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

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.

Bifunctional ligands of the bradykinin B 2 and B 1 receptors: An exercise in peptide hormone plasticity

“…For instance, parathyroid hormone (PTH) binds to its receptor subtype with its C-terminus free in the extracellular space. Thus, the PTH sequence can be readily fused without a spacer to that of partner proteins to produce bifunctional ligands such as PTH 1-34 -EGFP and PTH-peroxidases [76,92].…”