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1 Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, D-37073 Göttingen, Germany
Reprint 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{at}em.mpg.de; fax: 49-551-3899-359.
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 (KD = 27 nM) and astressin (KI = 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 Thr23 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 Asn32 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
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 • EndoHf, endo-ß-acetylglucosaminidase H • PNGaseF, peptide-N-glycosidase F • WGA, wheat germ agglutinin • RP-HPLC, reversed phase-high performance liquid chromatography • NanoESMS, nano-electrospray mass spectrometry • rCRFR1-NT-Kif, rCRFR1-NT expressed in presence of kifunensine • rCRFR1-Kif, rCRFR1 expressed in presence of kifunensine • GPCR, G protein-coupled receptor
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