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, 10 (10), 2050-62

Functional and Protein Chemical Characterization of the N-terminal Domain of the Rat Corticotropin-Releasing Factor Receptor 1

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Functional and Protein Chemical Characterization of the N-terminal Domain of the Rat Corticotropin-Releasing Factor Receptor 1

B A Hofmann et al. Protein Sci.

Abstract

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) = 27 nM) and astressin (K(I) = 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.

Figures

Fig. 1.
Fig. 1.
Western blot analysis of rCRFR1 and rCRFR1-Kif and binding of rUcn to rCRFR1, rCRFR1-Kif, and rCRFR1-NT-Kif. Membrane preparations with a total protein content of 11 μg, which were obtained from HEK 293 cells producing either rCRFR1 (A) or rCRFR1-Kif (B) were applied. The absence or presence of PNGaseF is indicated. (C) Competitive binding was performed using [125I-Tyr0]-rUcn as radioligand and increasing concentrations (10 pM–3.16 μM) of rUcn. Data represent duplicates from two independent experiments. Binding curves were normalized by total binding in absence of competitor [B0].
Fig. 2.
Fig. 2.
Polyacrylamide gel analysis of rCRFR1-NT and rCRFR1-NT-Kif. For deglycosylation with PNGaseF, approximately 100 ng Ni-affinity-purified rCRFR1-NT (A) or 37.5 μL of medium containing rCRFR1-NT-Kif (B) were applied to SDS-PAGE followed by Western blot and immunodetection. The absence or presence of PNGaseF is indicated. (C) SDS-PAGE of affinity-purified rCRFR1-NT-Kif was performed by application of 37.5 μL medium of transfected HEK 293 cells (M), 37.5 μL supernatant after adsorption on Ni-affinity resin (S1), and 30 μL of the third elution fraction (E). Proteins were detected by silver staining.
Fig. 3.
Fig. 3.
Edman degradation of 32 kD and 35 kD rCRFR1-NT-Kif. The yields of the respective amino acid residues of 29 cyles of Edman degradation of rCRFR1-NT-Kif are shown. (A) shows the data of 32 kD and 35 kD species starting with Ser24 and (B) shows the data of 32 kD and 35 kD species starting with Leu25. The initial yields of the 32 kD protein species were 47.0 pmol and 26.4 pmol for the sequences starting with Ser and Leu, respectively.
Fig. 4.
Fig. 4.
NanoES mass spectrum of rCRFR1-NT-Kif before and after EndoHf deglycosylation. rCRFR1-NT-Kif and EndoHf-deglycosylated rCRFR1-NT-Kif (c = ∼0.2 μg/μl) were dissolved in 50% methanol containing 1% acetic acid. (A) shows the ES mass spectrum of the heterogeneous glycoprotein. After deglycosylation, 5 distinct charge states (+ 11 to + 7) can be seen (B). The deconvoluted mass spectrum (C) represents the protein chain starting with amino acid Ser24 (*) and carrying three to five N-acetylhexosamine (HexNAc) residues, and the protein chain starting with amino Leu25 (#) and carrying four to five HexNAc residues.
Fig. 5.
Fig. 5.
HPLC chromatograms of the tryptic digests of rCRFR1-NT-Kif. After deglycosylation with PNGaseF and HPLC purification, rCRFR1-NT-Kif was digested using the endoprotease trypsin. (A) shows the chromatograms of the peptide map derived from reduced and alkylated rCRFR1-NT-Kif, whereas (B) shows the peptide map of non-reduced rCRFR1-NT-Kif. Cys-containing fragments are indicated by square brackets. The signals of the disulfide-linked peptides are marked by gray shading. Assignment of the fragments was carried out on the basis of the calculated and observed molecular masses of the peptides. Several signals were assigned to either rCRFR1-NT-Kif(30–57) or rCRFR1-NT-Kif(86–96) as a result of incomplete conversion of Asn32 and Asn90 into Asp residues caused by partial glycosylation of these residues.
Fig. 6.
Fig. 6.
Disulfide bridge arrangement of rCRFR1-NT-Kif. (A) and (B) show the disulfide-linked peptides of the tryptic and AspN digest, respectively. (C) represents the derived disulfide bridging. In the amino acid sequences, X represents an asparagine or aspartate residue depending on the extent of glycosylation at the corresponding position. The solid lines represent disulfide bridges directly deduced from the proteolytic digests. The dotted lines connect fragments without an unambiguous assignment of a single disulfide bridge. The dashed line represents the disulfide bridge which was concluded from the results of Cys derivatization. The 23-amino acid long signal peptide is marked by a black background and the 24 amino acid long signal peptide by a gray background.

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