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.     

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).     

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.     

Synthesis and biological activity of N-terminal lipidated and/or fluorescently labeled conjugates of astressin as corticotropin releasing factor antagonists

This report describes the synthesis of eight N-terminally modified astressin analogs and their biochemical evaluation as corticotropin releasing factor (CRF) antagonists. The lipidated astressin derivatives were tested on rat CRF receptor type 1 and 2alpha and were found to be active as CRF antagonists (rCRFR1: pA(2)=7.5-8.3; rCRFR2alpha: pA(2)=7.5-9.0) with nearly equal activities as compared to unmodified astressin (rCRFR1: pA(2)=8.3+/-0.09; rCRFR2alpha: pA(2)=8.7+/-0.08).     

Binding differences of human and amphibian corticotropin-releasing factor type 1 (CRF(1)) receptors: identification of amino acids mediating high-affinity astressin binding and functional antagonism

The corticotropin-releasing factor (CRF) type 1 receptors (CRF(1)) from human (hCRF(1)) and Xenopus (xCRF(1)) differ from one another by their agonist- and antagonist-binding preference. While the agonist-binding site of the xCRF(1) receptor has been mapped, the amino acids that mediate binding of the potent peptide antagonist astressin are unknown. By constructing receptor chimeras followed by site-directed mutagenesis, the astressin-binding site of the xCRF(1) receptor was located between residues 76 and 83. This region partially overlaps with the agonist-selective domain of the xCRF(1) receptor (residues 76-89). Mutagenesis of the amphibian residues Gln(76), Gly(81) and Val(83) to the human sequence (Arg(76)Asn(81)Gly(83)) generated a receptor mutant that bound astressin with even higher affinity than the native hCRF(1) receptor. An amino acid doublet (Glu(70)Tyr(71)) that is conserved in the xCRF(1) and hCRF(2(a)) receptor after incorporation into the hCRF(1) receptor sequence was found to facilitate antagonist binding up to 15-fold higher. In agreement with the binding data, astressin was a more potent functional antagonist at receptors expressing the Glu(70)Tyr(71) motif. These data show that the agonist- and antagonist-binding sites of the hCRF(1) receptor partially overlap and that two amino acids within the N terminus of the hCRF(1) receptor negatively influence binding and functional antagonism of astressin.     

Peptide ligand binding properties of the corticotropin-releasing factor (CRF) type 2 receptor: pharmacology of endogenously expressed receptors, G-protein-coupling sensitivity and determinants of CRF2 receptor selectivity

The CRF2 receptor is involved in stress responses, cardiovascular function and gastric motility. Endogenous agonists (urocortin (UCN) 2, UCN 3) and synthetic antagonists (astressin2-B, antisauvagine-30) are selective for CRF2 over the CRF1 receptor. Peptide ligand binding properties of the CRF2 receptor require further investigation, including ligand affinity for endogenously expressed receptors, the effect of receptor-G-protein coupling on ligand affinity, and the molecular basis of ligand selectivity. Ligand affinity for rat CRF(2a) in olfactory bulb and CRF(2b) in A7r5 cells was similar to that for the cloned human CRF(2a) receptor (within three-fold), except for oCRF (9.4- and 5.4-fold higher affinity in olfactory bulb and A7r5 cells, respectively). Receptor-G-protein uncoupling reduced agonist affinity only 1.2- to 6.5-fold (compared with 92-1300-fold for the CRF1 receptor). Ligand selectivity mechanisms were investigated using chimeric CRF2/CRF1 receptors. The juxtamembrane receptor domain determined selectivity of antisauvagine-30, the N-terminal-extracellular domain contributed to selectivity of UCN 3, and both domains contributed to selectivity of UCN 2 and astressin2-B. Therefore ligands differ in the contribution of receptor domains to their selectivity, and CRF2-selective antagonists bind the juxtamembrane domain. These findings will be important for identifying the CRF2 receptor in tissues and for developing ligands targeting the receptor, both of which will be useful in identifying the emerging physiological functions of the CRF2 receptor.     

Expression, purification, and characterization of a soluble form of the first extracellular domain of the human type 1 corticotropin releasing factor receptor.

The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.     

Photoaffinity labeling of parathyroid hormone receptors in clonal rat osteosarcoma cells

A photoreactive derivative of a sulfur-free bovine parathyroid hormone (PTH) analogue, [Nle8,N-epsilon-(4-azido-2-nitrophenyl)Lys13,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NAP-NlePTH), was purified from the products of the reaction of [Nle8,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NlePTH) with 4-fluoro-3-nitro-phenylazide and was used to identify binding components of the PTH receptor in clonal rat osteosarcoma cells (ROS 17/2.8). The purified analogue, NAP-NlePTH, is a fully active agonist in three different ROS 17/2.8 cell bioassays: 1) specific binding to saturable PTH receptors; 2) stimulation of cyclic AMP accumulation; and 3) inhibition of cellular alkaline phosphatase activity; this analogue gave dose response curves parallel to and 25-33% as potent as its parent molecule, NlePTH. Radioiodinated NAP-NlePTH (125I-labeled NAP-NlePTH) retained maximal receptor-binding potency. Radioligand saturation studies in intact cells showed that the Kd of PTH receptors for the photoligand was slightly less than that for 125I-labeled NlePTH (2.8 and 0.8 nM, respectively), but that the Bmax was essentially identical for both radioligands (8 fmol/10(5) cells). Photoaffinity labeling of ROS 17/2.8 cells revealed several 125I-labeled macromolecular components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One predominant 125I-labeled band, having an apparent Mr of 80,000 daltons (including Mr = 4,347 ligand; hereafter referred to as the Mr = 80,000 protein), was consistently demonstrated in both reducing and nonreducing conditions. Its labeling was completely inhibited by coincubation with NlePTH (10 nM) at 26-fold molar excess to the photoligand, but not by biologically inactive PTH fragments or unrelated hormone. Labeling of several other macromolecular components persisted in the presence of NlePTH (1 microM). Only the labeling of the Mr = 80,000 protein showed saturation kinetics for photoaffinity labeling; the dose of 125I-labeled NAP-NlePTH (0.8 nM) to half-saturate labeling of the Mr = 80,000 protein was close to the Kd (2.8 nM) of specific binding of the photoligand to receptors in intact ROS 17/2.8 cells. Pretreatment of the cells with NlePTH and dexamethasone led to the predicted proportional decrease or increase, respectively, in labeling of the Mr = 80,000 protein. Our data, using a highly purified photoactive derivative of PTH, having carefully defined chemical and biological properties, show a plasma membrane component of Mr = 80,000 in ROS 17/2.8 cells that possesses the affinity, binding capacity, and physiological characteristics of the PTH receptor.     

Sauvagine cross-links to the second extracellular loop of the corticotropin-releasing factor type 1 receptor

Contact sites between the corticotropin-releasing factor receptor type 1 (CRFR1), the sauvagine (SVG) radioligands [Tyr(0),Gln(1)]SVG ((125)I-YQS) and [Tyr(0),Gln(1), Leu(17)]SVG ((125)I-YQLS) were examined. (125)I-YQLS or (125)I-YQS was cross-linked to CRFR1 using the chemical cross-linker, disuccinimidyl suberate (DSS), which cross-links the epsilon amino groups of lysine residues that have a molecular distance of 11.4 A. DSS specifically and efficiently cross-linked (125)I-YQLS and (125)I-YQS to CRFR1. CRFR1 contains 5 putative extracellular lysine residues (Lys(110), Lys(111), Lys(113), Lys(257), and Lys(262)) that can cross-link to the 4 lysine residues (Lys(16), Lys(22), Lys(25), and Lys(27)) of the radioligands. Identification of the CNBr-cleaved fragments of CRFR1 cross-linked to (125)I-YQLS or (125)I-YQS established that the second extracellular loop of CRFR1 cross-links to Lys(16) of YQS. Additionally, site-directed mutagenesis (changing Lys to Arg in CRFR1 individually and in combination) revealed that Lys(257) in the second extracellular loop of CRFR1 is an important cross-linking site. In conclusion, it was shown that in SVG-bound CRFR1, Lys(257) of CRFR1 lies in close proximity (11.4 A) to Lys(16) of SVG.     

CRF receptor type 1 mediates continual hypoxia-induced CRF peptide and CRF mRNA expression increase in hypothalamic PVN of rats

We demonstrated previously that hypoxia activated CRF and CRF mRNA in PVN, and CRF receptor 1 (CRFR1) mRNA in rat pituitary. The aim of the study is to test whether the hypoxia-activated CRF and CRF mRNA is associated with triggering CRFR1. Rats were exposed to hypobaric hypoxia at altitude of 2 and 5 km. CRF and CRF mRNA were assayed by immunostaining and in situ hybridization. CRFR1 mRNA was assayed by RT-PCR. Results showed that 5 km continual hypoxia increased CRF and CRF mRNA in PVN, CRFR1 mRNA in pituitary, and plasma corticosterone. The hypoxia-increased CRF, CRF mRNA, CRFR1 mRNA, and corticosterone were blocked by CRFR1 antagonist (CP-154,526), suggesting that CRFR1 in PVN and pituitary are responsible for the hypoxia-increased CRF and CRF mRNA in PVN.     

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.     

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.     

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.     

Peripheral CRF activates myenteric neurons in the proximal colon through CRF(1) receptor in conscious rats

Corticotropin-releasing factor (CRF) injected peripherally induces clustered spike-burst activity in the proximal colon through CRF(1) receptors in rats. We investigated the effect of intraperitoneal CRF on proximal colon ganglionic myenteric cell activity in conscious rats using Fos immunohistochemistry on the colonic longitudinal muscle/myenteric plexus whole mount preparation. In vehicle-pretreated rats, there were only a few Fos immunoreactive (IR) cells per ganglion (1.2 +/- 0.6). CRF (10 microg/kg ip) induced Fos expression in 19.6 +/- 2.1 cells/ganglion. The CRF(1)/CRF(2) antagonist astressin (33 microg/kg ip) and the selective CRF(1) antagonist CP-154,526 (20 mg/kg sc) prevented intraperitoneal CRF-induced Fos expression in the proximal colon (number of Fos-IR cells/ganglion: 2.7 +/- 1.2 and 1.0 +/- 1.0, respectively), whereas atropine (1 mg/kg sc) had no effect. Double labeling of Fos with protein gene product 9.5 revealed the neuronal identity of activated cells that were encircled by varicose fibers immunoreactive to vesicular acetylcholine transporter. Fos immunoreactivity was mainly present in choline acetyltransferase-IR nerve cell bodies but not in the NADPH-diaphorase-positive cells. These results indicate that peripheral CRF activates myenteric cholinergic neurons in the proximal colon through CRF(1) receptor.     

Interaction of human parathyroid hormone-related peptide with parathyroid hormone receptors in clonal rat osteosarcoma cells

Synthetic peptides corresponding to the amino-terminal region of the human parathyroid hormone-related peptide (hPTHrp) were used to characterize the interaction of hPTHrp with parathyroid hormone (PTH) receptors in clonal rat osteosarcoma cells (ROS 17/2.8). Both hPTHrp-(1-34) and [Tyr40]hPTHrp-(1-40) showed full agonist activity in stimulating cyclic AMP accumulation in ROS cells; human PTHrp-(1-34) was approximately 2.5-fold as potent as hPTH-(1-34). Both [Tyr-40]hPTHrp-(3-40) and hPTH-(3-34) inhibited the cyclic AMP increase induced by either hPTHrp or PTH with parallel dose-inhibition curves. Binding to intact ROS cells of a 125I-labeled [Tyr40]hPTHrp-(1-40) (125I-[Tyr40]hPTHrp-(1-40)) which retains full biological activity was time- and temperature-dependent and reversible. Binding of 125I-[Tyr40]hPTHrp-(1-40) and 125I-labeled [Nle8, Nle18, Tyr34]bovine PTH-(1-34)NH2 to ROS cells was competed for, to the same extent and with the comparable potency, by either unlabeled hPTHrp or PTH peptides. The binding capacity and affinity of receptors in ROS cells were strikingly similar for hPTHrp and PTH. Affinity cross-linking with either radioligand resulted in high affinity, specific labeling of an apparently identical macromolecule centering at Mr = 80,000, which was detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both reducing and nonreducing conditions. The data indicate that hPTHrp and PTH, their amino-terminal fragments at least, interact with the identical receptors with regard to affinity, capacity, specificity, and physicochemical characteristics in osteoblastic ROS 17/2.8 cells.     

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.     

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 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.     

The parathyroid hormone-like peptide associated with humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS 17/2.8 cells

[Tyr36]human adenylate cyclase stimulating peptide (1-36)-NH2, an amino-terminal analog of a tumor peptide which is associated with hypercalcemia of malignancy, and [Nle8, Nle18, Tyr34]bovine parathyroid hormone (PTH)-(1-34)-NH2 both bind with similar affinities to receptors on rat osteosarcoma cells, ROS 17/2.8, when either of the peptides is used as the radioligand. Pretreatment of the cells with either peptide down-regulates available binding sites for either radioligand and desensitizes the cAMP accumulation stimulated by either peptide. Prior exposure of the cells to dexamethasone increases these responses to both peptides. Photoderivatized radioiodinated [Tyr36]human adenylate cyclase-stimulating peptide (1-36)-NH2 and [Nle8, Nle18, Tyr34]bovine PTH-(1-34)-NH2 both specifically label a Mr = 80,000 membrane protein on ROS 17/2.8 cells. The intensity of labeling this receptor band by either photoprobe is reduced by co-incubation with either peptide over the same dose range. Equivalent dose-dependent down-regulation of receptors which bind both photoprobes is also found when ROS 17/2.8 cells are preincubated with either peptide. Dexamethasone increases the intensity of receptor labeling. Our findings strongly indicate that both peptides recognize the same plasma membrane receptor on ROS 17/2.8 cells. Although the physiological function(s) of human adenylate cyclase-stimulating peptide is unknown, these results could explain why its biological actions on mineral ion metabolism so closely simulate those of PTH and raise interesting questions about the general biological and evolutionary significance of the use of the same receptor by chemically distinct peptides.     

Design, synthesis and pharmacological characterization of new highly selective CRF(2) antagonists: development of 123I-K31440 as a potential SPECT ligand

Novel analogs of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF(2)), have been synthesized and characterized in vitro and in vivo. The N-terminal amino acid D-phenylalanine in aSvg-30 was replaced by a D-tyrosine residue for specific radioactive labeling with 123I. Additionally, Met(17) of aSvg-30 was substituted by norleucine and the N-terminus of the peptide was acetylated to increase in vivo metabolic stability. The aSvg-30 analogs were tested for their ability to displace [125I-Tyr(0)]Svg in binding experiments and to inhibit Svg-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for the human CRF(1) (hCRF(1)), hCRF(2alpha) and hCRF(2beta) receptor. Ac-[D-Tyr(11), His(12), Nle(17)Svg(11-40), named K31440, showed high specific binding to hCRF(2alpha) (K(i) = 1.48 +/- 0.34 nM) and hCRF(2beta) (K(i) = 2.05 +/- 0.61 nM) but not the hCRF(1) receptor (K(i) = 288 +/- 13 nM) and decreased Svg-stimulated cAMP activity in hCRF(2)-expressing cells in a similar fashion as aSvg-30. In biodistribution studies specific uptake of 123I-K31440 was detected after 1 h in small intestine of BALB/c nude mice. These data demonstrate that 123I-K31440 may serve as a useful tool to detect native CRF(2) receptors and elucidate their role in gastrointestinal disorders and diseases such as irritable bowel syndrome or cancer.