'-triphosphate (GTP) and its precursor guanosine-5'-diphosphate (GDP) but was not detected in the presence of the hydrolysis-resistant GTP analogs, guanyl-5'-imidodiphosphate [Gpp(NH)p] and guanosine-5'-gamma-thiotriphosphate (GTP-gamma-S). The rank order of potency for CRF analogs and fragments in stimulating adenylate cyclase activity was comparable to their affinities for CRF binding sites in mouse spleen homogenates. The putative receptor antagonist, alpha helical ovine CRF(9-41), did not stimulate adenylate cyclase activity but did attenuate the stimulation by various concentrations of rat/human CRF. In summary, these data demonstrate the functional nature of CRF receptors in mouse spleen as evidenced by CRF stimulation of cAMP production and suggest that this peptide may play a physiological role in regulating immune function.     

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.