Joan Vaughan

Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.

Urocortin II: A member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors

Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain poly(A)+ RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1–10 μg of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.

Chemical and biological characterization of the inhibin family of protein hormones.

Publisher Summary This chapter discusses the chemical and biological characterization of the inhibin family of protein hormones, which is a family of peptides isolated from the follicular fluid or rete testis fluid on the basis of their ability to inhibit the secretion of the follicle-stimulating hormone (FSH) by cultured rat anterior pituitary cells. It also reviews the possible roles of inhibin and fibre-reinforced plastic (FRP)/activin in placenta, brain, and bone marrow. Inhibin-related dimers are broadly distributed anatomically and have powerful activities in several biological systems where inhibin and FRP/activin often exhibit opposite effects. While the physiologic roles of inhibin to regulate FSH secretion in the female rat and immature male rat are strongly supported, the significance of these hormones within the gonad, brain, placenta, and bone marrow have yet to be placed in in vivo context. Although the panoply of functions of inhibin and FRP/activin are certainly incompletely understood at this time, this family has already demonstrated a powerful mechanism for the generation of signal diversity whereby differential subunit association can result in the generation of dimers with opposing biological actions in multiple tissues.

Urocortin expression in rat brain: evidence against a pervasive relationship of urocortin-containing projections with targets bearing type 2 CRF receptors.

Histochemical and axonal transport methods were used to clarify the central organization of cells and fibers that express urocortin (UCN), a recently discovered corticotropin‐releasing factor (CRF)‐related neuropeptide, which has been proposed as an endogenous ligand for type 2 CRF receptors (CRF‐R2). Neurons that display both UCN mRNA and peptide expression were found to be centered in the Edinger‐Westphal (EW), lateral superior olivary (LSO), and supraoptic nuclei; lower levels of expression are seen in certain cranial nerve and spinal motoneurons and in small populations of neurons in the forebrain. Additional sites of UCN mRNA and peptide expression detected only in colchicine‐treated rats are considered to be minor ones. UCN‐immunoreactive projections in brain are predominantly descending and largely consistent with central projections attributed to the EW and LSO, targeting principally accessory optic, precerebellar, and auditory structures, as well as the spinal intermediate gray. Although neither the EW nor LSO are known to project to the forebrain, UCN‐ir neurons in the EW were identified that project to the lateral septal nucleus, which houses a prominent UCN‐ir terminal field. Although substantial UCN‐ir projections were observed to several brainstem cell groups that express CRF‐R2, including the dorsal raphe and interpeduncular nuclei and the nucleus of the solitary tract (NTS), most prominent seats of CRF‐R2 expression were found to contain inputs immunopositive for piscine urotensin I, but not rat UCN. The results define a central UCN system whose organization suggests a principal involvement in motor control and sensorimotor integration; its participation in stress‐related mechanisms would appear to derive principally by virtue of projections to the spinal intermediolateral column, the NTS, and the paraventricular nucleus. Several observations, including the lack of a pervasive relationship of UCN‐ir projections with CRF‐R2‐expressing targets, support the existence of still additional CRF‐related peptides in mammalian brain. J. Comp. Neurol. 415:285–312, 1999.

Purification and partial characterization of inhibin from porcine follicular fluid.

Inhibin, a protein of gonadal origin that suppresses the basal secretion of follicle stimulating hormone by anterior pituitary cells has been purified from porcine follicular fluid. Using several RP-HPLC steps and gel filtration under denaturing conditions, we obtained a fraction approximately ten thousand fold purified which showed one band on SDS PAGE and in the same experiment two bands after reduction (MW ca 14K and ca 18K) suggesting a molecular weight of 32K for inhibin. Edman degradation of isolated inhibin and carboxymethylated chain A indicated that the first 6 residues were H-Ser-Thr-Ala-Pro-Leu-Pro-; by subtraction, the first 3 residues of chain B could be deduced to be H-Gly-Leu-Glu-. EC50 was ca 0.3 ng/ml or 10 pM in our in vitro pituitary cell culture assay. Antibodies to residues 1-6 were raised which could immunoneutralize purified inhibin activity in an in vitro assay.

[38] Assay of corticotropin releasing factor

Publisher Summary The corticotropin releasing factor (CRF) can be assayed by a variety of biological and immunological techniques. The chapter describes two methods: an in vitro assay employing primary anterior pituitary cell cultures and a radioimmunoassay (RIA) procedure. The chapter presents the acid extraction method for tissue fragments and a Bond Elute method for aqueous samples for extraction of CRF. The cell culture assay is the exclusive assay used for the isolation and subsequent characterization of ovine CRF and has, thereby, been useful for the detection and quantification of CRF in crude or partially purified extracts. The method has been applied for a wide range of studies investigating the mode of action of CRF and its interactions with other physiological modulators such as glucocorticoids, catecholamines, and neurohypophysial peptides. This method is useful for the assay of both agonist and antagonist analogs of CRF and of highly purified CRF-like peptides of various mammalian and nonmammalian species.

Detection and purification of inhibin using antisera generated against synthetic peptide fragments

Publisher Summary Inhibin is a hormone whose best established physiological role is selective suppression of the release of follicle-stimulating hormone (FSH) from the pituitary. Subsequent to the identification of inhibin, two laboratories isolated a protein from porcine follicular fluid that selectively released FSH from pituitary cell cultures. Characterization of this protein––named FSH releasing protein (FRP)/activin––showed that it was a homo- or heterodimer of inhibin β subunits. This chapter also describes the production of antibodies to the α, βA, and βB subunits of inhibin; development of a radioimmunoassay (RIA) specific for inhibin, using an antibody directed to the α subunit; use of these antibodies for Western blot analysis; and a method for concentrating inhibin and FRP from biological fluids. It also discusses a method that was developed for the rapid isolation of inhibin from ram rete testis fluid (RTF) using immunoaffinity chromatography with an antibody directed against the α subunit.

Hormonal regulation of inhibin production by cultured Sertoli cells

The hormonal regulation of inhibin production by cultured rat Sertoli cells was examined using a specific radioimmunoassay (RIA) which detects the N-terminal portion of the porcine inhibin alpha chain. FSH, but not hCG or prolactin caused a dose-dependent increase in inhibin production (EC50 for FSH = 2.4 ng/ml); both secreted and intracellular levels of inhibin were increased, but the secreted form represented one-half to two-thirds of the total. The FSH-stimulated production of inhibin was augmented by addition of a phosphodiesterase inhibitor, and could be mimicked by cholera toxin, forskolin, or dibutyryl cAMP, all of which are known to increase intracellular cAMP levels. Inclusion of either dihydrotestosterone or estradiol in the cultures had no effect on inhibin production, both in the presence and absence of FSH. Examination of the conditioned media from forskolin-treated Sertoli cells by gel filtration chromatography revealed a single peak of bioactive and immunoreactive inhibin, at a molecular weight of approximately 32,000, similar to that observed for the porcine and bovine follicular fluid inhibins. Thus, FSH activated the cAMP pathway to stimulate Sertoli cell production of inhibin which in turn suppresses pituitary FSH release to form a closed-loop feedback system.

corticotropin-releasing Factor (crf) Family of Ligands and Their Receptors

Corticotropin-releasing factor (CRF) was recognized biologically in the 1950s and was first isolated from ovine hypothalamus and characterized as a 41 amino acid peptide in 1981. Subsequently, rat and human CRF were identified and found to be identical to one another, while differing from ovine CRF by seven residues. A variety of experimental observations indicate that CRF is the key neuroregulator of the hypothalamic-pituitary-adrenal cortical (HPA) axis. The actions of CRF on adrenocorticotropic hormone (ACTH) secretion are potentiated by vasopressin and blunted by glucocorticoid negative feedback. The broad central and peripheral distribution of the peptide and its receptors supports the notion that CRF is an important local regulator within the central nervous, immune, and other systems. Further, CRF mediates numerous complementary stress-related endocrine, immune, autonomic, and behavioral responses. Antagonists of CRF, such as α-helical CRF or astressin, block many stress-induced physiologic and pathophysiologic responses in experimental animals, and perturbations of the CRF system or the HPA have been reported in human affective disorders. The effects of CRF within the central nervous system may be anatomically and temporally limited by a high-affinity binding protein (CRF-BP). The actions of CRF are mediated by seven transmembrane-domain G-protein-coupled receptors (CRF-R) derived from two genes (R1 and R2), each of which has alternative splice variants. We have identified a novel urotensin and CRF-like peptide, urocortin, in the rat brain and the human genome. Urocortin has a high affinity for CRF-R1 and R2 as well as for CRF-BP. Synthetic urocortin has potent biological actions on both CRF-R1 (pituitary ACTH release) as well as CRF-R2 (vasodilation, reduction of vascular permeability) mediated events. This novel peptide appears to be an endogenous ligand for CRF-type 2 receptors.

Urocortin 2-Deficient Mice Exhibit Gender-Specific Alterations in Circadian Hypothalamus–Pituitary–Adrenal Axis and Depressive-Like Behavior

Gender differences in hypothalamus–pituitary–adrenal (HPA) axis activation and the prevalence of mood disorders are well documented. Urocortin 2, a recently identified member of the corticotropin-releasing factor family, is expressed in discrete neuroendocrine and stress-related nuclei of the rodent CNS. To determine the physiological role of urocortin 2, mice null for urocortin 2 were generated and HPA axis activity, ingestive, and stress-related behaviors and alterations in expression levels of CRF-related ligands and receptors were examined. Here we report that female, but not male, mice lacking urocortin 2 exhibit a significant increase in the basal daily rhythms of ACTH and corticosterone and a significant decrease in fluid intake and depressive-like behavior. The differential phenotype of urocortin 2 deficiency in female and male mice may imply a role for urocortin 2 in these gender differences.