A tagged parathyroid hormone derivative as a carrier of antibody cargoes transported by the G protein coupled PTH1 receptor

Authors: Charest-Morin, XavierCharest-Morin, XavierFortin, Jean-PhilippeFortin, Jean-PhilippeLodge, RobertLodge, RobertAllaeys, IsabelleAllaeys, IsabellePoubelle, PatricePoubelle, PatriceMarceau, FrançoisMarceau, François
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 187 nM estimated with ELISAs for PTH). PTH-myc CM induced cyclic AMP formations (10 min), with a minor loss of potency relative to authentic PTH1–84, and c-Fos expression (1–3 h). 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.
Document Type: Article de recherche
Issue Date: 12 August 2014
Open Access Date: 20 November 2017
Document version: AM
Permalink: http://hdl.handle.net/20.500.11794/15924
This document was published in: Peptides, Vol. 60, 71–79 (2014)
Alternative version: 10.1016/j.peptides.2014.08.001
Collection:Articles publiés dans des revues avec comité de lecture

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