Production and evaluation of parathyroid hormone receptor1 ligands with intrinsic or assembled peroxidase domains

Parathyroid hormone (PTH) can be C-terminally extended without significant affinity loss for the PTH 1 receptor (PTHR 1 ). We developed fusion protein ligands with enzymatic activity to probe PTHR 1 s at the cell surface. Two fusion proteins were generated by linking PTH to the N-terminus of either...

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Bibliographic Details
Published in:Scientific reports 2017-10, Vol.7 (1), p.1-14, Article 13099
Main Authors: Charest-Morin, Xavier, Poubelle, Patrice E., Marceau, François
Format: Article
Language:English
Subjects:
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Antibodies
Biotin
Cell fusion
Cell surface
Drug screening
Enzymatic activity
Fusion protein
Horseradish peroxidase
Humanities and Social Sciences
Ligands
multidisciplinary
Myc protein
N-Terminus
Osteoblasts
Parathyroid
Parathyroid hormone
Peroxidase
Phenols
Science
Science (multidisciplinary)
Soybeans
Streptavidin
Vitamin D
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Summary:Parathyroid hormone (PTH) can be C-terminally extended without significant affinity loss for the PTH 1 receptor (PTHR 1 ). We developed fusion protein ligands with enzymatic activity to probe PTHR 1 s at the cell surface. Two fusion proteins were generated by linking PTH to the N-terminus of either horseradish peroxidase (PTH-HRP) or the genetically modified soybean peroxidase APEX2 (PTH-APEX2). Alternatively, myc-tagged PTH (PTH-myc) was combined with antibodies, some of which HRP-conjugated, in the extracellular fluid. The three PTH-fusion proteins were produced as conditioned mediums (CM) by transfected producer HEK 293a cells. Binding of receptor-bound enzymatic ligands was revealed using widely available substrate/co-substrate systems. The stimulation of recipient HEK 293a expressing PTHR 1 s with the PTH-myc/antibodies combination or with PTH-APEX2 supported the histochemical or luminescent detection of recombinant PTHR 1 s (TrueBlue TM or luminol-based reagent). The PTH-HRP construction was the most sensitive and supported all tested peroxidase co-substrates (TrueBlue TM , tetramethylbenzidine (TMB), luminol, biotin-phenol with streptavidin-Qdots); the 3 latter schemes identified endogenous PTHR 1 in the osteoblastic HOS cell line. The specificity of the fusion protein binding to PTHR 1 was determined by its competition with an excess of PTH 1–34 . Bifunctional ligands possessing enzymatic activity detect intact receptors with various possible applications, including the screening of drugs that compete for receptor binding.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-017-13548-0