Purification and characterization of the human KDEL receptor.
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.
Scheel
A A
AA
MRC Laboratory of Molecular Biology, Cambridge, U.K.
Pelham
H R
HR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D018000:Receptors, Peptide / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016324:Sequence Tagged Sites; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection
C064459:KDEL receptor; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C073762:lysyl-aspartyl-glutamyl-leucine