Analysis of Chemokine Receptor Trafficking by Site-Specific Biotinylation.
Chemokine receptors undergo internalization and desensitization in response to ligand activation. Internalized receptors are either preferentially directed towards recycling pathways (e.g. CCR5) or sorted for proteasomal degradation (e.g. CXCR4). Here we describe a method for the analysis of receptor internalization and recycling based on specific Bir A-mediated biotinylation of an acceptor peptide coupled to the receptor, which allows a more detailed analysis of receptor trafficking compared to classical antibody-based detection methods. Studies on constitutive internalization of the chemokine receptors CXCR4 (12.1% ± 0.99% receptor internalization/h) and CCR5 (13.7% ± 0.68%/h) reveals modulation of these processes by inverse (TAK779; 10.9% ± 0.95%/h) or partial agonists (Met-CCL5; 15.6% ± 0.5%/h). These results suggest an actively driven internalization process. We also demonstrate the advantages of specific biotinylation compared to classical antibody detection during agonist-induced receptor internalization, which may be used for immunofluorescence analysis as well. Site-specific biotinylation may be applicable to studies on trafficking of transmembrane proteins, in general.
Department of Cellular and Molecular Immunology, University of Göttingen, Göttingen, Niedersachsen, Germany.
Marcel
M
Liebick
Department of Cellular and Molecular Immunology, University of Göttingen, Göttingen, Niedersachsen, Germany.
Christian
C
Schläger
Department of Cellular and Molecular Immunology, University of Göttingen, Göttingen, Niedersachsen, Germany.
Martin
M
Oppermann
D000577:Amides / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D001491:Basophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D019921:Biotinylation; D065100:CCR5 Receptor Antagonists / Q000494:pharmacology; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D018946:Chemokine CCL5 / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D021381:Protein Transport / Q000187:drug effects; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D051381:Rats; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054380:Receptors, CXCR5 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D000577:Amides; D000911:Antibodies, Monoclonal; C483024:CCL5 protein, human; D065100:CCR5 Receptor Antagonists; C527276:CXCR4 protein, human; C095085:CXCR5 protein, human; D018946:Chemokine CCL5; D029968:Escherichia coli Proteins; D000644:Quaternary Ammonium Compounds; C108335:RANTES, Met-; D019718:Receptors, CXCR4; D054380:Receptors, CXCR5; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D001710:Biotin; C119369:TAK 779; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli