title: A tagged parathyroid hormone derivative as a carrier of antibody cargoes transported by the G protein coupled PTH1 receptor.
abstract: Based on the known fact that the parathyroid hormone (PTH) might be extended at its C-terminus with biotechnological protein cargoes, a vector directing the secretion of PTH1-84 C-terminally fused with the antigenic epitope myc (PTH-myc) was exploited. The functional properties and potential of this analog for imaging PTH1R-expressing cells were examined. The PTH-myc construct was recombinantly produced as a conditioned medium (CM) of transfected HEK 293a cells (typical concentrations of 187nM estimated with ELISAs for PTH). PTH-myc CM induced cyclic AMP formations (10min), with a minor loss of potency relative to authentic PTH1-84, and c-Fos expression (1-3h). Treatment of recipient HEK 293a cells transiently expressing PTH1R with PTH-myc CM (supplemented with a fluorescent monoclonal anti-myc tag antibody, either 4A6 or 9E10) allowed the labeling of endosomal structures positive for Rab5 and/or for β-arrestin1 (microscopy, cytofluorometry). Authentic PTH was inactive in this respect, ruling out a non-specific form of endocytosis like pinocytosis. Using a horseradish peroxidase-conjugated secondary antibody, the endocytosis of the PTH-myc-based antibody complex by endogenous PTH1R was evidenced in MG-63 osteoblastoid cells. The secreted construct PTH-myc represents a bona fide agonist that supports the feasibility of transporting cargoes of considerable molecular weight inside cells using arrestin and Rab5-mediated PTH1R endocytosis. PTH-myc is also transported into cells that express PTH1R at a physiological level. Such tagged peptide hormones may be part of a cancer chemotherapy scheme exploiting a modular cytotoxic secondary antibody and the receptor repertoire expressed in a given tumor.
authors.affiliation: Centre de recherche en Rhumatologie et Immunologie, CHU de Québec, Québec, QC, Canada G1V 4G2.
authors.forename: Xavier
authors.initials: X
authors.lastname: Charest-Morin
authors.affiliation: Pfizer's Cardiovascular and Metabolic Diseases Research Unit, Cambridge, MA 02139, USA.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Fortin
authors.affiliation: Laboratory of Human Retrovirology, Institut de recherches cliniques de Montréal, Montreal, QC, Canada H2W 1R7.
authors.forename: Robert
authors.initials: R
authors.lastname: Lodge
authors.affiliation: Centre de recherche en Rhumatologie et Immunologie, CHU de Québec, Québec, QC, Canada G1V 4G2.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Allaeys
authors.affiliation: Centre de recherche en Rhumatologie et Immunologie, CHU de Québec, Québec, QC, Canada G1V 4G2.
authors.forename: Patrice E
authors.initials: PE
authors.lastname: Poubelle
authors.affiliation: Centre de recherche en Rhumatologie et Immunologie, CHU de Québec, Québec, QC, Canada G1V 4G2. Electronic address: francois.marceau@crchul.ulaval.ca.
authors.forename: François
authors.initials: F
authors.lastname: Marceau
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D001692:Biological Transport; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004337:Drug Carriers / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D010281:Parathyroid Hormone / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000378:metabolism*
chemical_list: D000906:Antibodies; D004337:Drug Carriers; D010281:Parathyroid Hormone; D016271:Proto-Oncogene Proteins c-myc; D044168:Receptor, Parathyroid Hormone, Type 1; D000242:Cyclic AMP
keywords: Osteoblast; PTH(1) receptor; Parathyroid hormone; Receptor-mediated transport