Dept. of Anatomy, The University of Sydney and (*) St. Vincent's Institute of Medical Research, Fitzroy
Homeostatic control of serum calcium is regulated by parathyroid hormone (PTH). This activity is achieved both by stimulating calcium resorption in the kidney and by resorbing calcium in the bone matrix to prevent hypocalcaemia. The N-terminal 34 residues (of a total of 84) retain all the biological activity. Adenylate cyclase activity can be abolished in N-terminal deleted peptides such as PTH(3-34) while receptor binding remains largely unimpaired indicating that the activation site and the receptor binding site are located in separate domains within the segment 1-34. Two loci in the N-terminal domain have been identified with receptor binding capacity reportedly involving the segment 25-34 forming a major site and the segment 10-27 forming a minor site. In addition to forming a part of the receptor binding site, the segment 30-34 has been identified as possessing the capacity to exert a mitogenic effect on chondrocytes and osteoblasts in a cyclic AMP-independent manner. The foetal protein PTH-related Protein or PTHrP is expressed by a very large number of tumours and causes morbidity and mortality in at least 25% of cancer patients through generating hypercalcaemia of malignancy syndrome as the PTHrP binds to the PTH receptors in addition to its numerous roles in normal physiology. We have studied the structures of PTH and three active mutants of PTHrP in aqueous solvent and in dilute trifluoroethanol in an attempt to elucidate the structural features common to both molecules. This work has confirmed that the structure in the presence of TFE is largely stabilized, exhibiting an N-terminal helix and a hydrophobic core in the C-terminal domain with a more rigid C-terminal helix. The backbone conformations of PTH and PTHrP were expected to be essentially identical when bound to the G-coupled glycoprotein receptors, despite the likely receptor binding sites having no sequence homology. The major structural feature evident is [[alpha]]-helix extending from residues Glu4 to Lys13 and Val21 to Gln29 with a turn incorporating Asn16 to Glu19 resulting in the C-terminal domain appearing quite globular in water, while the turn is replaced by an extension to the C-terminal helix in TFE only in PTH. Replacement of the turn in the Ala15 mutant with N-terminal helix did not affect receptor binding nor did the orientation of the C-terminal helix around the Arg20/Arg21 hinge confirming that the amphipathic C-terminal helix is responsible for receptor binding and is thus the focus of drug design measures.