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.     

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).     

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.     

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.     

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 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.     

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.     

Activation of corticotropin-releasing factor 2 receptor inhibits Purkinje neuron P-type calcium currents via Goα-dependent PKC epsilon pathway

Corticotropin-releasing factor (CRF) receptors have been demonstrated to be widely expressed in the central nervous system and in many peripheral tissues of mammalians. However, it is still unknown whether CRF receptors will function in cerebellar Purkinje neurons. In the present study, we investigated the expression profile of CRF receptors in rat cerebellum and identified a novel functional role of CRFR2 in modulating Purkinje neuron P-type Ca²⁺ currents (P-currents). We found that CRFR2α mRNA, but not CRFR1 and CRFR2β, was endogenously expressed in rat cerebellum. Activation of CRFR2 by UCN2 inhibited P-currents in a concentration-dependent manner (IC₅₀ ~ 0.07 µM). This inhibitory effect was abolished by astressin2B, a CRFR2 antagonist, and was blocked by GDP-β-S, pertussis toxin, or a selective antibody raised against the G_oα. Inhibition of phospholipase C (PLC) blocked the inhibitory action of UCN2. The application of diacylglycerol (DAG) antagonist, 1-hexadecyl-2-acetyl-sn-glycerol, as well as inhibition of either protein kinase C or its epsilon isoform (PKCε) abolished the UCN2 effect while 1-oleoyl-2-acetyl-sn-glycerol (EI-150), a membrane-permeable DAG analogue, occluded UCN2-mediated inhibition. In addition, UCN2 significantly increases spontaneous firing frequency of Purkinje neurons in cerebellar slices. In summary, activation of CRFR2 inhibits P-currents in Purkinje neurons via G_oα-dependent PLC/PKCε pathway, which might contribute to its physiological functions in the cerebellum.     

Identification of Amino Acids in the N-Terminal Domain of Corticotropin-Releasing Factor Receptor 1 that Are Important Determinants of High-Affinity Ligand Binding

Abstract : The aim of the present study was to identify the N-terminal regions of human corticotropin-releasing factor (CRF) receptor type 1 (hCRF-R1) that are crucial for ligand binding. Mutant receptors were constructed by replacing specific residues in hCRF-R1 with amino acids from the corresponding position in the N-terminal region of the human vasoactive intestinal peptide receptor type 2 (hVIP-R2). In cyclic AMP stimulation and CRF binding assays, it was established that two regions within the N-terminal domain were crucial for the binding of CRF receptor agonists and antagonists : one region mapping to amino acids 43-50 and a second amino acid sequence extending from position 76 to 84 of hCRF-R1. Recently, it was found that the latter sequence plays a very important role in determining the high ligand selectivity of the Xenopus CRF-R1 (xCRF-R1). Replacement of amino acids 76-84 of hCRF-R1 with residues from the same segment of the hVIP-R2 N terminus markedly reduced the binding affinity of CRF ligands. Mutation of Arg⁷⁶ or Asn⁸¹ but not Gly⁸³ of hCRF-R1 to the corresponding amino acids of xCRF-R1 or hVIP-R2 resulted in 100-1,000-fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N-terminal domain of CRF-R1 in high-affinity ligand binding.     

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.     

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.     

Distinct conformations of the corticotropin releasing factor type 1 receptor adopted following agonist and antagonist binding are differentially regulated

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.     

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 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.     

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.     

Type 1 corticotropin-releasing factor receptors in the ventromedial hypothalamus promote hypoglycemia-induced hormonal counterregulation

Type 2 corticotropin-releasing factor (CRF) receptors (CRFR2) within the ventromedial hypothalamus (VMH), a key glucose-sensing region, play a major role in regulating the hormonal counterregulatory responses (CRRs) to acute hypoglycemia. The VMH expresses both subtypes of CRF receptors, CRFR1 and CRFR2. The objective of this study was to examine the role of the CRFR1 receptor in the VMH in the regulation of the CRR to acute hypoglycemia. To compare the hormonal CRR to hypoglycemia, awake and unrestrained Sprague-Dawley rats were bilaterally microinjected to the VMH with either 1) aECF, 2) CRF (1 pmol/side), 3) CRFR1 antagonist Antalarmin (500 pmol/side), or 4) CRF + Antalarmin prior to undergoing a hyperinsulinemic hypoglycemic (2.8 mM) clamp. A second series of studies also incorporated an infusion of [(3)H]glucose to allow the calculation of glucose dynamics. In addition the effect of CRFR1 antagonism in the paraventricular nucleus (PVN) was studied. Activation of VMH CRFR1 increased, whereas inhibition of CRFR1 suppressed hypoglycemia-induced CRRs. Inhibition of VMH CRFR1 also increased peripheral glucose utilization and reduced endogenous glucose production during hypoglycemia, whereas VMH CRF reduced peripheral glucose utilization. In contrast CRFR1 inhibition in the PVN blunted corticosterone but not epinephrine or glucagon CRR to hypoglycemia. In contrast to CRFR2 activation, CRFR1 activation within the VMH amplifies CRRs to acute hypoglycemia. The balance between these two opposing CRFRs in this key glucose-sensing region may play an important role in determining the magnitude of CRRs to acute hypoglycemia.     

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.     

Separate locations of urocortin and its receptors in mouse testis: function in male reproduction and the relevant mechanisms.

Urocortin (UCN), a newly identified corticotrophin-releasing-factor (CRF) related peptide, has been demonstrated to play important roles in female reproductive system. However, few studies were reported about its effects on male reproduction. This study aimed to investigate the expression profile of UCN and CRF receptors (CRFR) in mouse testis and functions of UCN in male reproduction. Expression of UCN and CRFR mRNA was detected by RT-PCR. Localization of UCN peptide was determined by immunohistochemistry and double-immunostaining. We found that both UCN mRNA and peptide were obviously expressed in mature spermatozoa, whereas CRFR1 and CRFR2 were expressed respectively in spermatocytes and spermatogonia. Double-immunostaining results showed that UCN expression decreased with acrosome reaction (AR) proceeding. UCN significantly inhibited AR initiated by progesterone with chlortetracycline staining and decreased spermatozoa motility concentration-dependently. Pre-incubation of spermatozoa with astressin, a CRFR antagonist, did not affect these inhibitions. In addition, flow cytometry showed that UCN concentration-dependently decreased intracellular Ca(2+) [Ca(2+)](i) in spermatozoa. In summary, UCN located in mouse spermatozoa and exerted inhibitory effects on male reproductive functions including motility and AR. UCN's inhibition on [Ca(2+)](i) via T-type calcium channels might be responsible for these effects.     

The multimerization of hantavirus nucleocapsid protein depends on type-specific epitopes

Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.