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 malignancy. It is also expressed by a wide variety of nonmalignant tissues, in which it appears to play distinct paracrine and/or autocrine roles. The human PTHrP gene encodes three cDNA-predicted initial translational products of 139, 141, and 173 amino acids. Most human cell lines contain mRNAs encoding all three PTHrP isoforms. The physiological rationale for the existence of these three highly similar transcripts is unknown. In order to determine whether the protein products derived from these three transcripts differ, we transfected Chinese hamster ovary (CHO) cells and rat insulinoma (RIN) cells individually with cDNAs encoding human PTHrP(1-139), PTHrP(1-141), and PTHrP(1-173). Cell extracts and conditioned medium were then chromatographed using reversed-phase HPLC and analyzed using region-specific PTHrP immunoassays. As we had previously observed in SKRC-1 (renal cell carcinoma) and RIN(1-141) cells, multiple amino-terminal PTHrP species as well as a separate midregion PTHrP species were identified in all six cell lines. In addition, both CHO and RIN cell lines transfected with the PTHrP(1-139) construct contained a previously unrecognized carboxyterminal fragment that reacted 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 secretory form, rather than a biosynthetic intermediate or a degradation product. Surprisingly, RIN and CHO cells transfected with PTHrP(1-141) or -(1-173) contained little of this carboxy-terminal fragment, suggesting 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-terminal species, indicating the existence of cell-specific protein processing. These studies indicate that the posttranslational processing of PTHrP is highly complex. Specifically, (a) multiple amino-terminal PTHrP 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 apparatus, as both constitutive and regulated secretory cell types are capable of performing this cleavage; (c) a previously unrecognized carboxy-terminal fragment of PTHrP is secreted; and (d) processing of PTHrP appears to be both isoform- and cell-specific. Complete structural determination of each of these fragments is critical to understanding PTHrP physiology and pathophysiology.