PTH receptor-stimulating
proteins may be a common mediator of
humoral hypercalcemia of malignancy (HHM). Such
proteins exhibit
adenylate cyclase-stimulating activity (ACSA) in PTH-sensitive assays, and ACSA has been used to follow their purification.
Acid/
urea tumor extracts from a murine
squamous carcinoma model of HHM were previously shown to have very high ACSA, which was partially, but incompletely, inhibited by the PTH antagonist Nle8,18,Tyr34-bovine PTH-(3-34)
amide. ACSA from murine
tumor extracts has now been further purified using
solvent fractionation and reverse phase HPLC. Approximately half of the ACSA is attributable to a family of three
proteins (peaks IA, IB, and IC) with properties characteristic of the PTH receptor-stimulating
protein extracted from rat Leydig cell and human HHM
tumors. The ACSA in these three peaks of murine
tumor extract elutes in the same region as human
tumor ACSA on reverse phase HPLC, has a dose-response curve parallel to that of PTH, and is fully inhibited by the PTH-(3-34) antagonist in both the renal cortical and rat
osteosarcoma (ROS)
adenylate cyclase assays. The remaining half of the ACSA from murine
tumor extracts elutes as a single peak (peak II) at a higher
acetonitrile concentration on reverse phase HPLC. In the renal cortical assay, its dose-response curve differs from that of PTH, its ACSA is not affected by the PTH-(3-34) antagonist, and it potentiates PTH- or peak I-stimulated
adenylate cyclase activity. In the PTH-sensitive intact cell ROS assay, peak II exhibits no ACSA. We conclude that the potent ACSA of murine
tumor acid/
urea extract results in large part from amplification of the PTH-specific ACSA (peak I) by peak II. Peak II is a distinct
protein, not previously reported in
tumor extracts, that may act as a postreceptor step in the
adenylate cyclase system.