Transmembrane Topology of the Mammalian KDEL Receptor

Singh, Paramjeet; Tang, Bor Luen; Wong, Siew Heng; Hong, W.

doi:10.1128/mcb.13.10.6435-6441.1993

Cited by 11 publications

(2 citation statements)

References 26 publications

(29 reference statements)

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“…PDI also has a C-terminal tetrapeptide sequence, Lys-Asp-Glu-Leu (KDEL), which functions as an endoplasmic reticulum retention signal in yeast and mammalian cells (22). In cells, PDI binds by this signal to a known 23-26 kDa, six-or seven-transmembrane domain receptor (23,24) that facilitates its recycling from the Golgi apparatus back to the endoplasmic reticulum. Our results show that platelet PDI catalyzes the formation of a ternary vitronectin-thrombin-AT complex and that thrombospondin competes for the thrombin-AT.…”

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confidence: 99%

Protein Disulfide Isomerase Catalyzes the Formation of Disulfide-Linked Complexes of Vitronectin with Thrombin−Antithrombin

et al. 1999

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In this study, purified preparations of platelet protein disulfide isomerase (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to demonstrate that PDI catalyzes formation of vitronectin-thrombin-AT complexes. Complex formation requires reduced glutathione (GSH) and can be prevented by N-ethymaleimide, and the formed complex is dissociated by reducing agents such as mercaptoethanol. No vitronectin-thrombin complex formed in the absence of AT, indicating that the thrombin-AT complex is an obligate intermediate in the reaction. Under optimal conditions, the majority of the thrombin-AT is incorporated into the complex in 60 min. Thrombospondin-1, known to form disulfide-linked complexes with thrombin-AT [Milev, Y., and Essex, D. W. (1999) Arch. Biochem. Biophys. 361, 120-126], competes with vitronectin for thrombin-AT in the low-Ca(2+) environment that favors the active form of thrombospondin. The results presented here may also explain previous studies showing that vitronectin-thrombin-AT complexes form better in plasma (which contains PDI) than with purified proteins (where PDI was not used). We were able to purify a PDI from plasma that was immunologically identical to the platelet enzyme. We used the scrambled RNase assay to show that added purified PDI can function in a plasma environment. Complex formation in plasma was inhibited by inhibitors of PDI. PDI was released from the platelet surface in a soluble form at high pH (around the physiologic range), suggesting a source of the plasma PDI. In summary, these studies indicate that PDI functions to form disulfide-linked complexes of vitronectin with thrombin-AT.

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Protein Disulfide Isomerase Catalyzes the Formation of Disulfide-Linked Complexes of Vitronectin with Thrombin−Antithrombin

et al. 1999

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“…The sequence of human Erd2.1 is shown; residues in black are identical between the Erd2 genes of H. sapiens 1, H. sapiens 2(Lewis & Pelham, 1992b;Hsu et al, 1992), S. cereVisiae(Semenza et al, 1990), K. lactis, A. thaliana(Lee et al, 1993), P. falciparum(Elmendorf & Haldar, 1993), C. elegans 1 (Accession Number Q09473 and D. Banfield, personal communication), C. elegans 2 (Accession Number P48583), and D. melanogaster (D. Banfield, personal communication). An alternative topology of the KDEL receptor has recently been proposed bySingh et al (1993).…”

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Purification and Characterization of the Human KDEL Receptor

Scheel

Pelham

1996

Biochemistry

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Retention of soluble endoplasmic reticulum (ER) proteins is ensured by their continuous retrieval from subsequent compartments in the secretory pathway. Soluble ER proteins which escape to the Golgi apparatus bind to the KDEL receptor, a seven-transmembrane receptor, and are then returned to the endoplasmic reticulum. We have overexpressed the human KDEL receptor in insect cells using the baculovirus system. Infected cells accumulate large amounts of functional receptor as judged by a ligand binding assay. A hexahistidine-tagged version of the receptor could be purified in a single step to near homogeneity with high yield. After reconstitution of purified KDEL receptor into liposomes, a similar affinity and pH dependence for the binding of KDEL peptides was observed compared to the receptor in its natural environment, indicating that purified KDEL receptor is sufficient for specific and pH-sensitive binding of KDEL ligands. Determination of the receptor affinity in different lipid environments revealed that the receptor affinity is only slightly influenced by its lipid environment, suggesting that regulation of the receptor affinity by its surrounding lipids does not play a crucial role for the sorting of KDEL proteins.

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E5 oncoprotein retained in the endoplasmic reticulum/cis Golgi still induces PDGF receptor autophosphorylation but does not transform cells.

1995

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The E5 oncoprotein encoded by bovine papillomavirus type 1 is a homodimeric, hydrophobic polypeptide which is localized predominantly in Golgi membranes and which transforms several cell types apparently by inducing tyrosine phosphorylation of the platelet‐derived growth factor receptor (PDGF‐R). While the precise mechanism of receptor activation is unknown, E5 associates with several cellular proteins, including PDGF‐R and the 16K V‐ATPase protein, and induces the preferential phosphorylation of immature, Endo H‐sensitive forms of the receptor. To evaluate whether E5 accumulation in the Golgi was requisite for receptor phosphorylation and cell transformation, we sequestered the E5 protein in the endoplasmic reticulum (ER)/cis Golgi by appending the ER retention KDEL sequence to its C‐terminus. In transient assays and in cell lines, E5/KDEL protein and E5/KDEL* protein (a defective variant of KDEL), were stable and formed homodimers normally. E5/KDEL*, similar to wt E5, localized to the Golgi and was transformation‐proficient. In contrast, E5/KDEL failed to concentrate in the Golgi and was transformation‐incompetent. Despite these critical defects, however, E5/KDEL formed stable complexes with immature PDGF‐R and 16K and, even more unexpectedly, induced the phosphorylation of both mature and immature PDGF‐R on tyrosine residues to the same level as wt E5. These data demonstrate that E5 can bind and induce PDGF‐R phosphorylation in the ER/cis Golgi, but that successful mitogenic signalling (and consequent cell transformation) requires the translocation of E5/receptor complexes to distal Golgi compartments.

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Transmembrane Topology of the Mammalian KDEL Receptor

Cited by 11 publications

References 26 publications

Protein Disulfide Isomerase Catalyzes the Formation of Disulfide-Linked Complexes of Vitronectin with Thrombin−Antithrombin

Protein Disulfide Isomerase Catalyzes the Formation of Disulfide-Linked Complexes of Vitronectin with Thrombin−Antithrombin

Purification and Characterization of the Human KDEL Receptor

E5 oncoprotein retained in the endoplasmic reticulum/cis Golgi still induces PDGF receptor autophosphorylation but does not transform cells.

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