Frank M. Dautzenberg

The CRF peptide family and their receptors: yet more partners discovered

Abnormal signaling at corticotropin-releasing factor CRF1 and CRF2 receptors might contribute to the pathophysiology of stress-related disorders such as anxiety, depression and eating disorders, in addition to cardiac and inflammatory disorders. Recently, molecular characterization of CRF1 and CRF2 receptors and the cloning of novel ligands--urocortin, stresscopin-related peptide/urocortin II, and stresscopin/urocortin III--have revealed a far-reaching physiological importance for the family of CRF peptides. Although the physiological roles of the CRF2 receptor remain to be defined, the preclinical and clinical development of specific small-molecule antagonists of the CRF1 receptor opens new avenues for the treatment of psychiatric and neurological disorders.

International Union of Pharmacology. XXXVI. Current Status of the Nomenclature for Receptors for Corticotropin-Releasing Factor and Their Ligands

Receptors for corticotropin-releasing factor (CRF) are members of a family of G protein-coupled receptors (“Family B”) that respond to a variety of structurally dissimilar releasing factors, neuropeptides, and hormones (including secretin, growth hormone-releasing factor, calcitonin, parathyroid hormone, pituitary adenylate cyclase-activating polypeptide, and vasoactive intestinal polypeptide) and signal through the cyclic AMP and/or calcium pathways. To date, three genes encoding additional CRF-like peptides (urocortins) have been identified in mammals. The urocortins and CRF bind with differential ligand selectivity at the two mammalian CRF receptors. This report was prepared by the International Union of Pharmacology Subcommittee on CRF Receptors, to summarize the current state of CRF receptor biology and to propose changes in the classification and nomenclature of CRF ligands and receptors.

Corticotropin Releasing Factor (CRF) Receptor Signaling in the Central Nervous System: New Molecular Targets

Corticotropin-releasing factor (CRF) and the related urocortin peptides mediate behavioral, cognitive, autonomic, neuroendocrine and immunologic responses to aversive stimuli by activating CRF(1) or CRF(2) receptors in the central nervous system and anterior pituitary. Markers of hyperactive central CRF systems, including CRF hypersecretion and abnormal hypothalamic-pituitary-adrenal axis functioning, have been identified in subpopulations of patients with anxiety, stress and depressive disorders. Because CRF receptors are rapidly desensitized in the presence of high agonist concentrations, CRF hypersecretion alone may be insufficient to account for the enhanced CRF neurotransmission observed in these patients. Concomitant dysregulation of mechanisms stringently controlling magnitude and duration of CRF receptor signaling also may contribute to this phenomenon. While it is well established that the CRF(1) receptor mediates many anxiety- and depression-like behaviors as well as HPA axis stress responses, CRF(2) receptor functions are not well understood at present. One hypothesis holds that CRF(1) receptor activation initiates fear and anxiety-like responses, while CRF(2) receptor activation re-establishes homeostasis by counteracting the aversive effects of CRF(1) receptor signaling. An alternative hypothesis posits that CRF(1) and CRF(2) receptors contribute to opposite defensive modes, with CRF(1) receptors mediating active defensive responses triggered by escapable stressors, and CRF(2) receptors mediating anxiety- and depression-like responses induced by inescapable, uncontrollable stressors. CRF(1) receptor antagonists are being developed as novel treatments for affective and stress disorders. If it is confirmed that the CRF(2) receptor contributes importantly to anxiety and depression, the development of small molecule CRF(2) receptor antagonists would be therapeutically useful.

Role of CRF Receptor Signaling in Stress Vulnerability, Anxiety, and Depression

Markers of hyperactive central corticotropin releasing factor (CRF) systems and CRF‐related single nucleotide polymorphisms (SNPs) have been identified in patients with anxiety and depressive disorders. Designing more effective antagonists may now be guided by data showing that small molecules bind to transmembrane domains. Specifically, CRF1 receptor antagonists have been developed as novel anxiolytic and antidepressant treatments. Because CRF1 receptors become rapidly desensitized by G protein‐coupled receptor kinase (GRK) and β‐arrestin mechanisms in the presence of high agonist concentrations, neuronal hypersecretion of synaptic CRF alone may be insufficient to account for excessive central CRF neurotransmission in stress‐induced affective pathophysiology. In addition to desensitizing receptor function, GRK phosphorylation and β‐arrestin binding can shift a G protein‐coupled receptor (GPCR) to signal selectively via the extracellular signal‐regulated kinase/mitogen‐activated protein kinase (ERK‐MAPK) or Akt pathways independent of G proteins. Also, Epac‐dependent CRF1 receptor signaling via the ERK‐MAPK pathway has been found to potentiate brain‐derived neurotrophic factor (BDNF)‐stimulated TrkB signaling. Thus, genetic or acquired abnormalities in GRK and β‐arrestin function may be involved in the pathophysiology of stress‐induced anxiety and depression.

7TM receptors: the splicing on the cake

Within a given family of seven transmembrane domain (7TM) receptors, functional diversity is most often afforded by the existence of multiple receptor subtypes, each encoded by a distinct gene. However, it is now clear that the existence of introns in genes encoding some members of a receptor family provides scope for additional diversity by virtue of splicing events that result in the formation of different receptor mRNAs and consequently distinct receptor isoforms. A large number of 7TM receptor splice variants have now been shown to exist. In this article, the current data on alternatively spliced variants for hormone and neurotransmitter 7TMs are reviewed, their potential physiological importance considered and some of the issues pertaining to the classification and nomenclature of receptor isoforms produced in this way are addressed.

Molecular biology of the CRH receptors— in the mood

Dysfunctioning of corticotropin-releasing hormone (CRH) and its receptors (CRH(1) and CRH(2)) has been linked to the development of stress-related disorders, such as mood and eating disorders. The molecular characterization of CRH(1) and CRH(2) receptors and their splice variants has generated detailed information on their pharmacology, tissue distribution and physiology. While mammalian CRH(1) receptors nonselectively bind CRH analogs, the ligand specificity of CRH(2) is narrower. CRH(1) receptors are predominantly expressed in the brain and pituitary, whereas CRH(2) receptor expression is limited to particular brain areas and to some peripheral organs. Molecular approaches to block CRH(1) receptor expression in the brain argue in favor of its involvement in the regulation of some aspects of the stress response. The CRH(2alpha) receptor may be more important for motivational types of behavior essential for survival, such as feeding and defense.(1)

Cloning of a Human Renal p–Aminohippurate Transporter, hROAT1

For several decades, p-aminohippurate (PAH) has served as a model substrate to study the renal excretion of amphiphilic organic anions [1]. PAH is freely filtered in the glomeruli and also efficiently secreted in the proximal tubules. In all species investigated so far, the uptake of PAH across the basolateral membrane of proximal tubule cells occurs by exchange with intracellular α-ketoglutarate through a PAH/ α-ketoglutarate antiporter [1–3]. The dicarboxylate α-ketoglutarate is recycled by a Na +-coupled transport mechanism and the Na +-ions are pumped out of the cell by the Na +, K+-ATPase. Overall, the uptake of one PAH molecule requires the expenditure of one ATP molecule. Detailed investigations on substrate specificity revealed that the PAH transporter in the basolateral membrane of rat proximal tubule cells accepts a large variety of amphiphilic anions, uncharged compounds and even some organic cations [4]. The specificities of three basolateral organic anion transporters (PAH/ α-ketoglutarate antiporter, Na +-dicarboxylate symporter, sulfate/anion antiporter) and of the organic cation transporter localized in the same membrane partially overlap, ensuring excretion of most water-soluble xenobiotics with the urine. The proteins mediating PAH/ α-ketoglutarate antiport at the basolateral membrane of rat [5, 6] and winter flounder [7] proximal tubule cells have recently been identified by an expression cloning strategy using Xenopus laevisoocytes. The cloned renal organic anion transporters, termed OAT1/ROAT1 for rat and fROAT for flounder, are between 551 and 562 amino acids long with 12 putative transmembrane domains. Rat and flounder ROATs show an amino acid identity of 47% and are related to the organic cation transporters, OCT1 and OCT2 [8]. Following expression of cRNA derived from rat OAT1/ROAT1 and flounder fROAT in X. laevisoocytes, a probenecid-inhibitable, saturable uptake of PAH could be demonstrated [5–7]. PAH uptake was cis-inhibited by α-ketoglutarate and glutarate in the bath, and trans-stimulated by these dicarboxylates loaded previously into the oocytes, indicating that OAT1/ROAT1 and fROAT most likely represent the PAH/ α-ketoglutarate antiporter of the basolateral membrane of proximal tubule cells. In line with this assumption is the broad substrate specificity: besides PAH and α-ketoglutarate, OAT1 transported labeled methotrexate, cAMP, cGMP, PGE 2, and urate [5]. Direct experimental evidence is lacking for the presence of a PAH/α-ketoglutarate antiporter in the basolateral membrane of human renal proximal tubule cells. However, given the evolutionary conservation of PAH/ α-ketoglutarate antiport in the basolateral membrane, we assumed that a human homologue to OAT1/ROAT1 and fROAT exists. Here we report the PCR cloning of hROAT1 1, which is highly homologous to rat OAT1/ROAT1. Basic Renal Research

Synthesis of (1S, 3aS)-8-(2,3,3a, 4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro [4.5]decan-4-one, a potent and selective orphanin FQ (OFQ) receptor agonist with anxiolytic-like properties

The development of 8-(2,3,3a,4,5, 6-hexahydro-1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4. 5]decan-4-ones 3 starting from (RS)-8-acenaphten-1-yl-1-phenyl-1,3, 8-triazaspiro[4.5]decan-4-one 1 is reported. The synthesis and the binding affinities at human OFQ and opioid (micro, kappa, delta) receptors of the stereoisomers 3a-f are described. In vitro the most selective compound, (1S,3aS)-8-(2,3,3a,4,5, 6-hexahydro-1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4. 5]decan-4-one 3c, was found to act as a full agonist at the OFQ receptor in the GTPgamma(35)S binding test. It turned out to be selective versus a variety of other neurotransmitter systems. When tested in vivo following intraperitoneal injection, compound 3c was found to decrease neophobia in a novel environment and to exhibit dose-dependent anxiolytic-like effects in the elevated plus-maze procedure, thus confirming the effects observed following intracerebroventricular infusion of the OFQ peptide in rat.

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.

125I-Antisauvagine-30: a novel and specific high-affinity radioligand for the characterization of corticotropin-releasing factor type 2 receptors

Corticotropin-releasing factor (CRF) receptors type 1 (CRF(1)) and type 2 (CRF(2)) differ from each other in their pharmacological properties. The human and ovine CRF versions bind to CRF(1) receptors with significantly higher affinity than to CRF(2) receptors. Recently antisauvagine-30, an N-terminally truncated version of the CRF analog sauvagine, was characterized as a specific antagonist to mouse CRF(2B). We have synthesized the radiolabeled version (125)I-antisauvagine-30 and tested it for its affinity at human CRF(1) (hCRF(1)), hCRF(2A), Xenopus CRF(1) (xCRF(1)) and xCRF(2) receptors. In control binding studies (125)I-labeled hCRF, sauvagine and astressin were also bound to these receptors. (125)I-antisauvagine-30 exclusively bound to hCRF(2A) and xCRF(2) but not to hCRF(1) and xCRF(1) receptors. (125)I-antisauvagine-30 binding to hCRF(2A) and xCRF(2) receptors was saturable and of high affinity (hCRF(2A): K(d)=125 pM; xCRF(2): K(d)=1.1 nM). In displacement binding experiments using (125)I-antisauvagine-30 as radioligand several CRF analogs bound to hCRF(2A) and xCRF(2) receptors with similar rank orders as reported with other CRF radioligands. Finally, preliminary studies using (125)I-antisauvagine-30 binding to membrane homogenates prepared from different rat brain structures showed that the peptide bound specifically to brain areas expressing CRF(2) receptors. These data demonstrate that (125)I-antisauvagine-30 is the first high-affinity ligand to specifically label CRF(2) receptors.