INTRODUCTION

Parathyroid hormone (PTH) is a major regulator of calcium and phosphate homeostasis. It is known that the delivery route of administration significantly determines its effect on bone¹. The continuous infusion of PTH causes bone loss, but intermittent injections of low doses repress osteoblast apoptosis and stimulate the differentiation of bone lining cells and mesenchymal cells to osteoblasts, resulting in bone mass increase^2,3. The anabolic effect of PTH on bone has prompted considerable interest in the development of novel derivatives of PTH and PTH-related peptide (PTHrP). Successful PTH analogue development is viewed with considerable importance for the

treatment of osteoporosis.

PTH is composed of 84 amino acids, but its full biological effects are limited to its N-terminal region, PTH(1-34). However, this 34-amino acid- peptide has limited effectiveness when administered by parenterally, nasal sprays and pulmonary inhalation⁴. To resolve this problem, wider and deeper studies have been conducted on the structures and functions of PTH and PTH/PTHrP receptor using site-directed mutagenesis⁵, photoaffinity labeling⁶, NMR⁷, and crystallographic analysis⁸. However, many questions remain regarding the nature of the ligand-receptor interaction, particularly the interaction between short length PTH analogues and the PTH/PTHrP receptor.

Recently, Jin et al. reported that hPTH(1-34) crystallizes as a slightly bent, long helical dimer⁹. Whereas recent NMR analysis of hPTH(1-34) revealed that it forms an N-terminal helix and a C-terminal helix connected by a highly flexible region in physiologic solution¹⁰. According to the report of Jin et al., the N-terminal region of PTH(1-34) binds to a pocket consisting of the extra cellular portion of TM3, TM4, and TM6 and the second and third extra cellular loops of the PTH/PTHrp receptor¹¹. The middle region of PTH(1-34) is sandwiched between the first extra cellular loop and the N-terminal extra cellular region of the receptor adjacent to TM1, and the C-terminal region of hPTH(1-34) interacts extensively with the putative binding domain of the PTH/PTHrp receptor. After comparing their crystallographic data with published NMR data upon PTH(1-34), Jin et al.

proposed that only one orientation of hPTH(1-34) satisfied all the known ligand receptor interactions. Furthermore, it was recently reported that Tip39

is an endogenous ligand of the PTH2 receptor^12,13, and that the NMR structure of Tip39 helps to explain the manner in which PTH(1-34) binds to the PTH/PTHrp receptor¹⁴.

PTH and PTHrP interact with the PTH/PTHrP receptor (or PTH1R), which is a member of the B family of G-protein coupled receptors¹⁵. Residues 25-34 of PTH(1-34) are important for binding to the PTH/PTHrP receptor, whereas the first 13 amino acids of the N-terminus are essential for receptor binding and activation¹⁶. In terms of the short length PTH analogues, Gardella et al. itemized the residues of chemically synthesized PTH(1-14) and PTH(1-13) analogues that could be substituted by other amino acids¹⁷. On the other hand, Monticelli et al. performed a computer simulation study using a PTH1R/[Arg¹¹]PTH(1-11) tethered system and proposed a mechanism involving the binding of the PTH N-terminus residue with receptors. These workers also found that the 1^st, 2^nd, 4^th, 5^th, 7^th, and 8^th residues of PTH are very important components of receptor binding/activation¹⁸. However, too many degrees of freedom remained within the PTH N-terminus are unknown^19,20, and in particular, its structure and the elements facilitating its receptor binding remained to be defined.

Shimizu et al. showed that PTH(1-14)NH2 stimulated cAMP formation weakly, to ca. 1/1000 of that of hPTH(1-34), in LLC-PK cells stably expressing a high level of the hPTH/PTHrp receptor²¹. Furthermore, they substituted PTH residues and analyzed the functional tolerability of each residue substitution. Finally, they proposed that the short amino-terminal peptides of PTH could possibly be optimized to significantly increase signaling potency by modifying the interactions involving receptor regions containing the

transmembrane domains and the extra cellular loops^22-24. However, although a part has just been explored and is being tested by some research group recently, such shorter and more potent PTH analogues have not been fully developed to date. Furthermore, few studies have been conducted upon the structures and functions of derivatives of PTH(1-11), PTH(1-12) or PTH(1-13)^21,25-26.

PTH(1-14) is regarded as the basic entity required for receptor activation, but the functionality of PTH(1-14), required for PTH/PTHrp receptor activation, is retained in the first 9 amino acids²⁴. Accordingly, little work has been reported upon the relationships between the structures and functions of amino acids 9 to 14 N-terminal PTH analogues^21,25-26. Moreover, in previous studies, the substitution of the 1^st∼6^th amino acids residues was found to cause loss of the receptor activating function^27,28. To better understand the ligand and receptor interactions of the short N-terminal PTH fragment activating PTH/PTHrP receptor, and to develop a potent analogue, it is systemically synthesized more than forty-three short-length amino terminal PTH analogues substituted at the 7^th, 8^th, 10^th, and 11^th amino-acids whilst preserving the 2^nd∼6^th amino acids residues. The biological activity of these analogues were examined using a cAMP-generating assay, in LLC-PK1 cell lines stably transfected with the wild-type human PTH1 receptor, and their conformational characteristics were determined by NMR to study the relationships between the biological activity of the short-length PTH analogues and the solubilized NMR structures.