Bioactive N-terminal undecapeptides derived from parathyroid hormone: the role of alpha-helicity.

The N-terminal 1-34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N-terminal fragments presenting the principal activating domain such as PTH(1-11) and PTH(1-14) with helicity-enhancing substitutions yield potent analogues with PTH(1-34)-like activity. To further investigate the role of alpha-helicity on biological potency, we designed and synthesized by solid-phase methodology the following hPTH(1-11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix-promoting C(alpha)-tetrasubstituted alpha-amino acids alpha-amino isobutyric acid (Aib), 1-aminocyclopentane-1-carboxylic acid (Ac(5)c) and 1-aminocyclohexane-1-carboxylic acid (Ac(6)c): Ac(5)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (I); Aib-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (II); Ac(6)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (III); Aib-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (IV); Aib-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (V); S-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (VI), S-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (VII); Ac(5)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (VIII); Ac(6)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (IX); Ac(5)c-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (X); Ac(6)c-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (XI). All analogues were biologically evaluated and conformationally characterized in 2,2,2-trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I-V, which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand-stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III, VI and VII are very weakly active and analogues IV, V, VIII-XI are inactive. The most potent analogue, I exhibits biological activity 3500-fold higher than that of the native PTH(1-11) and only 15-fold weaker than that of the native sequence hPTH(1-34). Remarkably, the two most potent analogues, I and II, and the very weakly active analogues, VI and VII, exhibit similar helix contents. These results indicate that the presence of a stable N-terminal helical sequence is an important but not sufficient condition for biological activity.

Differential transcriptional effects of PTH and estrogen during anabolic bone formation.

The aim of this study was to compare transcriptional regulation in vivo during anabolic bone formation induced by either estradiol (E2) treatment or intermittent parathyroid hormone[1-34] (PTH) therapy. We utilized an ovariectomized (OVX) mouse model of osteoporosis and transcriptional profiling to identify genes upregulated by either high-dose E2 or PTH. Five weeks post-OVX, the mice were administered either E2 and/or PTH, or vehicle for 4 weeks. Femoral bones were analyzed by microCT and histomorphometry to confirm the anabolic effect of each treatment. OVX vehicle-treated control mice lost metaphyseal trabecular bone, with significant decrease in trabecular number, thickness, and connectivity. Both E2 and PTH treatments increased trabecular and cortical bone indices above the level of the sham operated controls, fully restoring both bone volume and bone mineral density (BMD). Moreover, PTH/E2 combination treatment led to significantly greater increase in cancellous bone and BMD than would be expected from the additive effects of the separate treatments. To determine whether PTH and E2 treatments were stimulating similar bone anabolic mechanisms, or were activating distinct signaling pathways, we compared patterns of gene expression using transcriptional profiling after either E2 or PTH treatment. After 4, 11, and 24 days of treatment, total RNA was collected from both the distal femoral metaphysis and diaphysis. Transcriptional profiling was performed using Affymetrix GeneChip probe arrays, comprised of approximately 36,000 full-length mouse genes and EST clusters from the UniGene database. Several markers of osteoblast activity, including c-fos, RANKL, PHEX, and PTHR1, were consistently upregulated by PTH in both skeletal sites. PTH treatment also increased expression of Cathespin K, consistent with the predicted increase in osteoclast activity. E2 treatment upregulated a largely distinct set of genes, including TGFbeta3, and BMP1, as well as several genes critical for cell cycle control, including Cyclin D1 and CDK inhibitor 1A. Overall, comparison of transcriptional profiles suggest that anabolic responses in bone to PTH and high-dose E2 treatment after OVX-induced osteoporosis involve largely distinct patterns of gene regulation, each resulting in restoration of bone mass.

Conformational and biological characterization of human parathyroid hormone hPTH(1-34) analogues containing beta-amino acid residues in positions 17-19.

The N-terminal 1-34 fragment of parathyroid hormone (PTH) elicits the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(18) upon biological function, we synthesized and characterized the following human (h) PTH(1-34) analogues containing beta-amino acid residues: [analogues: see text]. Biological activity and binding affinity of analogue I are one order of magnitude lower than those of the parent compound. In analogue II, both binding affinity and biological activity are partially recovered. Analogues III and V have no binding affinity and very low biological activity. Both bioactivity and binding affinity are partially recovered in analogue IV. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, 2D-nuclear magnetic resonance and molecular dynamics calculations. The results confirmed the presence in all analogues of two helical segments located at the N-terminal and C-terminal sequences. The insertion of beta-amino acid residues around position 18 does not cause appreciable conformational differences in the five analogues. The differences in biological activity and binding affinity among the five analogues cannot be related to structural differences in the membrane mimetic environment reported in this study. Our results stress the importance of the side-chain functionalities in the sequence 17-19 for biological function.

Structure-function relationship studies of bovine parathyroid hormone [bPTH(1-34)] analogues containing alpha-amino-iso-butyric acid (Aib) residues.

The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.

Structure-function studies of analogues of parathyroid hormone (PTH)-1-34 containing beta-amino acid residues in positions 11-13.

The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1-34), each containing single or double substitutions with beta-amino acid residues around the putative hinge located at position 12: I. [Nle(8,18),beta-Ala(11,12),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); II. [Nle(8,18),beta-Ala(12,13),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); III. [Nle(8,18),beta-Ala(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); IV. [Nle(8,18),beta-hLeu(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); V. [Nle(8,18),beta-Ala(12), Nal(23),Tyr(34)]bPTH-(1-34)NH(2); VI. [Nle(8,18),beta-Ala(13), Nal(23),Tyr(34)]bPTH-(1-34)NH(2) (beta-hLeu = beta-homo-leucine; beta-Ala = beta-alanine; Nal = L-2-naphthyl-alanine; Nle = norleucine). Analogues I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue II, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analogue IV, and completely restored in analogues V and VI. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the alpha-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of beta-Ala dyads at positions 11,12 and 12,13 in analogues I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analogue IV, compared to the structurally similar analogue III, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues V and VI, the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single beta-amino acid residue in positions 11, 12, or 13 in analogues III-VI does not favor a disordered structure in this portion of the sequence.

Parathyroid hormone 1 receptor: insights into structure and function.

The parathyroid hormone (PTH) and parathyroid hormone type 1 receptor (PTH1-Rc) are major players in regulating blood calcium homeostasis. PTH1-Rc is a G protein-coupled receptor (GPCR) and it is also activated by PTH-related protein (PTHrP), which has potent effects in several target tissues including bone, via endocrine, paracrine, and autocrine mechanisms in prenatal and adult stages of life, and in several diseases. Another layer of complexity is added by the recently discovered PTH2-Rc subtype displaying unique tissue distribution and ligand specificity, and the potential presence in mammals of a third receptor subtype. Understanding of the mechanisms regulating PTH1-Rc gene expression, receptor desensitization, endocytosis, recycling, and intracellular signaling is advancing and provides insights to understand PTH1-Rc's role in both normal and pathophysiology. In addition, development of an experimentally based model of the PTH-PTH1-Rc complex by combining photoaffinity crosslinking, mutagenesis, conformational analysis, and molecular modeling sheds light, at the atomic resolution, on the molecular mechanisms involved in ligand binding, receptor activation, and coupling to intracellular effectors. Taken together, these lines of investigation offer not only a better understanding of GPCR mechanisms of action in general, but also contribute to the treatment of PTH/PTHrP-PTH1-Rc-related disease states and to the rational development of novel mechanism-based drugs to treat them.

Identification of a contact domain between echistatin and the integrin alpha(v)beta(3) by photoaffinity cross-linking.

The integrin alpha(v)beta(3) is the major receptor mediating the attachment of osteoclasts to the extracellular matrix in bone and plays a critical role in bone resorption and bone remodeling. Most of the ligands interacting with the alpha(v)beta(3) receptor contain an Arg-Gly-Asp (RGD) motif. Recently, we have identified two small RGD peptides, containing a benzophenone moiety at either the carboxyl or amino terminus, that photo-cross-linked within the beta(3)[99-118] [Bitan, G., et al. (1999) Biochemistry 38, 3414-3420] or the beta(3)[167-171] [Bitan, G., et al. (2000) Biochemistry 39, 11014-11023] sequence, respectively, of the alpha(v)beta(3) receptor in a selective fashion. Here, we report the synthesis of a photoreactive analogue of echistatin (a 49-amino acid peptide), a potent RGD-containing antagonist of the alpha(v)beta(3) receptor both in vitro and in vivo. This bioactive analogue is substituted at position 45 with a p-benzoyl moiety (pBz(2)), located within the flexible C-terminal domain and removed 20 amino acid residues from the R(24)GD(26) triad. This C-terminal domain was reported to contribute to receptor binding affinity by acting as an auxiliary binding site. The radiolabeled (125)I-[Arg(35),Lys(45)(N(epsilon)-pBz(2))]-echistatin photo-cross-links effectively to a site within the beta(3)[209-220] sequence. Residues in this domain have been reported to be part of the metal ion-dependent adhesion site (MIDAS). Receptor fragments overlapping this domain were reported to bind to fibrinogen and block fibrinogen binding to alpha(IIb)beta(3), the platelet integrin receptor. Taken together, position 45 in echistatin, located within an auxiliary binding site in echistatin, cross-links to a site distinct from the two previously reported sites, beta(3)[99-118] and beta(3)[167-171], which cross-link to photophores flanking the RGD triad. These cross-linking data support the hypothesis that the ligand-bound conformation of the integrin beta(3) subunit differs from the known conformation of I domains.

Human parathyroid hormone 1-34 reverses bone loss in ovariectomized mice.

The experimental work characterizing the anabolic effect of parathyroid hormone (PTH) in bone has been performed in nonmurine ovariectomized (OVX) animals, mainly rats. A major drawback of these animal models is their inaccessibility to genetic manipulations such as gene knockout and overexpression. Therefore, this study on PTH anabolic activity was carried out in OVX mice that can be manipulated genetically in future studies. Adult Swiss-Webster mice were OVX, and after the fifth postoperative week were treated intermittently with human PTH(1-34) [hPTH(1-34)] or vehicle for 4 weeks. Femoral bones were evaluated by microcomputed tomography (microCT) followed by histomorphometry. A tight correlation was observed between trabecular density (BV/TV) determinations made by both methods. The BV/TV showed >60% loss in the distal metaphysis in 5-week and 9-week post-OVX, non-PTH-treated animals. PTH induced a approximately 35% recovery of this loss and a approximately 40% reversal of the associated decreases in trabecular number (Tb.N) and connectivity. PTH also caused a shift from single to double calcein-labeled trabecular surfaces, a significant enhancement in the mineralizing perimeter and a respective 2- and 3-fold stimulation of the mineral appositional rate (MAR) and bone formation rate (BFR). Diaphyseal endosteal cortical MAR and thickness also were increased with a high correlation between these parameters. These data show that OVX osteoporotic mice respond to PTH by increased osteoblast activity and the consequent restoration of trabecular network. The Swiss-Webster mouse model will be useful in future studies investigating molecular mechanisms involved in the pathogenesis and treatment of osteoporosis, including the mechanisms of action of known and future bone antiresorptive and anabolic agents.

Aib-rich peptides containing lactam-bridged side chains as models of the 3(10)-helix.

Aib-rich side chain lactam-bridged oligomers with n =1, 2, 3, were designed and synthesized as putative models of the 3(10)-helix. These peptides were conformationally characterized in aqueous solution containing SDS micelles by CD, NMR, and computer simulations. The lactam bridge between the side chains of L-Glu and L-Lys in (i) and (i+3) positions was introduced in order to enhance the conformational preference toward the right-handed 3(10)-helix. The NMR results clearly indicate that there is an increase of 3(10)-helix formation upon chain elongation. In the dimer and trimer (n = 2 and n = 3, respectively, in the structure reported above) the observed NOE connectivities are compatible with the 3(10)-helical arrangement, confirmed by the temperature coefficients of the amide proton resonances which suggest the presence of a hydrogen-bonded structure. The phi and psi dihedral angles of the structures obtained by molecular dynamics calculations are also compatible with the 3(10)-helix. Identification of the hydrogen-bond pattern indicate that C=O(i)- - -HN(i+3) hydrogen bonds, typical of the 3(10)-helical conformation, are highly probable in all low-energy structures. The CD spectra of these Aib-rich lactam-bridged oligopeptides, obtained in the same solvent system used for NMR experiments, provide important insight into the spectroscopic characteristics of the 3(10)-helix.

The specificity of interactions between nuclear hormone receptors and corepressors is mediated by distinct amino acid sequences within the interacting domains.

The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms interact with the nuclear corepressors [NCoR (nuclear corepressor protein) and SMRT (silencing mediator for retinoid and thyroid hormone receptors)] in the absence of ligand to silence transcription. NCoR and SMRT contain C-terminal nuclear hormone receptor (NHR) interacting domains that each contain variations of the consensus sequence I/L-x-x-I/V-I (CoRNR box). We have previously demonstrated that TRbeta1 preferentially interacts with NCoR, whereas RARalpha prefers SMRT. Here, we demonstrate that this is due, in part, to the presence of a novel NCoR interacting domain, termed N3, upstream of the previously described domains. An analogous domain is not present in SMRT. This domain is specific for TR and interacts poorly with RAR. Our data suggest that the presence of two corepressor interacting domains are necessary for full interactions with nuclear receptors in cells. Interestingly, mutation of N3 alone specifically decreases binding of NCoR to TR in cells but does not decrease NCoR-RAR interactions. In addition, while the exact CoRNR box sequence of a SMRT interacting domain is critical for recruitment of SMRT by RAR, the CoRNR box sequences themselves do not explain the strong interaction of the N2 domain with TRbeta1. Additional regions distal to the CoRNR box sequence are needed for optimal binding. Thus, through sequence differences in known interacting domains and the presence of a newly identified interacting domain, NCoR is able to preferentially bind TRbeta1. These preferences are likely to be important in corepressor action in vivo.

Mitogenic G(i) protein-MAP kinase signaling cascade in MC3T3-E1 osteogenic cells: activation by C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)] and attenuation of activation by cAMP.

In osteogenic and other cells the mitogen-activated protein (MAP) kinases have a key role in regulating proliferation and differentiated functions. The osteogenic growth peptide (OGP) is a 14 mer mitogen of osteogenic and fibroblastic cells that regulates bone turnover, fracture healing, and hematopoiesis, including the engraftment of bone marrow transplants. It is present in the serum and extracellular fluid either free or complexed to OGP-binding proteins (OGPBPs). The free immunoreactive OGP consists of the full length peptide and its C-terminal pentapeptide OGP(10-14). In the present study, designed to probe the signaling pathways triggered by OGP, we demonstrate in osteogenic MC3T3 E1 cells that mitogenic doses of OGP(10-14), but not OGP, enhance MAP kinase activity in a time-dependent manner. The OGP(10-14)-induced stimulation of both MAP kinase activity and DNA synthesis were abrogated by pertusis toxin, a G(i) protein inhibitor. These data offer direct evidence for the occurrence in osteogenic cells of a peptide-activated, mitogenic Gi protein-MAP kinase-signaling cascade. Forskolin and dBu(2)-cAMP abrogated the OGP(10-14)-stimulated proliferation, but induced only 50% inhibition of the OGP(10-14)-mediated MAP kinase activation, suggesting additional MAP kinase-dependent, OGP(10-14)-regulated, cellular functions. Finally, it is demonstrated that OGP(10-14) is the active form of OGP, apparently generated proteolytically in the extracellular milieu upon dissociation of OGP-OGPBP complexes.

Ligand-integrin alpha v beta 3 interaction determined by photoaffinity cross-linking: a challenge to the prevailing model.

Integrin alpha(V)beta(3) plays a crucial role in angiogenesis, apoptosis, and bone remodeling, mainly by interacting with matrix proteins through recognition of an Arg-Gly-Asp (RGD) motif. Recently, a small cyclic RGD-containing alpha(V)beta(3)-ligand possessing a C-terminal photoreactive group was photo-cross-linked within beta(3)[99-118], in the N-terminus of the beta(3) chain [Bitan G et al. (1999) Biochemistry 38, 3414-3420]. In this paper, a photoreactive group at the N-terminus of the RGD-ligand is shown to interact within beta(3)[167-171], approximately 60 residues C-terminal to the previously identified domain. On the basis of these findings, a model of the putative I-like domain of the beta(3) subunit, homologous to alpha(M)-, alpha(L)-, and alpha(2)-I-domains, reveals that the beta(3)[99-118] and beta(3)[167-171] contact sites are close to each other and are on the opposite side relative to the metal ion-dependent adhesion site (MIDAS) motif. These observations contradict the prevailing model that proposes proximity between metal- and RGD-binding sites on the I-like domain. Our data suggest that either the I-like domain structure predicted for beta(3) is incorrect, or there is no spatial proximity between the RGD-binding site and the MIDAS motif in the I-like domain. Our results indicate that the current models for ligand-receptor interaction should be revisited.

Structure-bioactivity of C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)].

The amino acid sequence of osteogenic growth peptide (OGP) consists of 14 residues identical to the C-terminal tail of histone H4. Native and synthetic OGP are mitogenic to osteoblastic and fibroblastic cells and enhance osteogenesis and hematopoiesis in vivo. The C-terminal truncated pentapeptide of OGP, H-Tyr-Gly-Phe-Gly-Gly-OH [OGP(10-14)], is a naturally occurring osteoblastic mitogen, equipotent to OGP. The present study assesses the role of individual amino acid residues and side chains in the OGP(10-14) mitogenic activity which showed a very high correlation between osteoblastic and fibroblastic cell cultures. Truncation of either Tyr10 or its replacement by Ala or D-Ala resulted in substantial, but not complete, loss of activity. Nevertheless, only a small loss of activity was observed following removal of the Tyr10 amino group. No further loss occurred consequent to the monoiodination of desaminoTyr10 on meta-position. However, a marked decrease in proliferative activity followed removal of the Tyr10 phenolic or the Phe12 aromatic group. Loss of activity of a similar magnitude also occurred subsequent to replacing Gly11 with L- or D-Ala. Approximately 50% loss of mitogenic activity occurred subsequent to truncation of Gly14 or blocking the C-terminal group as the methyl ester. All other modifications of the C-terminus and L- or D-Ala substitution of Gly13 resulted in 70-97% decrease in activity. Collectively, these data suggest that the integrity of the pharmacophores presented by Tyr and Phe side chains, as well as the Gly residues at the C-terminus, are important for optimal bioactivity of OGP(10-14).

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrp) chimeric peptides.

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane-embedded G protein-coupled receptor (PTH1 Rc) present on the surface of cells in target tissues such as bone and kidney. This binding occurs in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34) PTH/PTHrP hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH in terms of bioactivity. The sites of incompatibility were identified at positions 5 in PTHrP and 19 in PTH. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two segmental hybrids: PTHrP(1-27)-[Tyr(34)]bPTH(28-34)-NH(2) (hybrid I) and PTHrP(1-18)-[Nal(23), Tyr(34)]bPTH(19-34)-NH(2) (hybrid II). Hybrid I is as active as PTH(1-34)NH(2) and more than two orders of magnitude more active than hybrid II. The conformational properties of the hybrids were studied in water/trifluoroethanol (TFE) mixtures and in aqueous solutions containing dodecylphosphocholine (DPC) micelles by CD, two-dimensional nmr and computer simulations. Upon addition of TFE to the aqueous solution, both hybrids undergo a coil-helix transition. The helix content in 1:1 water/TFE obtained by CD data is about 75% for both hybrids. In the presence of DPC, helix formation is observed at detergent concentrations above critical micellar concentration and the maximum helix content is of approximately 35 and approximately 30% for hybrid I and II, respectively. Combined nmr analysis, distance geometry, and molecular dynamics calculations suggest that, in both solvent systems, the biologically active hybrid I exhibits two flexible sites, centered at residues 12 and 19, connecting helical segments. The flexibility point at position 19 is not present in the poorly active hybrid II. Our findings support the hypothesis, proposed in our previous work, that in bioactive PTH analogues the presence and location of flexibility points between helical segments are essential for enabling them to fold into the bioactive conformation upon interaction with the PTH1 receptor.

Mapping the bimolecular interface of the parathyroid hormone (PTH)-PTH1 receptor complex: spatial proximity between Lys(27) (of the hormone principal binding domain) and leu(261) (of the first extracellular loop) of the human PTH1 receptor.

In an effort to characterize the bimolecular interface between parathyroid hormone (PTH) and its human receptor PTH1-Rc (hPTH1-Rc), we previously identified two contact sites in the receptor: one for position 1 and another for position 13 (located at the ends of the principal activation domain) in PTH(1-34). The present study reports a third, novel "contact site" between hPTH1-Rc and Lys(27) of PTH(1-34). Lys(27) is located in the principal binding domain of the hormone (residues 25-34). The photoreactive PTH(1-34) analogue K27 contains a benzophenone (BP) moiety on Lys(27). The analogue binds to stably transfected HEK 293/C-21 cells (which express a high level of recombinant hPTH1-Rc) and stimulates adenylyl cyclase activity with a potency similar to PTH(1-34). In addition, (125)I-K27 cross-links effectively and specifically to the hPTH1-Rc. Enzymatic (Glu-C and Lys-C) and chemical (CNBr and BNPS-skatole) digestions of the photoconjugate between (125)I-K27 and hPTH1-Rc were performed. In addition, photoconjugates involving the bioactive mutants [L261M]- and [R262K]-hPTH1-Rc, transiently expressed in COS-7 cells, were also digested. The data obtained clearly identify L(261) or R(262) of the first extracellular loop of hPTH1-Rc as the contact site for Lys(27) in the hormone. On the basis of (i) the similarity in molecular mass between the CNBr digest of the (125)I-K27-[L261M]hPTH1-Rc conjugate and free (125)I-K27 and (ii) the failure to cross-link (125)I-K27 to a bioactive mutant receptor [L261A]hPTH1-Rc, we conclude that L(261) is the cross-linking site. These results provide the first demonstration of an interaction between the principal binding domain of PTH and the first extracellular loop of hPTH1-Rc. Revealing proximity of Lys(27) (in PTH) to L(261) (in hPTH1-Rc) provides additional insight into the nature of the ligand-receptor bimolecular interface and clearly illustrates that the extracellular loops of the receptor contribute to the specificity of the PTH-PTH1-Rc interaction. Taken together with previous studies, the new findings add important constraints on the possible positioning of the C-terminal helix of PTH (which contains the principal binding domain) relative to the first extracellular loop and the distal C-terminal helix of the large extracellular amino terminal domain of the PTH1-Rc.

Characterization of parathyroid hormone/receptor interactions: structure of the first extracellular loop.

The structural features of the first extracellular loop (ECL1) of the parathyroid hormone receptor (PTH1R) in the presence of dodecylphosphocholine micelles have been determined using high-resolution NMR techniques. The structure of the receptor fragment, PTH1R(241-285), includes three alpha-helices for residues 241-244, 256-264, and 275-284. The first and third correspond to the end and the beginning of transmembrane helices 2 and 3, respectively. Centrally located in the second helix is L(261), found to cross-link to Lys(27) of parathyroid hormone, PTH(1-34) [Greenberg, Z., Bisello, A., Mierke, D. F., Rosenblatt, M., and Chorev, M. (2000) Biochemistry 39, 8142-8152]. On the basis of nitroxide radical-induced relaxation studies, the central helix is found to associate with the surface of the membrane mimetic. These data, in conjunction with previous results indicating a preference of PTH for the lipid surface, suggest a membrane-associated pathway for the initial recognition and binding of PTH to its G-protein-coupled receptor. Using the structural features of ECL1 as determined here, along with the structure of the PTH(1-34), the intermolecular interactions consistent with the contact point between L(261)(receptor)-Lys(27)(ligand) are identified.

Design and evaluation of benzophenone-containing conformationally constrained ligands as tools for photoaffinity scanning of the integrin alphaVbeta3-ligand bimolecular interaction.

Integrins are cell-surface adhesion molecules involved in mediating cell-extracellular matrix interactions. High-resolution structural data are not available for these heterodimeric receptors. In order to generate tools for photoaffinity scanning of the RGD-binding site of human integrin alphaVbeta3. new conformationally constrained ligands were designed. The ligands were based on five different cyclic peptidic or peptidomimetic scaffolds with high affinity for alphaVbeta3. A single photoreactive group (a benzophenone moiety) was introduced at different positions relative to the RGD triad. In addition, an 125I or a biotin group was introduced as a reporting tag. Twenty-four cyclic ligands were prepared and their binding affinity for alphaVbeta3 was determined. In most cases, the modifications resulted in a 5- to 500-fold decrease in affinity relative to the unmodified scaffold. Analogs representing three of the five families were screened for their cross-linking efficiency. Ligands with submicromolar affinities cross-linked efficiently and specifically to the integrin receptor, whereas ligands with weaker affinities gave specific cross-linking, but with lower efficiency. Almost all of the screened ligands cross-linked predominantly to the beta3 subunit.