Abstract
Parathyroid hormone (PTH) can be C-terminally extended without significant affinity loss for the PTH1 receptor (PTHR1). We developed fusion protein ligands with enzymatic activity to probe PTHR1s 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 PTHR1s with the PTH-myc/antibodies combination or with PTH-APEX2 supported the histochemical or luminescent detection of recombinant PTHR1s (TrueBlueTM or luminol-based reagent). The PTH-HRP construction was the most sensitive and supported all tested peroxidase co-substrates (TrueBlueTM, tetramethylbenzidine (TMB), luminol, biotin-phenol with streptavidin-Qdots); the 3 latter schemes identified endogenous PTHR1 in the osteoblastic HOS cell line. The specificity of the fusion protein binding to PTHR1 was determined by its competition with an excess of PTH1–34. Bifunctional ligands possessing enzymatic activity detect intact receptors with various possible applications, including the screening of drugs that compete for receptor binding.
Highlights
Parathyroid hormone (PTH), is an important endocrine mediator involved in the regulation of calcium and phosphate concentrations[1]
The three fusion proteins (PTH-APEX2, PTH-HRP and PTH-myc; predicted sequences in Fig. 1) were all produced as conditioned mediums (CMs)
Using a rational design strategy based on the literature and on our previous findings, two new fusion proteins with peroxidase activity were produced for the PTHR1
Summary
Parathyroid hormone (PTH), is an important endocrine mediator involved in the regulation of calcium and phosphate concentrations[1]. PTH is secreted from the parathyroid gland in response to the lowering of blood Ca2+ concentrations and will increase Ca2+ concentrations by stimulating the PTHR13,4 This receptor binds parathyroid hormone-related protein (PTHrP) as well. Class B GPCRs are organised in two domains: one extracellular domain involved in the affinity and specificity of ligand binding and a transmembrane domain required for the activation of the receptor[10]. This two domain model suggests that the N-terminal domain of PTH will interact with the extracellular domain of the receptor whereas the C-terminal domain of the hormone will interact with the transmembrane domain. This led to the prediction that PTH could be prolonged at its C-terminal terminus to construct bifunctional receptor ligands
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