INTRODUCTION

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All known extracellular biological activity of human parathyroid hormone (hPTH)¹ is located in the NH₂ terminus of this 84-amino acid peptide hormone (1). hPTH-(1-37) is the naturally occurring bioactive hormone extractable from human blood (2, 3), and hPTH-(1-34) is known to maintain normocalcemia in blood via adenylate cyclase activation. To increase calcium flow into blood, the hormone acts directly on bone and kidney and indirectly on the intestine (1). In addition to the cyclic adenosine monophosphate (cAMP) pathway, involvement of the phosphatidylinositol hydrolysis signaling pathway is postulated for these functions (4). The receptor binding region mediating the calcium regulatory activity is located within sequence His-14 to Phe-34 (5, 6). The complete NH₂-terminal part of hPTH-(1-34) is required for stimulation of the cAMP-dependent pathway (4), and the minimum sequence affecting bone and kidney comprises amino acids 2-27 (1, 7). Adenylate cyclase activity is lost on deletion of the first NH₂-terminal amino acid, whereas receptor binding capacity is not influenced, indicating that the activation region for cAMP production and the receptor binding region are located in two distinct domains (4, 8). Adenylate cyclase activity measured in vitro does, however, not reflect the sequence-activity relationship indicated by various in vivo assays (4). hPTH-(2-34) is nearly inactive in an in vitro bioassay of cAMP stimulation, but in vivo the calcium level in blood is regulated with identical efficiency by hPTH-(2-34) and hPTH-(1-34) (Ref. 4 and references therein). This indicates that hPTH utilizes other second messengers in addition to cAMP for signal transduction and possibly additional receptors in vivo (9). Furthermore, hPTH is stimulating cell proliferation in skeletal derived cell cultures (10, 11) as well as DNA synthesis in chondrocytes (12). Different sequence regions of the peptide are responsible for these functions; for stimulation of DNA synthesis, amino acids Asp-30 to Phe-34 are postulated as an indispensable region, but flanking residues seem to be required in addition for this function (12).

hPTH stimulates an increase of bone formation and axial bone mass after periodic administration of the hormone (13). Thus, hPTH is useful in the treatment of patients with hypoparathyroidism and, moreover, in the treatment of osteoporotic patients. Therefore, it would be highly desirable to construct a stable mimetic of this peptide hormone. Thus, recent studies focused on the determination of the three-dimensional structure of NH₂-terminal peptides in solution by nuclear magnetic resonance (NMR) spectroscopy. In particular, hPTH-(1-34) is an intensely studied hormone fragment as it contains all functional domains (14-17). From most experiments it was concluded that hPTH-(1-34) does not form secondary structure elements in the absence of TFE (14, 16, 18), but helix formation in TFE-free solution is nevertheless observed for hPTH-(1-34), residues 4-13 and 21-29 (19), and for hPTH-(1-37), residues 5-10 and 17-28 (20). In TFE-containing solution hPTH-(1-34) displays helical regions from Ser-3 to Gly-12 and from Ser-17 to Lys-26 (16, 18), but no tertiary interactions for hPTH-(1-34) are found under these conditions. It is commonly known that TFE stabilizes secondary structures, in particular helices (21-26), but bears the risk of weakening hydrophobically stabilized tertiary structure domains (24), an effect also observed for hPTH-(1-34).²

Since hPTH is of considerable medical importance, drugs mimicking this structure could be useful as therapeutics. In a first step in this direction, we determine here the structures of the NH₂-terminally truncated fragments hPTH-(2-37), hPTH-(3-37), and hPTH-(4-37) in comparison with the biologically active fragment hPTH-(1-37) (20) under near physiological conditions to elucidate a possible correlation between the loss of calcium regulatory activity after stepwise truncation of NH₂-terminal amino acids and structural features of the peptides.

	MATERIALS AND METHODS

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Peptide Synthesis-- Synthesis of hPTH fragments was carried out using a PerSeptive 9050 automated peptide synthesizer on preloaded Fmoc-L-Leu-PEG-PS or Fmoc-L-Leu-TentaGelS PHB resin (loading 0.2 mmol/g, PerSeptive, Wiesbaden, Rapp Polymere, Tübingen, Germany) (20, 27, 28). Acylations with a 4-fold excess of Fmoc amino acids in N,N-dimethylformamide were performed in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate/N,N-diisopropylethylamine/1-hydroxybenzotriazole for 30 min. The following protective groups were used: Ser-(tert-butyl), Glu-(O-tert-butyl), Gln-(triphenylmethyl), His-(triphenylmethyl), Asn-(triphenylmethyl), Lys-(tert-butyloxycarbonyl), Arg-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), and Trp-(tert-butyloxycarbonyl). Fmoc groups were cleaved in 10 min with 20% piperidine in N,N-dimethylformamide. The peptides were deprotected and cleaved from the resin with trifluoroacetic acid/ethanedithiol/water, 94:3:3, for 120 min. After filtration and precipitation of the crude peptide by addition of cold tert-butyl methyl ether, the peptide was lyophilized from 10% acetic acid and purified by preparative reversed phase-high performance liquid chromatography (Vydac C18, 300A, 10 mm, 25 × 250 mm, flow rate 10 ml/min; buffer A, 0.6% trifluoroacetic acid in water; buffer B, 0.5% trifluoroacetic acid in acetonitrile/water, 4:1, detection at 230 nm). Pure fractions were pooled, and the final product was checked by reversed phase-high performance liquid chromatography (Vydac C18) and capillary zone electrophoresis (Biofocus 3000, Bio-Rad, München, Germany). Electrospray mass spectrometry (Sciex API III, Perkin-Elmer, Langen, Germany), gas phase sequencing (473A Protein Sequencer, Applied Biosystems/Perkin-Elmer, Weiterstadt, Germany), and amino acid analysis (Aminoquant 1090L, Hewlett Packard, Waldbronn) showed correct mass, amino acid sequence, and composition.

Biological Activity-- In vitro biological activity of the synthetic hPTH-(1-37), hPTH-(2-37), hPTH-(3-37), and hPTH-(4-37) fragments was tested by observation of the stimulation of the cAMP generation in osteogenic cells (rat osteosarcoma cells) compared with synthetic hPTH-(1-34) fragment. ROS 17/2.8 cells were grown in 25-cm² plastic flasks at 37 °C in a humidified atmosphere of air/CO₂ in Ham's F12/Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 50 mg of streptomycin/ml, and 50 units of penicillin/ml. The medium was changed on alternate days. The cells reached confluence within 3-4 days and were plated into 24-well dishes for experiments. Assays were performed on confluent cultures 1-2 days after change in medium. cAMP measurements were as follows. The cells were preincubated with 1 mM 3-isobutyl-1-methylxanthine for 15 min. The cells were then incubated for an additional 5 min in the presence of the agonists (hPTH-(1-34), hPTH-(1-37), hPTH-(2-37), hPTH-(3-37), and hPTH-(4-37)). Incubation with forskolin was used as positive control. Supernatant was aspirated, and cAMP was extracted after addition of 70% chilled ethanol, evaporation, and redilution of the cells in cAMP buffer. The samples were kept at 20 °C until cAMP levels were determined by a specific radioimmunoassay (29).

CD Spectroscopy-- CD spectra were recorded at 25 °C in 0.1-mm cells from 250 to 190 nm at 20 nm/min on a Jasco J 600A CD spectropolarimeter. Peptide concentrations ranged from 270 to 310 µM in 50 mM phosphate buffer, pH 6.0, with 270 mM sodium chloride in 30 µl volume. The reference sample contained buffer without peptide. Eight scans were accumulated from samples and reference, respectively.

NMR Spectroscopy-- Two-dimensional NMR spectra were obtained on a commercial Bruker AMX600 spectrometer at 298 K with standard methods (31, 32). For hPTH-(4-37) an additional set of spectra was measured at 288 K to resolve frequency degeneracy. The measurements were carried out in 50 mM phosphate buffer with 270 mM sodium chloride. Peptide concentrations were 1.6 mM, pH 6.0 (hPTH-(2-37)), 2.1 mM, pH 6.0 (hPTH-(3-37)), and 1.9 mM, pH 5.8 (hPTH-(4-37)).

Restrained Molecular Dynamics Calculations-- Distance geometry and molecular dynamics (MD) calculations were performed with the XPLOR 3.1 program package (33) on an HP735 computer. The number of nontrivial interresidual NOESY cross-peaks used for structure calculation was 171 for hPTH-(2-37), 210 for hPTH-(3-37), and 159 hPTH-(4-37) (Table I). These cross-peaks were divided into three groups according to their following relative intensities: strong, 0.2 to 0.3 nm, medium, 0.2 to 0.4 nm, and weak, 0.2 to 0.5 nm. 0.05 nm was added to the upper distance limit for distances involving unresolved methyl or methylene proton resonances (pseudoatom approach).

Structure Analysis-- The final structures were analyzed with respect to stable idealized elements of regular secondary structure using the DSSP (definition of secondary structure of proteins) program package (35). To elucidate the stability of the structures, we calculated the local root mean square deviations with a five-amino acid window (36). For visualization of structure data the SYBYL 6.0 (TRIPOS Association), the RASMOL V 2.6 (37), and the MOLSCRIPT program packages (38) were used.

	RESULTS AND DISCUSSION

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Activity Tests-- The biological activities of hPTH-(1-37), hPTH-(2-37), and hPTH-(1-34) are virtually identical in the in vivo activity test of calcium homeostasis in blood using Parsons' Chicken Assay (30). Fragment hPTH-(3-37) shows less than 10% of this activity, and hPTH-(4-37) is inactive (Fig. 1a). In the in vitro activity test measuring only the cAMP production in cultured rat osteosarcoma cells, hPTH-(2-37) is much less active than hPTH-(1-37). hPTH-(3-37) and hPTH-(4-37) do not stimulate the adenylate cyclase (Fig. 1b).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Activity tests. a, in vivo activity test of hPTH-(1-37), hPTH-(2-37), hPTH-(3-37), and hPTH-(4-37) relative to hPTH-(1-34) as a standard using Parsons' Chicken Assay (30). Pure solvent without PTH served as control. The variation of the calcium level in blood is used as indicator for PTH activity. After subtraction of the control value (10.78 mg/dl Ca2+), data are expressed as percentage of the hPTH-(1-34) value (14.60 mg/dl Ca2+) and are plotted as the mean ± S.D. from eight separate experiments. b, in vitro activity test of hPTH-(1-37), hPTH-(2-37), hPTH-(3-37), and hPTH-(4-37) in comparison to synthetic hPTH-(1-34). The cAMP generation in rat osteosarcoma cells after stimulation with the different hPTH fragments is used as indicator for PTH activity. Data are expressed in picomoles/well as a response to the different hPTH fragments and the control, respectively, and are plotted as the mean ± S.D. from three separate preparations, each assayed in duplicate.

CD Spectroscopy-- To compare the overall content of helical structure of the different peptides, far UV CD spectroscopy was used (Fig. 2) with peptide concentrations ranging from 270 to 310 µM. The overall shape of the spectra of the different peptides indicates the presence of both -helical and random coil structural elements (40, 41). With the stepwise truncation of the NH₂-terminal amino acids the ellipticity at 222 nm changes to less negative values. The evaluation of the helix content of the different peptides by standard methods (42) shows the following approximate fractional helix contents: hPTH-(1-37), 29%; hPTH-(2-37), 24%; hPTH-(3-37), 23%; and hPTH-(4-37), 22%.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Far UV CD spectra of hPTH-(1-37) (---), hPTH-(2-37) (- - -), hPTH-(3-37) (-··-), and hPTH-(4-37) (···). Sample conditions are as follows: 50 mM phosphate buffer, pH 6.0, 270 mM NaCl, peptide concentration 270-310 µM, 0.1-mm cell.

Analysis of C- Proton Chemical Shifts-- To allow an initial mutual comparison of the truncated fragments and hPTH-(1-37), we used the chemical shift data available from our experiments to perform a secondary structure estimation based on the chemical shift index strategy (44, 45). The procedure depends on a direct correlation between the chemical shifts of C- proton resonances of consecutive amino acids and the local secondary structure: an upfield shift of the C- proton resonances relative to the corresponding "random coil" values indicates local -helical structure (negative value in Fig. 3), and a downfield shift of C- proton resonances compared with the corresponding random coil values indicates a local -sheet structure (positive value in Fig. 3). Only deviations from the random coil values by more than 0.1 ppm are taken into account for secondary structure estimation. For hPTH-(1-37) and hPTH-(2-37), the chemical shifts of C- proton resonances suggest two helical regions extending from Ser-17 to at least Gln-29 and around Glu-4 to His-9. In contrast, no indication of an NH₂-terminal helix is found for hPTH-(3-37) and hPTH-(4-37), although the helical region in the COOH-terminal part can clearly be derived (Fig. 3). No other elements of regular secondary structure were evidenced by this procedure.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Difference values of the observed C- proton chemical shifts relative to the random coil values of Wishart et al. (45). The threshold of ±0.1 ppm is indicated by dashed lines. a, hPTH-(1-37); b, hPTH-(2-37); c, hPTH-(3-37); d, hPTH-(4-37).

Analysis of Medium Range NOEs-- The NOEs observed for the various hPTH fragments were determined from the 200-ms NOESY spectra at 298 K (Table I and Fig. 4). The d_N(i,i + 3) and d(i,i + 3) NOESY cross-peaks fully corroborate the existence of two helical regions for hPTH-(1-37) and hPTH-(2-37). Indications for an NH₂-terminal helix for hPTH-(3-37), however, are weak and are entirely missing for hPTH-(4-37), thus confirming the results from the chemical shift index procedure. In particular, helix typical (i,i + 3) NOEs are clustered from Ile-5 to Leu-11 and Ser-17 to Phe-34 for hPTH-(1-37) and hPTH-(2-37), respectively. For hPTH-(1-37), two helical regions were found earlier, a short one from Ile-5 to Asn-10 and a longer one from Ser-17 through at least Leu-28 (20). For hPTH-(3-37) two weak helix typical NOEs are found in the NH₂-terminal region, and for hPTH-(4-37) no helix typical NOE could be found in the NH₂-terminal region, and frequency degenerations of possible (i,i + 3) NOEs were not present. In contrast, clear evidence of the COOH-terminal helix in these two fragments is found from Ser-17 to Phe-34 and His-32, respectively. To investigate whether the missing (i,i + 3) NOEs in the NH₂-terminal region of hPTH-(4-37) can be accounted for by the lower concentration of this peptide (1.9 mM), two-dimensional NMR spectra of a sample of hPTH-(1-37) with 1.8 mM concentration were measured with the same buffer, temperature, and spectrometer conditions. From the 200-ms NOESY spectrum of this sample, two d_N(i,i + 3) and five d(i,i + 3) NOEs in the NH₂-terminal region of hPTH-(1-37) could be assigned. Thus, the lower concentration cannot account for the missing (i,i + 3) NOEs in the NH₂-terminal region of hPTH-(4-37). The loss of the NH₂-terminal helix on removal of the first two amino acids was fully confirmed by the NOESY cross-peak patterns (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Patterns of sequential and medium range NOE cross-peaks versus peptide sequences. The thickness of the bars corresponds to the relative strength of the NOESY cross-peaks. An asterisk indicates that the NOE could not be unambiguously assigned because of frequency degenerations. a, hPTH-(1-37); b, hPTH-(2-37); c, hPTH-(3-37); d, hPTH-(4-37).

Relative NOE Intensities-- Relative intensities of sequential and medium range NOEs may be used to estimate the perfection and stability of helical structures, in addition to the upfield shift of the -proton resonances. For an ideal -helix the d_N(i,i + 1) and d_N(i,i + 3) distances should be nearly identical, whereas the d_NN(i,i + 1) distances should be shorter, yielding higher intensity NOEs (47-49). For all PTH fragments employed in our experiments, most of the sequential d_N(i,i + 1) NOEs are of higher intensity than the corresponding d_NN(i,i + 1) and d_N(i,i + 3) NOEs (Fig. 4), indicating that the helices are not ideal but are in an equilibrium with a more extended conformation, possibly a 3₁₀-helix. The helices of the PTH fragments are clearly more stable than nascent helices that do not show (i,i + 3) NOEs (48, 50). Simultaneous observation of d_N(i,i + 2) and d_N(i,i + 4) NOEs may arise from a mixture of 3₁₀- and -helix type structures (50). This effect was observed for the COOH-terminal region of the different PTH fragments. Karle and Balaram (51, 52) suggest that six-residue sequences are equally likely to form 3₁₀- or -helices. Both helices in the hPTH fragments we studied seem to represent an equilibrium between an -helix and 3₁₀-helical conformation, the NH₂-terminal helix having a higher tendency to a more extended 3₁₀-helical conformation. This phenomenon is also reflected by values of the upfield shift of the -proton resonances (Fig. 3).

Other NOEs-- For each fragment, four to six long range (|i  j| > 5) NOEs could be assigned (Table I). All fragments show several long range NOEs between Leu-15 and Trp-23. For hPTH-(1-37), five NOEs were found between Leu-15 and Trp-23 (20), four NOEs for hPTH-(2-37) and hPTH-(3-37), and five NOEs for hPTH-(4-37). Additionally, two NOEs between Leu-15 and Val-21 were observed in hPTH-(3-37). These NOEs indicate a spatial proximity between Leu-15 and Trp-23, probably due to hydrophobic interactions between these two residues. The observed NOEs are responsible for a clear restriction of the conformational space of the calculated structures and lead to a defined loop region around His-14 to Ser-17. Furthermore, due to the ring current field of the spatial neighboring aromatic ring system of Trp-23 the  proton resonances of Leu-15 are shifted upfield in comparison to the analogous resonances of other leucines for all four fragments.

Structure Calculation and Analysis-- 159-210 interresidual NOEs per fragment were collected from 200-ms NOESY spectra at 298 K and used in restrained MD calculations (Table I). For structure calculation of the NH₂-terminally truncated fragments, the combined distance geometry/simulated annealing protocol described earlier (20, 33) was used. For each fragment a family of 30 structures was calculated, and the 10 structures with lowest energy values and lowest number of NOE violations were selected from each group. To resolve frequency degenerations of proton resonances in the spectra of hPTH-(4-37) an additional set of spectra was obtained at 288 K. From this NOESY spectrum, 175 unambiguous interresidual NOEs could be assigned. Only NOEs were taken into account for structure calculation that were also observed, albeit ambiguously, in the NOESY spectrum at 298 K. For each of the four fragments the COOH-terminal helix extending from Met-18 to at least Leu-28 is found by DSSP analysis. For hPTH-(1-37) an NH₂-terminal helix from Gln-6 to His-9 exists. For hPTH-(2-37), five structures show an NH₂-terminal -helix around Leu-7; the others show turns or 3₁₀-helix in this region. None of the 10 calculated structures of hPTH-(3-37) displays an NH₂-terminal -helix, and only two structures exhibit a 3₁₀-helix from Glu-4 to Gln-6. No structure of hPTH-(4-37) shows an NH₂-terminal helix, and only in one case a turn is indicated by DSSP in this region. The extension of the COOH-terminal helix of hPTH-(4-37) is virtually identical to that of the corresponding helix in the other fragments. The loss of the NH₂-terminal helix after truncation of the first two amino acids is corroborated by the structure calculations.

Local RMSD Values-- To elucidate the stability of the structures in the helical regions and the defined loop, we calculated the local root mean square deviations (RMSD) using a five-amino acid window (36) (Fig. 5). The upper trace represents the local RMSD values for all heavy atoms, and the lower trace represents the values for the peptide backbone. The regions with defined structure show substantially reduced local RMSD values compared with the flexible regions at the termini and around Gly-12. For hPTH-(1-37) and hPTH-(2-37) two regions with local backbone RMSD values lower than 0.07 nm were found from Gln-6 to His-9 and Asn-16 to Lys-26 for hPTH-(1-37) and from Leu-7 to His-9 and Asn-16 to Asp-30 for hPTH-(2-37), respectively. Comparatively high RMSD values for the amino acids Leu-11 to Lys-13 for fragments hPTH-(1-37) and hPTH-(2-37) indicate a flexible hinge region between the NH₂-terminal helix and the loop region followed by the COOH-terminal helix. For hPTH-(3-37) and hPTH-(4-37) a decrease of the RMSD values is found in the region of the COOH-terminal helix from Ser-17 to Gln-29. Compared with the fragments hPTH-(1-37) and hPTH-(2-37) the NH₂-terminal region is structurally less well defined for the fragments hPTH-(3-37) and hPTH-(4-37) (Fig. 5).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Local RMSD values calculated with a five-amino acid window (36). The plot was calculated on the basis of the 10 final structures of each hPTH fragment. The upper trace describes the RMSD values for all heavy atoms, and the lower trace describes the values for the peptide backbone. a, hPTH-(1-37); b, hPTH-(2-37); c, hPTH-(3-37); d, hPTH-(4-37).

View larger version (36K): [in this window] [in a new window]   Fig. 6.   a, best fit superposition of the backbone atoms of His-14 to Leu-28 of the 10 final structures selected from the MD calculation of hPTH-(2-37). Only the backbone atoms of amino acids His-14 to Leu-28 are shown. b, same structures and superposition as in a, but all backbone atoms are shown to illustrate the inherent flexibility of the peptide. c, best fit superposition of the backbone atoms of Gln-6 to His-9 of the 10 final structures selected from the MD calculation of hPTH-(2-37). Only the backbone atoms of amino acids Val-2 to Lys-13 are shown. d, best fit superposition of the backbone atoms of His-14 to Leu-28 of the 10 calculated and selected structures of hPTH-(4-37). Only the backbone atoms of amino acids His-14 to Leu-28 are shown.

Helix Content-- From NMR and structure calculation data the following helix contents for the different peptides were estimated. The secondary structure analysis using the DSSP program (35) result in a helix content of 43% for hPTH-(1-37), 44% for hPTH-(2-37), 37% for hPTH-(3-37), and 32% for hPTH-(4-37). For this calculation only amino acids that reside in a helical conformation in more than 50% of the calculated structures were taken into account. Under the assumption that residues which contribute to medium and strong (i,i + 3) NOEs are part of helical structures (Fig. 4), the helix content is 59% for hPTH-(1-37), 55% for hPTH-(2-37), 43% for hPTH-(3-37), and 38% for hPTH-(4-37). The helix content according to the chemical shift indexing procedure is 46 and 50% for hPTH-(1-37) and hPTH-(2-37), respectively, 31% for hPTH-(3-37), and 32% for hPTH-(4-37) (Fig. 3). From the NMR results a clear decrease in the helix content is derived between hPTH-(2-37) and hPTH-(3-37). Assuming that the length of helical regions is reflected correctly by the combined NMR results, there is a significant underestimation of the helix content from CD spectra (22-29%), which is also reported for other peptides (54, 55). One explanation for the apparent lower helix content estimated from CD spectra is that the helical sequences are in helical conformation in 50-70% on time average in the case of PTH. Other explanations are the absolute length of the helices and the associated end group effects (56-60) as well as a possible contribution of the aromatic side chain of Trp-23 to the far UV CD signal (59, 61). Additionally, the shape and intensity of the CD signal depends on the geometry of a peptide helix. An ideal -helix has a stronger CD signal than a 3₁₀-helix (59) with a different shape (50, 58). These phenomena lead to a lower percentage of helicity estimated from the []₂₂₂ value. Thus, changes in the short NH₂-terminal helix could not be detected on the basis of the CD signal at 222 nm alone. The possibility of an equilibrium with a 3₁₀-helix is also reflected by the values of the upfield shifts of the C- proton resonances (Fig. 3).

Conclusion-- After deletion of the NH₂-terminal two or three amino acids, PTH's biological activity is lost, but its receptor binding ability remains unimpaired (8, 53). Table II summarizes the results of the activity tests and the structure calculations. All fragments show the loop region and the COOH-terminal helix. His-14 to Leu-28 (loop and COOH-terminal helix) comprises the major part of the receptor binding region that is known to reside within His-14 to Phe-34 (5, 6, 68). The NH₂-terminal helix, however, is present only in the in vivo bioactive fragments hPTH-(1-37) and hPTH-(2-37), but not in the inactive fragments hPTH-(3-37) and hPTH-(4-37) (Table II). This may indicate that the NH₂-terminal helix is correlated with the in vivo bioactivity of the PTH fragments concerning the calcium level in blood. Existence of the NH₂-terminal helix, however, cannot be connected to the ability to stimulate adenylate cyclase, as hPTH-(2-37) is nearly inactive in the cAMP assay. This result may imply different structural requirements for triggering the different signal transduction pathways (4), and may thus indicate the occurrence of different PTH receptors as discussed in the literature (69). To decide whether or not the in vivo biological activity is determined on a structural level by the NH₂-terminal helix or depends on a direct functional role of the first two amino acids, structure calculations and activity tests of stabilized PTH fragments are currently under investigation.