No abstract

Parathyroid hormone-like adenylate cyclase-stimulating proteins (hACSPs) have been implicated as one of the calcemic, bone-resorbing agents in patients with humoral hypercalcemia of malignancy. We report the synthesis of an amino-terminal hACSP fragment, Tyr' hACSP (1-36) amide. The synthetic hACSP is a potent agonist of renal membrane adenylate cyclase (K., 1.7 X 10-"') and of bone cell adenylate cyclase (K. 1 X 10-9 M). It is a potent bone-resorbing agent in vitro, stimulating 45Ca release from fetal rat long bones at a concentration of 10-' M. When infused via osmotic minipumps into rats, it is also a potent calcemic factor in vivo, inducing a rise in serum calcium from (mean ± SD) 10.6 ± 0.6 to 19.7 ± 3.2 mg/dl when infused at 1.4 pg/h and from 9.9 ± 0.7 to 11.4 ± 1.2 mg/dl when infused at 0.14 ,ug/h.These findings indicate that biologically active hACSP fragments can be synthesized. One such synthetic peptide possesses the in vitro and in vivo bioactivities demonstrated in native, tumor-derived hACSPs. It is also a potent calcemic, bone-resorbing agent.

Parathyroid hormone (PTH) and PTH-related proteins (PTHrP) interact with a common receptor in rat bone cells and in canine renal membranes with similar affinity, but PTHrP are substantially less potent than PTH in stimulating adenylate cyclase in canine renal membranes; in contrast, PTH and PTHrP are equipotent in stimulating adenylate cyclase in rat bone cells. This discrepancy has been largely viewed as reflecting differences in the relative efficiency of signal transduction of PTHrP between bone and kidney assay systems. To test the alternative (but not mutually exclusive) hypothesis that these differences could reflect interspecies differences in PTH receptors, we have characterized the bioactivity of amino-terminal PTHrP and PTH in rat and human renal cortical membranes (RCM) and compared them to results we previously reported in canine RCM. The stability of PTH and PTHrP peptides under binding and adenylate cyclase assay conditions was greater than 80% for each species. Competitive inhibition of [125I](Tyr36)hPTHrP-(1-36)NH2 binding to rat RCM by bPTH-(1-34) and (Tyr36)hPTHrP-(1-36)NH2 yielded nearly identical binding dissociation constants (3.7 and 3.6 nM, respectively), and binding to human RCM demonstrated slightly greater potency for PTHrP (0.5 nM) than for PTH (0.9 nM). Similarly, adenylate cyclase stimulating activity was equivalent for the two peptides in rat RCM, but PTHrP was twofold more potent than PTH in human RCM. Covalent photoaffinity labeling of protease-protected rat RCM yielded an apparent 80 kD receptor protein, and cross-linking of human RCM labeled an 85 kD receptor, indistinguishable in size from the canine renal PTH receptor. We conclude that rat, canine, and human renal cortical PTH receptors exhibit species specificity. The previously observed differences between rat bone cells and canine renal membranes in the efficiency of signal transduction by PTHrP may be explained, at least in part, by these species differences.

Tumors associated with humoral hypercalcemia of malignancy (HHM) contain parathyroid hormone-like adenylate cyclase-stimulating proteins (hACSPs). We previously purified a 17,000 MW hACSP from an HHM-associated breast carcinoma. This report describes the characterization of hACSPs from three additional HHM-associated tumors: two typical HHM-associated tumors (squamous carcinomas) and a third unusual tumor type (pheochromocytoma). Each tumor was extracted in acid-urea/ethanol-sodium chloride, and adenylate cyclase-stimulating activity (ACSA) was examined following reverse-phase and size-exclusion HPLC and isoelectric focusing. Each tumor contained a high molecular weight form of ACSA which co-eluted with the 17,000 molecular weight breast carcinoma hACSP in each of the three separation procedures. Each also contained a lower molecular weight form(s) of ACSA (6,000-9,000 molecular weight). Both forms were inhibited by Nle8,18Tyr34bPTH(3-34) amide. The high molecular weight form was not changed to the lower molecular weight form by a reducing agent. Some HHM-associated tumors contain two forms of hACSP, one with a molecular weight of 17,000 and another with a molecular weight of 6,000-9,000, which appears to be an amino-terminal cleavage product of the larger protein.