Topological characterization of the lymphoid-specific seven transmembrane receptor BLR1 by epitope-tagging and high level expression.
We have tagged the human lymphocyte-specific G-protein-coupled receptor BLR1 either with an amino-terminal or a carboxyl-terminal epitope-tag recognized by an anti-MYC monoclonal antibody. Flow cytometry was used to determine the efficiency of transient transfections and to establish human embryonic kidney 293 cell clones showing stable high level expression of BLR1. Analysis of permeabilized versus non-permeabilized transfected 293 cells demonstrated that BLR1 is an integral plasma membrane protein, topologically oriented therein as predicted for other members of this class of seven pass membrane receptors. In addition, BLR1 was expressed in 293 cells to high levels as a glycosylated membrane protein. The easily detectable and assayable expression of tagged G-protein-coupled receptors, as exemplified for BLR1 in 293 cells, provides a suitable system for further functional studies and offers an efficient screening tool for the identification of receptor-specific antibodies, ligands, or receptor-associated proteins.
Emrich
T
T
Institut für Biochemie der Ludwig-Maximilians-Universität München, Martinsried, FRG.
Förster
R
R
Lipp
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M
D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D000939:Epitopes; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D054380:Receptors, CXCR5; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000648:ultrastructure; D014162:Transfection
D015415:Biomarkers; C095085:CXCR5 protein, human; D000939:Epitopes; D008565:Membrane Proteins; D054380:Receptors, CXCR5; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins