ELIMINATION OF THE CARBOXY-TERMINAL SEQUENCES OF PARATHYROID HORMONE-RELATED PROTEIN-1-173 INCREASES PRODUCTION AND SECRETION OF THE TRUNCATED FORMS

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-arg inyl (R(37)), paired basic (RR(154-155)),,d related basic residue moti fs (RLKR(-4 to -1), RRR(19-21), KKKK88-91, KRK(96-98) KKKRR(102-106) a nd KKKK147-150). To analyze the primary structural determinants involv ed in the posttranslational processing and secretion of PTHrP 1-173, w e constructed a series of nonsense mutants that code for carboxy-termi nal truncated polypeptides. Since the basic residue motifs are probabl e sites of endoproteolysis, these sites and the residues downstream we re 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 mutan t constructs were transiently transfected into two cell Lines, COS-1 a nd SK-N-BE(2). The COS-1 cells have a constitutive secretory pathway, whereas the neuroblastoma-derived BE-2 cells have, in addition, a regu lated secretory pathway. PTHrP was measured in the conditioned media a nd cell extracts of the transfected cells with two peptide-specific RI As. In COS-1 cells, PTHrP truncation mutants 1-152, 1-146, 1-101, 1-95 , and 1-87 mere present relative to the 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 i ncreases above the quantities of PTHrP 1-173 were not as dramatic. As the carboxy-terminal sequences were eliminated, the amount of total an d secreted PTHrP increased, and the percentage found within the cell d ecreased. In COS-1 cells, 10.5% of the total PTHrP 1-173 was intracell ular, whereas only 1% of the total PTPrP 1-87 was intracellular. In BE -2 cells, 54% of the total PTHrP 1-173 and only 9% of the total 1-87 m utant were intracellular. In COS-1 cells, a time course analysis demon strated that PTHrP 1-87 and 1-95 were detectable in media 3 h after tr ansfection, whereas 1-173 was barely detectable after 24 h. Our studie s suggest that the carboxy-terminal sequence of PTHrP 1-173 is respons ible for the intracellular degradation of this polypeptide, which may be the endogenous cellular mechanism that regulates the amount of proc essed and secreted PTHrP.