title: Bradykinin B₂ receptor-mediated transport into intact cells: anti-receptor antibody-based cargoes.
abstract: Endocytosis of the bradykinin-stimulated B(2) receptors is parallel to the transport and subsequent degradation of the ligand. To implement biotechnological applications based on receptor-mediated transport, one strategy is to conjugate the agonist ligand to a cargo. Alternatively, we studied whether the B(2) receptor can transport large antibody-based cargoes into intact cells and characterized the ensuing endosomal routing. Myc-tagged B(2) receptors (coded by the vector myc-B(2)R) and a truncated construction devoid of the Ser-Thr phosphorylation domain (myc-B(2)R(trunc) vector) were coupled to anti-myc monoclonal antibodies that did not impair bradykinin binding or elicit calcium signaling in intact cells. Anti-myc antibodies, conjugated or not with secondary antibodies optionally coupled to Qdot nanomaterials, were transported into early endosome autoantigen 1-, and β-arrestin-positive vesicles in bradykinin-stimulated intact cells expressing receptors encoded by myc-B(2)R. Antibody-conjugated cargoes progressed into late-endosomes-lysosomes within 3h without evidence of autophagy. Receptors encoded by myc-B(2)R(trunc) did not support the ligand-controlled endocytosis of anti-myc antibodies. Aside from small ligand-conjugated cargoes, very large antibody-based cargoes can be transported by agonist-stimulated B(2) receptors into intact cells. The latter type of cargo requires a receptor competent for interaction with β-arrestins, enters the degradation pathway separately from the receptor as a function of time and has the potential to confer a qualitatively novel function to a receptor.
authors.affiliation: Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Québec QC, Canada G1V 4G2.
authors.forename: Marie-Thérèse
authors.initials: MT
authors.lastname: Bawolak
authors.forename: Robert
authors.initials: R
authors.lastname: Lodge
authors.forename: Guillaume
authors.initials: G
authors.lastname: Morissette
authors.forename: François
authors.initials: F
authors.lastname: Marceau
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism*; D019390:Arrestins / Q000378:metabolism; D001920:Bradykinin / Q000494:pharmacology; D000939:Epitopes / Q000276:immunology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D021381:Protein Transport / Q000187:drug effects; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000276:immunology; D043782:Receptor, Bradykinin B2 / Q000819:agonists / Q000378:metabolism*; D000071557:beta-Arrestins
chemical_list: D000911:Antibodies, Monoclonal; D019390:Arrestins; D000939:Epitopes; D016271:Proto-Oncogene Proteins c-myc; D043782:Receptor, Bradykinin B2; D000071557:beta-Arrestins; D001920:Bradykinin