PARATHYROID HORMONE-RELATED PROTEIN - EVIDENCE FOR ISOFORM-SPECIFIC AND TISSUE-SPECIFIC POSTTRANSLATIONAL PROCESSING

Parathyroid hormone-related protein (PTHrP) is expressed by malignant tumors and leads to the syndrome of humoral hypercalcemia of malignanc y. It is also expressed by a wide variety of nonmalignant tissues, in which it appears to play distinct paracrine and/or autocrine roles. Th e human PTHrP gene encodes three cDNA-predicted initial translational products of 139, 141, and 173 amino acids. Most human cell lines conta in mRNAs encoding all three PTHrP isoforms. The physiological rational e for the existence of these three highly similar transcripts is unkno wn. In order to determine whether the protein products derived from th ese three transcripts differ, we transfected Chinese hamster ovary (CH O) cells and rat insulinoma (RIN) cells individually with cDNAs encodi ng human PTHrP(1-139), PTHrP(1-141), and PTHrP(1-173). Cell extracts a nd conditioned medium were then chromatographed using reversed-phase H PLC and analyzed using region-specific PTHrP immunoassays. As we had p reviously observed in SKRC-1 (renal cell carcinoma) and RIN(1-141) cel ls, multiple amino-terminal PTHrP species as well as a separate midreg ion PTHrP species were identified in all six cell lines. In addition, both CHO and RIN cell lines transfected with the PTHrP(1-139) construc t contained a previously unrecognized carboxyterminal fragment that re acted with a PTHrP(109-138) antiserum. This carboxy-terminal fragment was physically distinct from the midregion fragment discovered earlier and was also present in conditioned medium, indicating that it is a s ecretory form, rather than a biosynthetic intermediate or a degradatio n product. Surprisingly, RIN and CHO cells transfected with PTHrP(1-14 1) or -(1-173) contained little of this carboxy-terminal fragment, sug gesting that isoform-specific protein processing exists for PTHrP. In addition, while RIN cells produced a single predominant amino-terminal species, CHO cells contained two, approximately equimolar, amino-term inal species, indicating the existence of cell-specific protein proces sing. These studies indicate that the posttranslational processing of PTHrP is highly complex. Specifically, (a) multiple amino-terminal PTH rP secretory forms, as well as a midregion form, are generated by cell lines containing each of the three PTHrP transcripts; (b) the Arg(37) cleavage that generates the midregion fragment occurs in the Golgi ap paratus, as both constitutive and regulated secretory cell types are c apable of performing this cleavage; (c) a previously unrecognized carb oxy-terminal fragment of PTHrP is secreted; and (d) processing of PTHr P appears to be both isoform- and cell-specific. Complete structural d etermination of each of these fragments is critical to understanding P THrP physiology and pathophysiology.