Structural and Physiologic Characterization of the Mid-region Secretory Species of Parathyroid Hormone-related Protein

Terence L Wu,Rupangi C Vasavada,Kai Yang,Thierry Massfelder,Michael Ganz,S Khawar Abbas,Anthony D Care,Andrew F Stewart

doi:10.1074/jbc.271.40.24371

Abstract

Parathyroid hormone-related protein (PTHrP) is initially translated as a preprohormone which is posttranslationally processed to yield a family of mature secretory forms. Most attention has focused on the amino-terminal portion of the molecule which is homologous to parathyroid hormone. It is clear, however, that a mid-region species of PTHrP is posttranslationally cleaved from the highly conserved mid-region of PTHrP, and that the amino terminus of this peptide is Ala38. The purposes of the current study were three: 1) to confirm that Arg37 immediately preceding Ala38 serves as a posttranslational processing site in the PTHrP precursor, 2) to determine the carboxyl terminus of the mid-region secretory species of PTHrP, and 3) to synthesize this authentic mid-region secretory form of PTHrP and determine whether it is biologically active. The results indicate that: 1) Arg37 is indeed a processing site in the PTHrP precursor; 2) three distinct mid-region PTHrP species are generated by posttranslational processing, PTHrP(38-94)amide, PTHrP(38-95), and most likely, PTHrP(38-101); and 3) synthetic mid-region PTHrP(38-94)amide is active in four different biological systems. These studies confirm the finding that PTHrP is a prohormone. More importantly, they define a novel, biologically active highly conserved mid-region secretory form of PTHrP.

Highlights

Parathyroid hormone-related protein (PTHrP) is initially translated as a preprohormone which is posttranslationally processed to yield a family of mature secretory forms
Fresh medium was added to the cells and was harvested after 90 min of exposure to the cells. These conditions were selected for we have previously shown that rat insulinoma (RIN) cells do not significantly degrade PTHrP within this time frame [7] and that the mid-region secretory form of PTHrP found in the medium under these conditions is chromatographically identical to that found within cells prior to secretion and prior to exposure to extracellular proteases
Site-directed Mutagenesis of Arg37 Prevents Prohormone Cleavage—We have previously reported that the mid-region secretory form of PTHrP begins at Ala38, findings which would suggest that Arg37 serves as a substrate for a prohormone convertase with monobasic specificity [7]

Summary

Introduction

Parathyroid hormone-related protein (PTHrP) is initially translated as a preprohormone which is posttranslationally processed to yield a family of mature secretory forms. Most attention has focused on the aminoterminal portion of the molecule which is homologous to parathyroid hormone It is clear, that a midregion species of PTHrP is posttranslationally cleaved from the highly conserved mid-region of PTHrP, and that the amino terminus of this peptide is Ala. Mid-region PTHrP peptides have been shown to be produced by a variety of cell types, to result from posttranslational processing of the preproPTHrP, and to be packaged into secretory granules prior to secretion via the regulated secretory pathway [7, 8, 12] Partial purification of this midregion secretory species of PTHrP has shown that it has an molecular mass, as assessed by SDS-PAGE, of approximately 7000 Da. Amino acid sequencing has shown that it begins at alanine 38 of the cDNA-predicted amino acid sequence (Fig. 1) [7]. Luparello et al [16] have shown that PTHrP[67– 86]amide inhibits the mitogenesis but

Methods

Results

Conclusion