Marilyn H. Perrin

Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse

Two G protein‐coupled receptors have been identified that bind corticotropin‐releasing factor (CRF) and urocortin (UCN) with high affinity. Hybridization histochemical methods were used to shed light on controversies concerning their localization in rat brain, and to provide normative distributional data in mouse, the standard model for genetic manipulation in mammals. The distribution of CRF‐R1 mRNA in mouse was found to be fundamentally similar to that in rat, with expression predominating in the cerebral cortex, sensory relay nuclei, and in the cerebellum and its major afferents. Pronounced species differences in distribution were few, although more subtle variations in the relative strength of R1 expression were seen in several forebrain regions. CRF‐R2 mRNA displayed comparable expression in rat and mouse brain, distinct from, and more restricted than that of CRF‐R1. Major neuronal sites of CRF‐R2 expression included aspects of the olfactory bulb, lateral septal nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, medial and posterior cortical nuclei of the amygdala, ventral hippocampus, mesencephalic raphe nuclei, and novel localizations in the nucleus of the solitary tract and area postrema. Several sites of expression in the limbic forebrain were found to overlap partially with ones of androgen receptor expression. In pituitary, rat and mouse displayed CRF‐R1 mRNA signal continuously over the intermediate lobe and over a subset of cells in the anterior lobe, whereas CRF‐R2 transcripts were expressed mainly in the posterior lobe. The distinctive expression pattern of CRF‐R2 mRNA identifies additional putative central sites of action for CRF and/or UCN. Constitutive expression of CRF‐R2 mRNA in the nucleus of the solitary tract, and stress‐inducible expression of CRF‐R1 transcripts in the paraventricular nucleus may provide a basis for understanding documented effects of CRF‐related peptides at a loci shown previously to lack a capacity for CRF‐R expression or CRF binding. Other such “mismatches” remain to be reconciled. J. Comp. Neurol. 428:191–212, 2000.

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.

Functional amyloids as natural storage of peptide hormones in pituitary secretory granules.

Plethora of Secretory Amyloids Protein aggregation and the formation of amyloids are associated with several dozen pathological conditions in humans, including Alzheimers disease, Parkinsons disease, and type II diabetes. In addition, a few functional amyloid systems are known: the prions of fungi, the bacterial protein curli, the protein of chorion of the eggshell of silkworm, and the amyloid protein Pmel-17 involved in mammalian skin pigmentation. Now Maji et al. (p. 328, published online 18 June) propose that endocrine hormone peptides and proteins are stored in an amyloid-like state in secretory granules. Thus, the amyloid fold may represent a fundamental, ancient, and evolutionarily conserved protein structural motif that is capable of performing a wide variety of functions contributing to normal cell and tissue physiology. Peptide and protein hormones are stored in secretory granules in a nonpathological amyloid conformation. Amyloids are highly organized cross–β-sheet–rich protein or peptide aggregates that are associated with pathological conditions including Alzheimer’s disease and type II diabetes. However, amyloids may also have a normal biological function, as demonstrated by fungal prions, which are involved in prion replication, and the amyloid protein Pmel17, which is involved in mammalian skin pigmentation. We found that peptide and protein hormones in secretory granules of the endocrine system are stored in an amyloid-like cross–β-sheet–rich conformation. Thus, functional amyloids in the pituitary and other organs can contribute to normal cell and tissue physiology.

Corticotropin-releasing factor receptors are widely distributed within the rat central nervous system: an autoradiographic study

Corticotropin-releasing factor (CRF) receptor-binding sites have been localized and quantified in the rat central nervous system (CNS) by autoradiography with an iodine-125-labeled analogue of ovine CRF substituted with norleucine and tyrosine at amino acid residues 21 and 32, respectively. High affinity and pharmacologically specific receptor- binding sites for CRF were found in discrete areas within the rat CNS. CRF receptors were highly concentrated in laminae 1 and 4 throughout the neocortex, the external plexiform layer of the olfactory bulb, the external layer of the median eminence, several cranial nerve nuclei in the brainstem including the facial, oculomotor, trochlear, vestibulocochlear, and trigeminal nuclei, the deep cerebellar nuclei, and the cerebellar cortex. Moderate concentrations of CRF receptors were present in the olfactory tubercle, caudate-putamen, claustrum, nucleus accumbens, nucleus of the diagonal band, basolateral nucleus of the amygdala, paraventricular nucleus of the hypothalamus, mammillary peduncle, inferior and superior olives, medullary reticular formation, inferior colliculus, and brainstem nuclei including tegmental, parabrachial, hypoglossal, pontine, cuneate, and gracilis nuclei, and in spinal cord. Lower densities of CRF binding were found in the bed nucleus of the stria terminalis, central and medial amygdaloid nuclei, and regions of the thalamus, hypothalamus, hippocampus, and brainstem. The distribution of CRF-binding sites generally correlates with the immunocytochemical distribution of CRF pathways and with the pharmacological sites of action of CRF. These data strongly support a physiological role for endogenous CRF in regulating and integrating functions in the CNS.

Corticotropin Releasing Factor Receptors and Their Ligand Family

ABSTRACT: The CRF receptors belong to the VIP/GRF/PTH family of G‐protein coupled receptors whose actions are mediated through activation of adenylate cyclase. Two CRF receptors, encoded by distinct genes, CRF‐R1 and CRF‐R2, and that can exist in two alternatively spliced forms, have been cloned. The type‐1 receptor is expressed in many areas of the rodent brain, as well as in the pituitary, gonads, and skin. In the rodent, one splice variant of the type‐2 receptor, CRF‐R2α, is expressed mainly in the brain, whereas the other variant, CRF‐R2β, is found not only in the CNS, but also in cardiac and skeletal muscle, epididymis, and the gastrointestinal tract. The poor correlation between the sites of expression of CRF‐R2 and CRF, as well as the relatively low affinity of CRF for CRF‐R2, suggested the presence of another ligand, whose existence was confirmed in our cloning of urocortin. This CRF‐like peptide is found not only in brain, but also in peripheral sites, such as lymphocytes. The broad tissue distribution of CRF receptors and their ligands underscores the important role of this system in maintenance of homeostasis. Functional studies of the two receptor types reveal differences in the specificity for CRF and related ligands. On the basis of its greater affinity for urocortin, in comparison with CRF, as well as its brain distribution, CRF‐R2 may be the cognate receptor for urocortin. Mutagenesis studies of CRF receptors directed toward understanding the basis for their specificity, provide insight into the structural determinants for hormone‐receptor recognition and signal transduction.

High affinity binding sites for a somatostatin-28 analog in rat brain

Abstract Using an iodinated analog of a large (28 residues) and biologically active form of somatostatin, 125 I[Leu 8 ,D-Trp 22 ,Tyr 25 ]SS-28, it was possible to demonstrate saturable and high affinity binding sites (dissociation constant = 0.46 ± 0.04 nM) in rat cortical membranes. Somatostatin, somatostatin-28, as well as two potent analogs, [D-Trp 8 ] somatostatin and [D-Trp 22 ] somatostatin-28, could completely displace the radiogland in the nanomolar range whereas the inactive analog Des-Trp 8 -somatostatin and the unrelated peptide GnRH showed no affinity for these binding sites; octa- and nona-peptide analogs of somatostatin were inactive. High binding was found in hippocampus, amygdala, tuberculum olfactorium, caudate-putamen and cortex; moderate binding in midbrain and hypothalamus, and no binding in the cerebellum. These results suggest that specific somatostatin receptors can be measured within the brain with 125 I[Leu 8 ,D-Trp 22 ,Tyr 25 ] SS-28 as radioligand.

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.

Structure of the N-terminal domain of a type B1 G protein-coupled receptor in complex with a peptide ligand

The corticotropin releasing factor (CRF) family of ligands and their receptors coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play additional roles within the cardiovascular, gastrointestinal, and other systems. The actions of CRF and the related urocortins are mediated by activation of two receptors, CRF-R1 and CRF-R2, belonging to the B1 family of G protein-coupled receptors. The short-consensus-repeat fold (SCR) within the first extracellular domain (ECD1) of the CRF receptor(s) comprises the major ligand binding site and serves to dock a peptide ligand via its C-terminal segment, thus positioning the N-terminal segment to interact with the receptors juxtamembrane domains to activate the receptor. Here we present the 3D NMR structure of ECD1 of CRF-R2β in complex with astressin, a peptide antagonist. In the structure of the complex the C-terminal segment of astressin forms an amphipathic helix, whose entire hydrophobic face interacts with the short-consensus-repeat motif, covering a large intermolecular interface. In addition, the complex is characterized by intermolecular hydrogen bonds and a salt bridge. These interactions are quantitatively weighted by an analysis of the effects on the full-length receptor affinities using an Ala scan of CRF. These structural studies identify the major determinants for CRF ligand specificity and selectivity and support a two-step model for receptor activation. Furthermore, because of a proposed conservation of the fold for both the ECD1s and ligands, this structure can serve as a model for ligand recognition for the entire B1 receptor family.

Corticotropin releasing factor receptor-mediated stimulation of adenylate cyclase activity in the rat brain

Corticotropin releasing factor (CRF)-stimulated adenylate cyclase activity and receptor binding were examined in rat brain homogenates using a potent synthetic CRF analog--[D-Tyr3,D-Pro4,Nle18,21,alpha-helical]CRF3-41 (alpha-hel CRF3-41). Binding of alpha-hel CRF3-41 in the rat brain was saturable, reversible, of high affinity and exhibited relevant peptide specificity. This analog also stimulated adenylate cyclase activity of various brain regions; the greatest magnitude of stimulation was in the cerebral cortex followed by the septum, cerebellum and thalamus. Adenylate cyclase stimulation in the cerebral cortex was concentration-dependent with an ED50 of 2.5 +/- 0.4 nM for alpha-hel CRF3-41 and an ED50 of 16 +/- 2 nM for ovine and rat CRF. Maximal stimulation was comparable for all peptides. Agonist-stimulated adenylate cyclase activity was competitively blocked by the CRF antagonists. The inactive CRF analog, ovine CRF1-39, at concentrations less than 1 microM, did not significantly stimulate adenylate cyclase. Adrenalectomy, which has been reported to modulate CRF receptor number and CRF-stimulated adenylate cyclase activity in the anterior pituitary, had no effect on CRF receptor binding or CRF-stimulated adenylate cyclase activity in the cerebral cortex. These results suggest that, as in the anterior pituitary, at least some of the physiological responses mediated by CRF receptors in the brain utilize the cyclic nucleotide regulatory pathway as a post-receptor mechanism.