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 Arg 76 or Asn 81 but not Gly 83 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. Key Words: Corticotropin-releasing factor receptor 1 mutagenesis-Ligand binding-Cyclic AMP-Corticotropinreleasing factor binding site. J. Neurochem. 72, 388 -395 (1999).The 41-amino acid peptide corticotropin-releasing factor (CRF) (for references, see Vale et al., 1981) is the main integrator of the stress response (Dunn and Berridge, 1990;Owens and Nemeroff, 1991). Two major subtypes of the CRF receptor (CRF-R) have been identified in vertebrates as belonging to the superfamily of G protein-coupled receptors (GPCRs): CRF-R type 1 (CRF-R1) and type 2 (CRF-R2). The cDNAs for CRF-R1 and CRF-R2 have been cloned from human Vita et al., 1993;Liaw et al., 1996), rat (Chang et al., 1993;Perrin et al., 1993;Lovenberg et al., 1995), mouse (Vita et al., 1993;Kishimoto et al., 1995;Perrin et al., 1995;Stenzel et al., 1995), chicken (Yu et al., 1996), and amphibian (Dautzenberg et al., 1997) tissues. Two functional splice variants, CRF-R2␣ and CRF-R2␤, have been found for CRF-R2. CRF-R2␣ cDNA encodes a protein of 411-413 amino acids (Lovenberg et al., 1995;Liaw et al., 1996;Dautzenberg et al., 1997), whereas the CRF-R2␤ protein comprises 430 -438 amino acids (Kishimoto et al., 1995;Lovenberg et al., 1995;Perrin et al., 1995;Stenzel et al., 1995;Valdenaire et al., 1997). Both splice variants differ in the N-terminal region and are ϳ70% identical to CRF-R1.High-affinity ligand binding is an important prerequisite for the signal transduction of GPCRs. Small nonpeptidic molecules can bind to the transmembrane domains (TMs) of their receptors (Dixon et al., 1987;Wheatley et al., 1988;Strader et al., 1989), whereas the contribution of extracellular domains (ECs) and TMs has been identified as parts of the binding site of GP...

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Rat corticotropin-releasing factor receptor 1 (rCRFR1) was produced either in transfected HEK 293 cells as a complex glycosylated protein or in the presence of the mannosidase I inhibitor kifunensine as a high mannose glycosylated protein. The altered glycosylation did not influence the biological function of rCRFR1 as demonstrated by competitive binding of rat urocortin (rUcn) or human/rat corticotropin-releasing factor (h/rCRF) and agonist-induced cAMP accumulation. The low production rate of the N-terminal domain of rCRFR1 (rCRFR1-NT) by transfected HEK 293 cells, was increased by a factor of 100 in the presence of kifunensine. The product, rCRFR1-NT-Kif, bound rUcn specifically (K D ‫ס‬ 27 nM) and astressin (K I ‫ס‬ 60 nM). This affinity was 10-fold lower than the affinity of full length rCRFR1. However, it was sufficiently high for rCRFR1-NT-Kif to serve as a model for the N-terminal domain of rCRFR1. With protein fragmentation, Edman degradation, and mass spectrometric analysis, evidence was found for the signal peptide cleavage site C-terminally to Thr 23 and three disulfide bridges between precursor residues 30 and 54, 44 and 87, and 68 and 102. Of all putative N-glycosylation sites in positions 32, 38, 45, 78, 90, and 98, all Asn residues except for Asn 32 were glycosylated to a significant extent. No O-glycosylation was observed.Keywords: Corticotropin-releasing factor; CRF receptor; human embryonic kidney cells; scintillation proximity assay; amino-terminal domain; binding domain; disulfide bond structure; glycosylation structure Corticotropin-releasing factor (CRF), a peptide 41 amino acids long , is released from the hypothalamus into the hypophyseal portal system and stimulates ACTH secretion from the pituitary as an endocrine response to stress. In addition to CRF, the CRFlike 40-amino acid peptide urocortin (Ucn) has been characterized (Vaughan et al. 1995;Donaldson et al. 1996). CRF and Ucn are distributed widely throughout the CNS of roReprint requests to: Dr. Klaus Eckart, Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Hermann Rein Str. 3, 37073 Göttingen, Germany; e-mail: eckart@em.mpg.de; fax: 49-551-3899-359.2 Present address: Schering AG, D-13342 Berlin, Germany. Parts of this work were presented at the meetings of the Society for Neuroscience in Miami (October 23-28 1999) and New Orleans (November 4-9, 2000), and the meeting of the Endocrine Society in Toronto (June 21-24 2000).Abbreviations: CRF, corticotropin-releasing factor; Ucn, urocortin; Svg, sauvagine; CRFR, CRF receptor; rCRFR, rat CRFR; hCRFR, human CRFR; mCRFR, mouse CRFR; xCRFR, Xenopus laevis CRFR; rCRFBP, rat CRF binding protein; NT, amino-terminal domain; Ast, astressin; EC, extracellular domain; HEK, human embryonic kidney; SPA, scintillation proximity assay; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; EndoH f , endo-␤-acetylglucosaminidase H; PNGaseF, peptide-N-glycosidase F; WGA, wheat germ agglutinin; RP-HPLC, reversed phase-high performance liquid chromatogr...

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