Alteration of the hormonal bioactivity of parathyroid hormone-related protein (PTHrP) as a result of limited proteolysis by prostate-specific antigen

Abstract

Objectives To discover whether the proteolytic activity of prostate-specific antigen (PSA) affects the structure and function of parathyroid hormone-related protein (PTHrP), as both are abundant components of human seminal plasma. Methods The ability of PTHrP to act as a substrate was studied by incubating a synthetic polypeptide, consisting of 34 amino acid residues of the amino-terminal domain of PTHrP, with purified PSA. The incubate was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-pressure liquid chromatography separation, amino-terminal peptide sequencing and mass spectrometry. The physiologic effect of the proteolytic activity of PSA on PTHrP was studied by measuring any alteration in PTHrP ( 1–34)-induced elevation of cyclic adenosine monophosphate (cAMP) production by UMR-106 rat osteosarcoma cells in culture. All cell culture experiments were performed with PSA and PTHrP (1–34) at physiologic concentrations. Results Our data show that PSA proteolytically cleaves PTHrP (1–34) after either residue 22 or 23, generating three peptide fragments. Both cleavages occur carboxy terminally of a phenylalanine residue. The cAMP production in rat osteosarcoma cells, induced by the amino-terminal portion of PTHrP (1–34), as a result of its structural similarity with parathyroid hormone (PTH), was abated by PSA in a dose- and timedependent fashion. In contrast, heat-inactivated PSA had no effect on cAMP production. Conclusions Our study demonstrates that PTHrP is a substrate for PSA. The cleavage of the amino-terminal portion of PTHrP completely disrupts its ability to interact with the PTH/PTHrP receptor and thus inhibits its PTH-like activity. The proteolytic processing of PTHrP by PSA may play an important role in the post-translational/post-secretional regulation of prostatic PTHrP activities, which are believed to include regulation of prostate growth and differentiation.