Corticotrophin-releasing factor receptors: from molecular biology to drug design.

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

Localization of agonist- and antagonist-binding domains of human corticotropin-releasing factor receptors.

The CRF receptors, CRFR1 and CRFR2, are members of the G protein-coupled receptor superfamily. Despite their considerable sequence similarity, CRFR1 and CRFR2 have quite different affinities for the peptide ligand rat/human CRF. Previous studies using chimeric receptors between human CRFR1 and CRFR2 have identified three potentially important regions in the second and third extracellular domains of CRF receptor for the binding of rat/human CRF. The present report further demonstrates that these same three regions also affect the binding of urocortin and sauvagine, two other members of the CRF peptide family, albeit to different extents. We also show that a fourth region in the third extracellular domain, Asp254, has been identified to be important for sauvagine but not CRF or urocortin binding. Thus, the three peptide ligands not only interact with a different set of regions on CRFR1 and CRFR2 but also differentially interact with some of the same regions. These data could, at least in part, account for the much higher affinity of CRFR2 for urocortin and sauvagine compared with rat/human CRF. We have also identified two amino acid residues, His199 in the third transmembrane domain and Met276 in the fifth transmembrane domain, that are important for binding the non-peptide high-affinity CRFR1 antagonist NBI 27914. Mutations of His199 and Met276 to the corresponding amino acids in CRFR2 each decreased the binding affinity of NBI 27914 for CRFR1 by 40- and 200-fold, respectively. This suggests that the transmembrane regions are critically important in forming the binding pocket for the nonpeptide antagonist.

Localization of ligand-binding domains of human corticotropin-releasing factor receptor: a chimeric receptor approach.

Two CRF receptors, CRFR1 and CRFR2, have recently been cloned and characterized. CRFR1 shares 70% sequence identity with CRFR2, yet has much higher affinity for rat/human CRF (r/hCRF) than CRFR2. As a first step toward understanding the interactions between rat/human CRF and its receptor, the regions that are involved in receptor-ligand binding and/or receptor activation were determined by using chimeric receptor constructs of the two human CRFR subtypes, CRFR1 and CRFR2, followed by generating point mutations of the receptor. The EC50 values in stimulation of intracellular cAMP of the chimeric and mutant receptors for the peptide ligand were determined using a cAMP-dependent reporter system. Three regions of the receptor were found to be important for optimal binding of r/hCRF and/or receptor activation. The first region was mapped to the junction of the third extracellular domain and the fifth transmembrane domain; substitution of three amino acids of CRFR1 in this region (Val266, Tyr267, and Thr268) by the corresponding CRFR2 amino acids (Asp266, Leu267, and Val268) increased the EC50 value by approximately 10-fold. The other two regions were localized to the second extracellular domain of the CRFR1 involving amino acids 175-178 and His189 residue. Substitutions in these two regions each increased the EC50 value for r/hCRF by approximately 7- to 8-fold only in the presence of the amino acid 266-268 mutation involving the first region, suggesting that their roles in peptide ligand binding might be secondary.

Heterogeneity between brain and pituitary corticotropin-releasing factor receptors is due to differential glycosylation.

Chemical affinity cross-linking studies have identified brain and pituitary CRF receptors with similar pharmacological characteristics but different mol wts (anterior pituitary, 75,000; brain, 58,000). In order to determine whether the heterogeneous nature of CRF receptors was inherent in the protein, we examined the glycoprotein nature of both types of CRF receptors using lectin affinity chromatography and treatments with exo- and endoglycosidases. CRF receptors in both the cerebral cortex and anterior pituitary adsorbed to and specifically eluted from Concanavalin-A- and wheat germ agglutinin-immobilized lectin affinity columns, indicating that both forms of the receptor are glycoproteins containing complex and high-mannose carbohydrate moieties. Cerebral cortical CRF receptors were sensitive to both neuraminidase and alpha-mannosidase treatment while pituitary CRF receptors were only affected by neuraminidase treatment, suggesting that CRF receptors in brain and pituitary differed slightly in the nature of their glycosylation units. After treatment of cerebral cortical or anterior pituitary CRF receptors with the endoglycosidase, N-glycanase, the mol wts were markedly decreased; the mol wt of the anterior pituitary CRF receptor was decreased from 75,000 to approximately 40,000-45,000 while in a corresponding manner, the cortical receptor was decreased from 58,000 to approximately 40,000-45,000. Limited proteolysis after deglycosylation with N-glycanase using the proteinases Staphylococcus aureus V8 (S. aureus V8) or papain, generated virtually identical peptide fragments from anterior pituitary- or cerebral cortex- labeled CRF receptor proteins. In summary, these data support the hypothesis that the ligand binding subunit of the CRF receptor in both brain and pituitary resides on a polypeptide of 40,000-45,000 and appears to be identical in both tissues. Differences observed in the mobility of the two proteins were found to be due to differences in the posttranslational modification of the proteins in the two tissues.

Corticotropin-releasing factor receptors in human small cell lung carcinoma cells: radioligand binding, second messenger, and northern blot analysis data.

Numerous peptides, growth factors, and receptors have been identified in small cell lung carcinoma (SCLC) cells. The present study was designed to examine the radioligand binding, second messenger, and messenger RNA (mRNA) characteristics of CRF receptors in a variety of SCLC lines and to compare their characteristics to CRF receptors in the mouse pituitary tumor AtT-20 cells. The human SCLC cell lines NCI-H69, H82, H146, H209, H345, H446, and H510A and control AtT-20 cells all demonstrated specific [125I]Tyr(o)-ovine CRF ([125I]oCRF) binding, which was linear with increasing protein concentrations, saturable, reversible, and of high affinity. NCI-H82 cells showed the highest level of specific [125I]oCRF binding (approximately 60% of the total binding). Scatchard analysis revealed a single homogeneous class of binding sites in NCI-H82 and AtT-20 cells, with Kd values of 263 +/- 48 and 285 +/- 75 pM, respectively, and binding capacities of 74 +/- 7 and 70 +/- 13 fmol/mg protein, respectively. [125I]oCRF-binding sites on NCI-H82 and AtT-20 cells had comparable pharmacological characteristics with the following rank order of inhibitory potencies: rat/human CRF approximately ovine CRF approximately bovine CRF > alpha-helical oCRF-(9-41) > bovine CRF-(1-41)OH >> vasoactive intestinal peptide, secretin, GH-releasing hormone. [125I]oCRF binding in the cell lines was inhibited by guanine nucleotides, suggesting a coupling of receptors to guanine nucleotide-binding proteins. The functional nature of the CRF receptor was demonstrated in second messenger studies in which rat/human CRF stimulated cAMP production in NCI-H82 and AtT-20 cells with comparable EC50 values of about 3 nM; the percent stimulation over basal activity was significantly higher in NCI-H82 cells (approximately 30-fold increase) than in AtT-20 cells (approximately 12-fold increase). Northern blot analysis of total RNA revealed the presence of a 2.6-kilobase mRNA band in NCI-H82 cells corresponding to the recently cloned human CRF receptor. In summary, the data demonstrate the presence of CRF receptors in SCLC cell lines with kinetic, pharmacological, second messenger, and mRNA characteristics comparable to those in pituitary and brain and suggest a possible role for CRF as a regulatory peptide in human SCLC.

Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: characterization of an active digitonin-solubilized receptor complex.

The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of [125I]Tyro-ovine CRF ([125I]oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for [125I] oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, [125I]oCRF labeled the same size receptor complex. These data suggest that either the guanine nucleotide-binding protein (Ns) is tightly associated with the CRF receptor after solubilization and is insensitive to guanine nucleotides, or that high affinity binding for soluble CRF receptors is not dependent on the coupling of a guanine nucleotide-binding protein. The solubilization of CRF receptors from membranes in digitonin should allow for the more complete molecular and functional characterization of CRF-mediated events and purification of the receptor.

Cloning and characterization of the human corticotropin-releasing factor-2 receptor complementary deoxyribonucleic acid.

Two CRF receptor subtypes (CRF1 and CRF2 receptors) with distinct brain localizations and pharmacological profiles have recently been cloned and characterized. For the CRF2 receptor subtype, at least 2 splice forms with different 5'-coding sequences (CRF2 alpha and CRF2 beta) have been identified in rat. In this article, we report the genomic structure and the corresponding complementary DNA (cDNA) sequence of the human CRF2 receptor. The gene coding for human CRF2 receptor consists of at least 12 exons and spans approximately 30 kilobases. The cDNA sequence in the protein-coding region is 94% identical to that of the reported rat CRF2 alpha receptor. At present, there is no evidence for the existence of a CRF2 beta receptor homolog in humans. The encoded receptor is 411 amino acids in length and is 70% identical to the human CRF1 receptor, with least sequence homology in the N-terminal extracellular domain (47% identical). Cells transfected with the full-length human CRF2 receptor cDNA responded to rat/human CRF and sauvagine by increasing the intracellular cAMP level, with EC50 values of approximately 20 and 1 nM, respectively. The CRF- and sauvagine-induced accumulation of intracellular cAMP could be competitively inhibited by the CRF receptor antagonist D-Phe-CRF. This pharmacological profile was comparable to that of the rat CRF2 alpha receptor. The relative abundance of the CRF2 receptor messenger RNA appears to be lower in humans than in rats for the tissues studied thus far.

Selective impairment of corticotropin-releasing factor1 (CRF1) receptor-mediated function using CRF coupled to saporin.

CRF is the main component in the brain neuropeptide effector system responsible for the behavioral, endocrine, and physiological activation that accompanies stress activation. Reduced CRF system activation plays a role in the etiology of a variety of psychiatric and metabolic disease states. We have developed a novel protein conjugate that joins native rat/human CRF to a ribosome-inactivating protein, saporin (CRF-SAP), for the purpose of targeted inactivation of CRF receptor-expressing cells. Cytotoxicity measurements revealed that CRF-SAP (1-100 nM) produced concentration-dependent and progressive cell death over time in CRF1 receptor-transfected L cells, but at similar concentrations had no effect on CRF2alpha receptor-transfected cells. The CRF-SAP-induced toxicity in CRF1-transfected cells was prevented by coincubation with the competitive CRF1/CRF2 receptor peptide antagonist, [D-Phe12]CRF-(12-41), or the selective nonpeptide CRF1 receptor antagonist, NBI 27914. Finally, in cultured rat pituitary cells that express native CRF1 receptors, CRF-SAP suppressed CRF-induced (1 nM) ACTH release. GnRH (1-10 nM) stimulated LH release was also assessed in the same pituitary cultures. Although there was a slight decrease in LH release from these cultures, this decrease was observed with CRF-SAP or SAP alone, suggesting that the response was nonspecific. Taken together, these results suggest the utility of CRF-SAP as a specific and subtype-selective tool for long term impairment of CRF1 receptor-expressing cells.

Preterm birth: associated neuroendocrine, medical, and behavioral risk factors.

Increased CRH secretion by the placenta of pregnant women has been associated with preterm birth. Certain indices of risk, both medical and psychosocial in nature, have been linked to preterm delivery. Levels of total, bound, and free CRH, CRH-binding protein (CRH-BP), and cortisol were measured prospectively in a large sample of pregnant Danish women who delivered preterm and term infants. Measures of maternal serum hormones were taken at 7--23 and 27--37 weeks gestation and, for those who delivered at term, at 37--43 weeks gestation. At 7--23 weeks gestation, maternal levels of total CRH (P = 0.01), bound CRH (P = 0.03), and CRH-BP (P = 0.01) were higher in the preterm than in the term group. At 27--37 weeks gestation, levels of total CRH (P < 0.0001), bound CRH (P < 0.0001), free CRH (P < 0.0001), and cortisol (P < 0.0001) were all higher in the preterm than the term group, whereas levels of CRH-BP (P < 0.0001) were lower in the preterm than in the term group. The best medical and behavioral factors associated with preterm delivery were, respectively, previous preterm delivery (P < 0.0001) and engagement in certain risk-taking behaviors (P = 0.008). The positive relations between preterm delivery and various adverse medical and socioeconomic variables with increases in placental secretion of CRH suggest that the latter may participate in the pathophysiology of preterm delivery.

Neuroprotective effects of the CRF1 antagonist R121920 after permanent focal ischemia in the rat.

The neuroprotective effects of a systemically active, highly selective, corticotropin-releasing factor-1 (CRF1) receptor antagonist, R121920 ((7-(dipropylamino)-2,5-dimethyl-3- [2-(dimethylamino)-5-pyridyl] pyrazolo [1,5-a] pyrimidine), was assessed in two rat models of permanent focal cerebral ischemia, where the middle cerebral artery (MCA) was occluded either through the subtemporal approach or using the intraluminal suture technique. R121920 rapidly crossed the blood-brain barrier after intravenous (IV) bolus administration (10 mg/kg), with peak brain concentrations at 5 minutes (2.26 +/- 0.40 microg/mL), which were approximately 2-fold greater than those in plasma (0.98 +/- 0.24 microg/mL). Treatment with R121920 (10 mg/kg IV followed by 5 mg/kg subcutaneously at hourly intervals for 4 hours) significantly (P < 0.001) reduced total (by 40%) and cortical (by 37%) infarct volume at 24 hours after subtemporal MCA occlusion (MCAO). In the intraluminal suture MCAO model, IV administration of R121920 (10 mg/kg) at the time of ischemia onset (and at multiple times thereafter) reduced both hemispheric infarct volume (by 34%, P < 0.001) and brain swelling (by 50%, P < 0.001) when assessed at 24 hours. In this model of focal ischemia, significant reduction (P < 0.05) in both outcome measures was obtained when R121920 administration was delayed up to 1 hour after MCAO. These results further define the antiischemic properties of selective CRF 1 antagonists in two experimental models of permanent focal cerebral ischemia.

Photoaffinity labelling of dopamine D2 receptors by [3H]azidomethylspiperone.

We have characterized the dopamine D2 receptor photoaffinity probe, [3H]azido-N-methylspiperone ([3H]AMS). In the absence of light, [3H]AMS bound reversibly and with high affinity (Kd 70 pM) to sites in canine striatal membranes and was competitively inhibited by dopaminergic agonists and antagonists with an appropriate D2 receptor specificity. Upon photolysis, [3H]AMS covalently incorporated into a peptide of Mr 92,000 as assessed by fluorography following SDS-polyacrylamide gel electrophoresis. Labelling of this peptide was specifically and stereoselectively blocked by D2 antagonists and agonists. Minor specifically labelled peptides of Mr 70,000-55,000 were observed under some conditions and were the result of proteolytic degradation of the peptide at Mr 92,000.

Glycoprotein nature of D2 dopamine receptors.

The glycoprotein nature of the ligand binding subunit of photoaffinity-labeled striatal D2 receptors was investigated. Upon photolysis, [125I]N-azidophenethylspiperone covalently incorporated into a major band of Mr 94000 with an appropriate pharmacological profile for D2 receptors as assessed by autoradiography following SDS-polyacrylamide gel electrophoresis. The exoglycosidase, neuraminidase, altered the electrophoretic mobility of the 94 kDa labeled band to 54 kDa with a slight modification in the binding affinity of [3H]spiperone. Endoglycosidase treatment (glycopeptidase-F) produced a further increase in the mobility of the 94 kDa peptide to approximately 43 kDa. A smaller specifically photolabeled D2 receptor peptide of 34 kDa does not contain terminal sialic acid but is an N-linked glycoprotein as assessed by lectin affinity chromatography and susceptibility to digestion by glycopeptidase-F to a peptide of approximately 23 kDa.

Recent advances with the CRF1 receptor: design of small molecule inhibitors, receptor subtypes and clinical indications.

Corticotropin-releasing factor (CRF) has been widely implicated as playing a major role in modulating the endocrine, autonomic, behavioral and immune responses to stress. The recent cloning of multiple receptors for CRF as well as the discovery of non-peptide receptor antagonists for CRF receptors have begun a new era of CRF study. Presently, there are five distinct targets for CRF with unique cDNA sequences, pharmacology and localization. These fall into three distinct classes, encoded by three different genes and have been termed the CRF1 and CRF2 receptors (belonging to the superfamily of G-protein coupled receptors) and the CRF-binding protein. The CRF2 receptor exists as three splice variants of the same gene and have been designated CRF2a CRF2b and CRF2g. The pharmacology and localization of all of these proteins in brain has been well established. The CRF1 receptor subtype is localized primarily to cortical and cerebellar regions while the CRF2a receptor is localized to subcortical regions including the lateral septum, and paraventricular and ventromedial nuclei of the hypothalamus. The CRF2b receptor is primarily localized to heart, skeletal muscle and in the brain, to cerebral arterioles and choroid plexus. The CRF2g receptor has most recently been identified in human amygdala. Expression of these receptors in mammalian cell lines has made possible the identification of non-peptide, high affinity, selective receptor antagonists. While the natural mammalian ligands oCRF and r/hCRF have high affinity for the CRF1 receptor subtype, they have lower affinity for the CRF2 receptor family making them ineffective labels for CRF2 receptors. [125I]Sauvagine has been characterized as a high affinity ligand for both the CRF1 and the CRF2 receptor subtypes and has been used in both radioligand binding and receptor autoradiographic studies as a tool to aid in the discovery of selective small molecule receptor antagonists. A number of non-peptide CRF1 receptor antagonists that can specifically and selectively block the CRF1 receptor subtype have recently been identified. Compounds such as CP 154,526 (12), NBI 27914 (129) and Antalarmin (154) inhibit CRF-stimulation of cAMP or CRF-stimulated ACTH release from cultured rat anterior pituitary cells. Furthermore, when administered peripherally, these compounds compete for ex vivo [125I]sauvagine binding to CRF1 receptors in brain sections demonstrating their ability to cross the blood-brain-barrier. In in vivo studies, peripheral administration of these compounds attenuate stress-induced elevations in plasma ACTH levels in rats demonstrating that CRF1 receptors can be blocked in the periphery. Furthermore, peripherally administered CRF1 receptor antagonists have also been demonstrated to inhibit CRF-induced seizure activity. These data clearly demonstrate that non-peptide CRF1 receptor antagonists, when administered systemically, can specifically block central CRF1 receptors and provide tools that can be used to determine the role of CRF1 receptors in various neuropsychiatric and neurodegenerative disorders. In addition, these molecules will prove useful in the discovery and development of potential orally active therapeutics for these disorders.

Effects of chronic antidepressant and benzodiazepine treatment on corticotropin-releasing-factor receptors in rat brain and pituitary.

We examined the effects of chronic treatment with antidepressants (imipramine or desipramine) or benzodiazepines (diazepam, alprazolam, or adinazolam) on modulation of corticotropin-releasing-factor (CRF) receptors in discrete areas of rat brain and in anterior pituitary. As previously reported, we found that chronic antidepressant treatment downregulated 5-HT2 serotonin and beta-adrenergic receptors in cerebral cortex. Although there was a trend toward increased CRF binding in brain stem, striatum, cerebellum, hypothalamus, and frontal cerebral cortex following antidepressant treatment, the changes were only statistically significant in brain stem in imipramine-treated rats. In addition, no significant changes were seen in CRF binding in other brain regions including parietal/temporal cerebral cortex, olfactory bulb, hippocampus, and anterior pituitary. Following chronic benzodiazepine treatment CRF receptor binding was significantly decreased in the frontal cerebral cortex and hippocampus; although there was a trend for CRF receptors to be decreased in other brain areas and increased in anterior pituitary, the changes were not statistically significant.

Normal pattern of labeling of cerebral cortical corticotropin-releasing factor (CRF) receptors in Alzheimer's disease: evidence from chemical cross-linking studies.

Recent data have demonstrated that in Alzheimer's disease, the concentrations of corticotropin-releasing factor (CRF) were reduced and that there were reciprocal increases in CRF receptors in affected cerebrocortical areas. In order to determine whether the increases in CRF receptors in Alzheimer's disease were due to altered molecular composition of the binding protein, we compared the labeling pattern of 125I-Tyr0-ovine CRF in temporal neocortex of Alzheimer's patients and age-matched controls using chemical cross-linking techniques. A similar pattern of 125I-Tyr0-ovine CRF labeling was seen in Alzheimer's and control brains, with the major CRF binding protein corresponding to an apparent molecular weight of 58,000 Da. These data indicate that the increased CRF receptor population in cerebral cortex in Alzheimer's disease comprises bona fide CRF receptor binding subunits with no apparent change in the molecular structure.

Previous stress alters corticotropin-releasing factor neurotransmission in the locus coeruleus.

Spontaneous and stress-evoked discharge of locus coeruleus neurons were characterized in rats with a history of stress. Rats exposed to one or five daily 30-min sessions of footshock were anesthetized with halothane and surgically prepared for locus coeruleus single-unit recording immediately following the last session. Locus coeruleus spontaneous discharge rate and discharge evoked by sciatic nerve stimulation were comparable between acutely and repeatedly stressed rats and controls. In contrast, locus coeruleus activation produced by intracerebroventricular administration of corticotropin-releasing factor (3 micrograms) or by hypotensive challenge (which requires endogenous corticotropin-releasing factor release in the locus coeruleus) was greatly attenuated in acutely stressed rats. The corticotropin-releasing factor dose-response curve was shifted to the right in acutely stressed rats compared with controls. In repeatedly stressed rats, the effects of 3 micrograms corticotropin-releasing factor on locus coeruleus discharge were similarly diminished. Although the maximum effect produced by corticotropin-releasing factor was decreased in these rats, the dose-response curve was shifted to the left, indicative of sensitization. Hypotensive challenge, which was ineffective in acutely stressed rats, increased locus coeruleus discharge of repeatedly stressed rats by a similar magnitude as in matched controls. The return of locus coeruleus responsiveness to hypotension in repeatedly stressed rats may be related to the sensitization to corticotropin-releasing factor. Finally, the protocol of repeated stress did not alter the affinity or density of corticotropin-releasing factor receptors in either the frontal cortex or brainstem. Taken together, the results suggest that a history of stress alters corticotropin-releasing factor neurotransmission in the locus coeruleus at the postsynaptic level. However, these effects are not reflected by corticotropin-releasing factor binding kinetics in brainstem. Stress-induced changes in corticotropin-releasing factor neurotransmitter function in the locus coeruleus may play a role in certain symptoms of stress-related psychiatric disorders.

Colorimetric assay for rapid screening of corticotropin releasing factor receptor ligands.

The corticotropin releasing factor (CRF) receptor is known to be coupled to Gs and transduces its signal through stimulation of cyclic AMP (cAMP) production. Here we describe the characterization of several stable CRF receptor-expressing LVIP2.0Zc cell lines that also contain an exogenous cAMP-responsive beta-galactosidase reporter gene construct. The CRF receptor activity was assayed by measuring the induction of beta-galactosidase in response to CRF. Rat/human and bovine CRF stimulated beta-galactosidase activity in a dose-dependent manner with EC50 values of approximately 0.1 nM; the biologically weak deamidated analog of bovine CRF was approximately 500-fold less potent. The CRF receptor antagonist, [d-Phe12,Nle21,38,Ala32]r/hCRF(12-41) produced a dose-dependent inhibition of CRF-stimulated beta-galactosidase activity, further demonstrating the pharmacological specificity of the interaction. The magnitude of the maximal response to CRF varied among individual cell lines. This variation was independent of the level of CRF receptor expression, but reflected differences in the intrinsic activity of adenylate cyclase. In contrast to most cAMP assay systems, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine decreased the CRF-induced beta-galactosidase activity when used in the context of the assay regimen described here. Since the assay can be easily performed in a high-throughput 96-well plate format, these cell lines provide an efficient way for the identification of CRF receptor agonists and antagonists.

Corticotropin-releasing factor (CRF) receptors in intermediate lobe of the pituitary: biochemical characterization and autoradiographic localization.

CRF receptors were characterized using radioligand binding and chemical affinity cross-linking techniques and localized using autoradiographic techniques in porcine, bovine and rat pituitaries. The binding of 125I-[Tyr0]-ovine CRF (125I-oCRF) to porcine anterior and neurointermediate lobe membranes was saturable and of high affinity with comparable KD values (200-600 pM) and receptor densities (100-200 fmoles/mg protein). The pharmacological rank order of potencies for various analogs and fragments of CRF in inhibiting 125I-oCRF binding in neurointermediate lobe was characteristic of the well-established CRF receptor in anterior pituitary. Furthermore, the binding of 125I-oCRF to both anterior and neurointermediate lobes of the pituitary was guanine nucleotide-sensitive. Affinity cross-linking studies revealed that the molecular weight of the CRF binding protein in rat intermediate lobe was identical to that in rat anterior lobe (Mr = 75,000). While the CRF binding protein in the anterior lobes of porcine and bovine pituitaries had identical molecular weights to CRF receptors in rat pituitary (Mr = 75,000), the molecular weight of the CRF binding protein in porcine and bovine intermediate lobe was slightly higher (Mr = 78,000). Pituitary autoradiograms from the three species showed specific binding sites for 125I-oCRF in anterior and intermediate lobes, with none being apparent in the posterior pituitary. The identification of CRF receptors in the intermediate lobe with comparable characteristics to those previously identified in the anterior pituitary substantiate further the physiological role of CRF in regulating intermediate lobe hormone secretion.

Homologous desensitization of human corticotropin-releasing factor1 receptor in stable transfected mouse fibroblast cells.

Previous radioligand binding and second messenger studies have shown that corticotropin-releasing factor (CRF) modulates its receptor following both in vivo and in vitro treatment. In the present study, we determined the sequence of events leading to CRF-induced downregulation and desensitization of cloned CRF receptors in murine fibroblast cells (Ltk-) stably transfected with CRF1 DNA (from human pituitary). Treatment of cells with rat/human CRF produced a dose- and time-dependent decrease in [125I]Tyr degrees-ovine CRF ([125I]oCRF) binding and a concomitant decrease in CRF-stimulated adenylate cyclase activity. Significant decreases in [125I]oCRF binding and agonist-stimulated cAMP production were evident minutes after CRF treatment with maximal (60-80%) reductions seen following 1 h of CRF treatment. Scatchard analysis revealed that the decrease in [125I]oCRF binding was due to the downregulation of the receptor with no significant alteration seen in the affinity of the ligand. Since the transfected cell line is engineered using an artificial promoter, we did not detect any significant changes in CRF1 receptor mRNA levels following CRF treatment for up to 24 h.

Brain corticotropin-releasing factor immunoreactivity and receptors in five inbred rat strains: relationship to forced swimming behaviour.

In the present work we studied the relationship between behaviour in the forced swimming test (FST), a test that presumably measures depressive-like behaviour in rodents, and central corticotropin-releasing factor (CRF) concentration and binding in five strains of rats. The strains were: Brown-Norway (BN), Fisher (FIS) 344, Lewis (LEW), spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The FST data corresponding to the pretest showed significant inter-strain differences in both struggling and immobility: BN and WKY rats displayed lower levels of struggling and longer periods of immobility, LEW and SHR rats showed intermediate levels, and FIS rats were the most active. The results of the pretest were roughly similar to those observed in the test, the activity of WKY being extremely low. The CRF binding revealed significant inter-strain differences in prefrontal cortex and hippocampus, but not in cerebellum, pons-medulla or hypothalamus: in the prefrontal cortex, BN and FIS rats showed greater CRF binding than LEW, SHR and WKY rats; in the hippocampus BN rats showed higher levels of CRF binding than the other strains. The study of CRF content in various brain areas revealed inter-strain differences in prefrontal cortex and pons-medulla, but not in parietal-temporal cortex or in hypothalamus (CRF concentrations in the hippocampus were not detectable): CRF content in the prefrontal cortex was higher in BN than in the other strains, although the differences with FIS were not statistically significant; in the pons-medulla, FIS and LEW showed significantly higher CRF content than the other strains. From the present results it appears that BN and WKY rats were more prone to adopt passive strategies in the FST, but they did not show higher brain CRF immunoreactivity or down-regulation of CRF receptors. Hence, although there were inter-strains differences in all variables studied, no evidence for a relationship between the FST behaviour and central CRF activity was found.