| 1. |
- Lorenzo, Carmen, et al.
(författare)
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Echinococcus granulosus antigen 5 is closely related to proteases of the trypsin family
- 2003
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 369:Pt 1, s. 191-198
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Tidskriftsartikel (refereegranskat)abstract
- Antigen 5 (Ag5) is a dominant secreted component of the larval stage of Echinococcus granulosus, and is highly immunogenic in human infections. Although the diagnostic value of Ag5 has been thoroughly evaluated, there has been little progress in its molecular characterization and the understanding of its biological role. In the present study, the Ag5 gene was cloned by reverse transcription-PCR on the basis of the amino acid sequences of tryptic fragments. The nucleotide sequence indicates that Ag5 is synthesized as a single polypeptide chain that is afterwards processed into single disulphide-bridged 22 and 38 kDa subunits. Whereas the 22 kDa component contains a highly conserved glycosaminoglycan-binding motif that may help to confine Ag5 in the host tissue surrounding the parasite, the 38 kDa subunit is closely related to serine proteases of the trypsin family. The sequences in the vicinity of the active-site histidine, aspartic acid and serine residues, and critical cysteine residues involved in disulphide formation, are well conserved, but the catalytic serine residue is replaced by threonine. Since there are no significant chemical differences between the O gamma atoms of these residues, we performed a series of enzymic assays to find out whether Ag5 is a catalytic molecule. Neither proteolytic activity nor binding to protease inhibitors could be detected using the native purified antigen. Thus it may be possible that Ag5 possesses a highly specific physiological substrate or, more likely, that trypsin-like folding has been recruited to fulfil novel functions.
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| 2. |
- Neuschäfer-Rube, Frank, et al.
(författare)
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Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin EP4-R essential for agonist-induced beta-arrestin1 recruitment that differs from the apparent principal phosphorylation site.
- 2004
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Ingår i: Biochemical Journal. - 0264-6021. ; 379:3, s. 573-585
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Tidskriftsartikel (refereegranskat)abstract
- hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a Gs-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced b-arrestin complex formation, which is stabilized by phosphorylation. Subsequently b-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with b-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370–382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389–Ser-390–Thr-391–Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of b-arrestin1. However, phosphorylation greatly enhanced the stability of the b-arrestin1–receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both b-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.
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| 3. |
- Neuschäfer-Rube, Frank, et al.
(författare)
-
Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site
- 2004
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 379:Pt 3, s. 573-585
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Tidskriftsartikel (refereegranskat)abstract
- hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.
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