Corticotropin‐releasing factor receptor type 1 from Tupaia belangeri

Abstract

A cDNA clone encoding corticotropin-releasing factor (CRF) type 1 (CRF-R1) has been isolated from the tree shrew Tupaia belangeri with a PCR-based approach. The full-length cDNA encoded a 415-amino-acid protein with highest sequence identity (Ϸ98%) to human CRF-R1 and slightly less identity to rat or mouse CRF-R1 (Ϸ97%). Only eight amino acids (residues 3, 4, 6, 35, 36 and 39 in the Nterminus, residue 232 in transmembrane domain 4 and residue 410 in the C-terminus) differed between tree shrew CRF-R1 (tCRF-R1) and human CRF-R1 (hCRF-R1). tCRF-R1 mRNA was detected by semiquantitative RT-PCR and RNase protection analysis in the pituitary and in brain areas such as amygdala, brainstem, cerebellum, cortex, olfactory bulb, and striatum. In peripheral organs, only weak expression of tCRF-R1 mRNA was observed in ovary, testis, and adrenal gland. Binding studies using human embryonic kidney 293 (HEK293) cells stably transfected with tCRF-R1 showed that the CRF agonists ovine CRF (K D ϭ 1.28 nM), human/rat CRF (K D ϭ 1.09 nM), urocortin (K D ϭ 0.37 nM) and sauvagine (K D ϭ 0.77 nM), respectively, were bound with significantly higher affinities than the CRF antagonist astressin (K D ϭ 12.4 nM). In agreement with the binding data half maximum effective EC 50 values of 0.83 nM (human/rat CRF), 1.41 nM (ovine CRF), 1.25 nM (rat urocortin) and 0.71 nM (sauvagine) were calculated when the cAMP production in HEK293 cells stably transfected with tCRF-R1 was stimulated with the four CRF analogues. These data underline the close relationship between human and tree shrew CRF-R1.Keywords : receptor cloning; receptor expression ; ligand binding; cyclic AMP.Corticotropin-releasing factor (CRF), a 41-amino-acid peptide, was originally identified by its ability to regulate the secretion of adrenocorticotropic hormone from the anterior pituitary [1]. The peptide is the main regulator of the stress response [2,3]. Two CRF receptor (CRF-R) subtypes have been identified in vertebrate species; CRF receptor type 1 (CRF-R1) cDNA, encoding a polypeptide of 415Ϫ420 amino acids has been cloned from human, rat [6,7] [9]. The receptor exists in at least two different functional splice variants, named CRF-R2A (411Ϫ413 residues) and CRF-R2β (430Ϫ438 amino acids). Both splice variants differ in the N-terminal region and show an overall identity to vertebrate CRF-R1 of Ϸ70% [9Ϫ15].CRF-R1 mRNA was mainly found in brain and the anterior pituitary [4Ϫ6, 9, 16Ϫ18]. In peripheral organs, CRF-R1 is only weakly expressed [5, 19Ϫ21]. In contrast to CRF-R1, CRF-R2A mRNA is limited to discrete regions of the rodent brain including hypothalamus, lateral septum and hippocampus but is almost Fax: ϩ41 61 6884484. E-mail: frank.dautzenberg@roche.com Abbreviations. CRF, corticotropin releasing factor; CRF-R, CRF receptor; oCRF, ovine CRF ; h/rCRF, human/rat CRF ; rUcn, rat urocortin; 5′-RACE and 3′-RACE, rapid amplification of cDNA ends. not detectable in the pituitary [17,18]. CRF-R2β is mainly expressed in peripheral organs such as heart, lung and skeletal muscle an...

Role of SAP97 Protein in the Regulation of Corticotropin-releasing Factor Receptor 1 Endocytosis and Extracellular Signal-regulated Kinase 1/2 Signaling

“…CRF mediates these responses by activating two distinct G protein-coupled receptors (GPCRs), CRF receptor 1 (CRFR1) and CRF receptor 2 (CRFR2) (3,4). In comparison with CRFR2, CRFR1 exhibits a significantly higher affinity for CRF and shows far greater expression within the brain and in the pituitary (5,6). The dysregulation of the hypothalamic-pituitary-adrenal axis is thought to be involved in the onset of psychiatric diseases, such as depression, and specific CRFR1 antagonists have recently been shown to demonstrate anxiolytic-and antidepressant-like effects (7,8).…”

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

“…The CRF 1 receptor, a 415-446 amino acid polypeptide, has been cloned from a variety of species including man Vita et al, 1993), tree shrew (Palchaudhuri et al, 1998), mouse (Vita et al, 1993), rat (Chang et al, 1993;Perrin et al, 1993), sheep (Myers et al, 1998), chicken (Yu et al, 1996), frog (Dautzenberg et al, 1997), and fish (Arai et al, 2001;Pohl et al, 2001). Although a larger number of splice variants of the CRF 1 receptor cDNA have been identified (Chang et al, 1993;Chen et al, 1993;Ross et al, 1994;Myers et al, 1998;Grammatopoulos et al, 1999) (Fig.…”

“…2), they have not been shown to encode functional receptors in vivo due to their low binding affinity or lack of activation in recombinant systems (reviewed in Dautzenberg et al, 2001a). The CRF 1 receptor is abundantly expressed in the central nervous system with major expression sites in cortex, cerebellum, hippocampus, amygdala, olfactory bulb, and pituitary (Potter et al, 1994;Chalmers et al, 1996;Palchaudhuri et al, 1998). In the periphery, CRF 1 receptor mRNA is expressed at low levels in the skin, ovary, testis, and adrenal gland (Vita et al, 1993;Nappi and Rivest, 1995;Palchaudhuri et al, 1998).…”

Molecular cloning and initial characterization of African green monkey (Cercopithecus aethiops) corticotropin releasing factor receptor type 1 (CRF1) from COS-7 cells

“…These diverse phenotypic effects are mediated through interaction with G protein coupled membrane bound CRF receptors (CRF1 and CRF2) (Perrin, Vale, 1999;Hillhouse, Grammatopoulos, 2006). The gene for mammalian CRF1 has been cloned in humans, rat, mouse, hamster, sheep, bovine, tree shrew, chicken and rhesus monkey (Chen et al, 1993;Vita et al, 1993;Yu et al, 1996;Myers et al, 1998;Palchaudhuri et al, 1998;Perrin, Vale, 1999;Pisarchik, Slominski, 2002;Oshida et al, 2004;Hillhouse, Grammatopoulos, 2006); and more specifically, the genes for human and rodent CRF1 contain 14 and 13 exons, respectively (Tsai-Morris et al, 1996;Sakai et al, 1998;Parham et al, 2004), and produce several alternatively spliced isoforms (reviewed in (Hillhouse, Grammatopoulos, 2006;Slominski et al, 2006b)). …”