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The endoproteolytic processing of polypeptides at basic residues into distinct biologically active peptides is a common theme in prohormone maturation and processing. PTH-related protein (PTHrP) 1-173 contains eight putative endoproteolytic consensus sites that include a mono-arginyl (R37), paired basic (RR154-155), and related basic residue motifs (RLKR-4 to -1, RRR19-21, KKKK88-91, KRK96-98, KKKRR102-106, and KKKK147-150). To analyze the primary structural determinants involved in the posttranslational processing and secretion of PTHrP 1-173, we constructed a series of nonsense mutants that code for carboxy-terminal truncated polypeptides. Since the basic residue motifs are probable sites of endoproteolysis, these sites and the residues downstream were serially eliminated, thereby creating PTHrP 1-152, 1-146, 1-101, 1-95, 1-87, 1-36, and 1-18. The wild type PTHrP 1-173 and nonsense mutant constructs were transiently transfected into two cell lines, COS-1 and SK-N-BE(2). The COS-1 cells have a constitutive secretory pathway, whereas the neuroblastoma-derived BE-2 cells have, in addition, a regulated secretory pathway. PTHrP was measured in the conditioned media and cell extracts of the transfected cells with two peptide-specific RIAs. In COS-1 cells, PTHrP truncation mutants 1-152, 1-146, 1-101, 1-95, and 1-87 were present relative to wild type isoform 1-173, at 4.4-, 3-, 19-, 12-, and 57-fold excess, respectively; a similar pattern was also detected with BE-2 transfected cells, although the relative increases above the quantities of PTHrP 1-173 were not as dramatic. As the carboxy-terminal sequences were eliminated, the amount of total and secreted PTHrP increased, and the percentage found within the cell decreased. In COS-1 cells, 10.5% of the total PTHrP 1-173 was intracellular, whereas only 1% of the total PTHrP 1-87 was intracellular. In BE-2 cells, 54% of the total PTHrP 1-173 and only 9% of the total 1-87 mutant were intracellular. In COS-1 cells, a time course analysis demonstrated that PTHrP 1-87 and 1-95 were detectable in media 3 h after transfection, whereas 1-173 was barely detectable after 24 h. Our studies suggest that the carboxy-terminal sequence of PTHrP 1-173 is responsible for the intracellular degradation of this polypeptide, which may be the endogenous cellular mechanism that regulates the amount of processed and secreted PTHrP.

Elimination of the carboxy-terminal sequences of parathyroid hormone-related protein 1-173 increases production and secretion of the truncated forms.