Kinetic and Thermodynamic Insights into Agonist Interactions with the Parathyroid Hormone Receptor-1 from a New NanoBRET Assay.

Polypeptides that activate the parathyroid hormone receptor-1 (PTHR1) are important in human physiology and medicine. Most previous studies of peptide binding to this receptor have involved the displacement of a radiolabeled ligand. We report a new assay format based on bioluminescence resonance energy transfer (BRET). Fusion of a NanoLuc luciferase (nLuc) unit to the N-terminus of the PTHR1 allows the direct detection of binding by an agonist peptide bearing a tetramethylrhodamine (TMR) unit. Affinity measurements from the BRET assay align well with results previously obtained via radioligand displacement. The BRET assay offers substantial operational benefits relative to affinity measurements involving radioactive compounds. The convenience of the new assay allowed us to explore several questions raised by earlier reports. For example, we show that although the first two residues of PTH(1-34) (the drug teriparatide) are critical for PTHR1 activation, these two residues contribute little or nothing to affinity. Comparisons among the well-studied agonists PTH(1-34), PTHrP(1-34), and "long-acting PTH" (LA-PTH) reveal that the high affinity of LA-PTH arises largely from a diminished rate constant for dissociation relative to the other two. A D-peptide recently reported to be comparable to PTH(1-34) as an agonist of the PTHR1 was found not to bind detectably to the receptor and to be a very weak agonist.

Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers.

Targeted therapeutics are needed for triple-negative breast cancer (TNBC). In this study, we investigated the activation of Src family of cytoplasmic tyrosine kinases (SFKs) and two SFK substrates-CUB-domain containing protein 1 (CDCP1) and protein kinase C δ (PKCδ)-in 56 formalin-fixed, paraffin-embedded (FFPE) TNBCs. Expression of SFK phosphorylated at Y416 (SFK_pY416+) in tumor cells was strongly associated with phosphorylation of CDCP1 and PKCδ (CDCP1_ pY743+ and PKCδ_pY311+), as assessed by immunohistochemistry, indicating increased SFK activity in situ. To enable biochemical analysis, protein extraction from FFPE tissue was optimized. Cleaved CDCP1 isoform (70 kDa) was expressed to a varying degree in all samples but only phosphorylated in TNBC tumor cells that expressed SFK_pY416. Interestingly, active SFK was found to be biphosphorylated (SFK_pY416+/pY527+). Biphosphorylated active SFK was observed more frequently in forkhead box protein A1 (FOXA1)- TNBCs. In addition, in SFK_pY416- samples, FOXA1+ TNBC tended to be SFK_pY527+ (classic inactive SFK), and FOXA1- TNBC tended to be SFK_pY527- (SFK poised for activation). Strong SFK_pY416 staining was also observed in tumor-infiltrating lymphocytes in a subset of TNBCs with high tumor-infiltrating lymphocyte content. This report will facilitate protein biochemical analysis of FFPE tumor samples and justifies the development of therapies targeting the SFK/CDCP1/PKCδ pathway for TNBC treatment.