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.

A role for N-cadherin in the development of the differentiated osteoblastic phenotype.

Cadherins are a family of cell surface adhesion molecules that play an important role in tissue differentiation. A limited repertoire of cadherins has been identified in osteoblasts, and the role of these molecules in osteoblast function remains to be elucidated. We recently cloned an osteoblast-derived N-cadherin gene from a rat osteoblast complementary DNA library. After in situ hybridization of rat bone and immunohistochemistry of human osteophytes, N-cadherin expression was localized prominently in well-differentiated (lining) osteoblasts. Northern blot hybridization in primary cultures of fetal rat calvaria and in human SaOS-2 and rat ROS osteoblast-like cells showed a relationship between N-cadherin messenger RNA expression and cell-to-cell adhesion, morphological differentiation, and alkaline phosphatase and osteocalcin gene expression. Treatment with a synthetic peptide containing the His-Ala-Val (HAV) adhesion motif of N-cadherin significantly decreased bone nodule formation in primary cultures of fetal rat calvaria and inhibited cell-to-cell contact in rat osteoblastic TRAB-11 cells. HAV peptide also regulated the expression of specific genes such as alkaline phosphatase and the immediate early gene zif268 in SaOS-2 cells. Transient transfection of SaOS-2 cells with a dominant-negative N-cadherin mutant (NCADdeltaC) significantly inhibited their morphological differentiation. In addition, aggregation of NCTC cells derived from mouse connective tissue stably transfected with osteoblast-derived N-cadherin was inhibited by either treatment with HAV or transfection with NCADdeltaC. Together, these results strongly support a role for N-cadherin, in concert with other previously identified osteoblast cadherins, in the late stages of osteoblast differentiation.

Photoaffinity cross-linking identifies differences in the interactions of an agonist and an antagonist with the parathyroid hormone/parathyroid hormone-related protein receptor.

Analogs of parathyroid hormone (PTH)-related protein (PTHrP), singularly substituted with a photoreactive L-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in human embryonic kidney cells stably expressing the recombinant human PTH/PTHrP receptor. Two of these analogs, in which the photoreactive residue is either in position 1 or 2 (Bpa(1)- and Bpa(2)-PTHrP, respectively) displayed high affinity binding. Bpa(1)-PTHrP also displayed high efficacy for the stimulation of increased cAMP levels. Surprisingly, Bpa(2)-PTHrP was found to be a potent antagonist, despite the presence of the principal activation domain (sequence 1-6). Analysis of the digestion profiles of the ligand-receptor photoconjugates revealed that both the agonist and the antagonist cross-link to the S-CH(3) group of Met(425) in transmembrane domain 6 of the human PTH/PTHrP receptor. However, the antagonist Bpa(2)-PTHrP also cross-links to a proximal site within the receptor domain Pro(415)-Met(425). Unlike the antagonist Bpa(2)-PTHrP, the potent agonist Bpa(2)-PTH, also bearing the Bpa residue in position 2, cross-links only to the S-CH(3) group of Met(425) (similar to Bpa(1)-PTHrP and Bpa(1)-PTH). Taken together, these results suggest that the antagonist Bpa(2)-PTHrP is able to distinguish between two distinct conformations of the receptor. The comparison between PTHrP analogs substituted by Bpa at two consecutive positions and across PTH and PTHrP reveals insights into the PTH/PTHrP ligand-receptor bimolecular interaction at the level of a single amino acid.

Endocytosis of ligand-human parathyroid hormone receptor 1 complexes is protein kinase C-dependent and involves beta-arrestin2. Real-time monitoring by fluorescence microscopy.

Endocytosis and intracellular trafficking of the human parathyroid hormone receptor subtype 1 (hPTH1-Rc) and its ligands was monitored independently by real-time fluorescence microscopy in stably transfected HEK-293 cells. Complexes of fluorescence-labeled parathyroid hormone (PTH)-(1-34) agonist bound to the hPTH1-Rc internalized rapidly at 37 degrees C via clathrin-coated vesicles, whereas fluorescent PTH-(7-34) antagonist-hPTH1Rc complexes did not. A functional C terminus epitope-tagged receptor (C-Tag-hPTH1-Rc) was immunolocalized to the cell membrane and, to a lesser extent, the cytoplasm. PTH and PTH-related protein agonists stimulated C-Tag-hPTH1-Rc internalization. Relocalization to the cell membrane occurred 1 h after removal of the ligand. Endocytosis of fluorescent PTH agonist-hPTH1-Rc complexes was blocked by the protein kinase C (PKC) inhibitor staurosporine but not by the specific protein kinase A inhibitor N-(2-(methylamino)ethyl)-5-isoquinoline-sulfonamide. Fluorescent PTH antagonist-hPTH1-Rc complexes were rapidly internalized after PKC activation by phorbol 12-myristate 13-acetate or thrombin, but not after stimulation of the cAMP/protein kinase A pathway by forskolin. In cells co-expressing the hPTH1-Rc and a green fluorescent protein-beta-arrestin2 fusion protein (beta-Arr2-GFP), PTH agonists stimulated beta-Arr2-GFP mobilization to the cell membrane. Subsequently, fluorescent PTH-(1-34)-hPTH1Rc complexes and beta-Arr2-GFP co-localized intracellularly. In conclusion, agonist-activated hPTH1-Rc internalization involves beta-arrestin mobilization and targeting to clathrin-coated vesicles. Our results also indicate that receptor occupancy, rather than receptor-mediated signaling, is necessary, although not sufficient, for endocytosis of the hPTH1-Rc. Activation of PKC, however, is absolutely required.

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrP) point-mutated hybrids.

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34)PTH/PTHrP segmental hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH leading to substantial reduction in potency. The sites of incompatibility were identified as positions 5 in PTH and 19 in PTHrP. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two point-mutated PTH/PTHrP 1-34 hybrids in which the arginine residues at positions 19 and 21 of the native sequence of PTHrP have been replaced by valine (hybrid V(21)) and glutamic acid (hybrid E(19)), respectively, taken from the PTH sequence. Hybrid V(21) exhibits both high receptor affinity and biological potency, while hybrid E(19) binds weakly and is poorly active. The conformational properties of the two hybrids were studied in aqueous solution containing dodecylphosphocholine (DPC) micelles and in water/2,2, 2-trifluoroethanol (TFE) mixtures. Upon addition of TFE or DPC micelles to the aqueous solution, both hybrids undergo a coil-helix transition. The maximum helix content in 1 : 1 water/TFE, obtained by CD data for both hybrids, is approximately 80%. In the presence of DPC micelles, the maximum helix content is approximately 40%. The conformational properties of the two hybrids in the micellar system were further investigated by combined 2D-nmr, distance geometry (DG), and molecular dynamics (MD) calculations. The common structural motif, consisting of two helical segments located at N- and C-termini, was observed in both hybrids. However, the biologically potent hybrid V(21) exhibits two flexible sites, centered at residues 12 and 19 and connecting helical segments, while the flexibility sites in the weakly active hybrid E(19) are located at position 11 and in the sequence 20-26. Our findings support the hypothesis that the presence and location of flexibility points between helical segments are essential for enabling the active analogs to fold into the bioactive conformation upon interaction with the receptor.

Development of a photoreactive parathyroid hormone antagonist to probe antagonist-receptor bimolecular interaction.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert their calciotropic activities by binding to a specific seven-transmembrane-helix-containing G protein-coupled receptor mainly located in bone and kidney cells. In order to map in detail the nature of hormone-receptor interaction, we are employing 'photoaffinity scanning' of the bimolecular interface. To this end, we have developed photoreactive benzophenone (BP)-containing PTH analogs which can be specifically and efficiently cross-linked to the human (h) PTH/PTHrP receptor. In this report, we describe the photocross-linking of a BP-containing PTH antagonist, [Nle8,18,D-2-Nal12,Lys13(epsilon-BP),2-Nal23,Tyr34]bPT H(7-34)NH2 (ANT) to the recombinant hPTH/PTHrP receptor stably expressed in human embryonic kidney cells (HEK-293, clone C-21). This photoreactive antagonist has high affinity for the hPTH/PTHrP receptor and inhibits agonist-induced cyclase activity and intracellular calcium release. The photo-induced cross-linking of the radioiodinated antagonist (125I-ANT) to the recombinant hPTH/PTHrP receptor followed by SDS-PAGE analysis reveals a single radiolabeled band of approximately 85kDa, similar to that observed after cross-linking of a radioiodinated BP-containing agonist. The formation of this covalent 125I-ANT - hPTH/PTHrP receptor conjugate is competed dose-dependently by a variety of unlabelled PTH- and PTHrP-derived agonists and antagonists. This is the first report of a specific and efficient photocross-linking of a radioiodinated PTH antagonist to the hPTH/PTHrP receptor. Therefore, it provides the opportunity to study directly the nature of the bimolecular interaction of PTH antagonist with the hPTH/PTHrP receptor.

Direct identification of two contact sites for parathyroid hormone (PTH) in the novel PTH-2 receptor using photoaffinity cross-linking.

Direct examination of the interacting sites between PTH and the human PTH2 receptor (PTH2R) was conducted by photoaffinity cross-linking followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. Photoreactive analogs of PTH, individually substituted with an L-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in cells stably expressing recombinant PTH2R. One highly bioactive analog, [Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]PTH-(1-34)NH 2 (Bpa1-PTH), was chosen for cross-linking studies. In addition, a PTH analog in which the photoreacive moiety is at the mid-region position 13 (K13) was demonstrated to be bioactive, then cross-linked to PTH2R. The minimal digestion-restricted domain containing the contact site ("contact domain") for 125I-Bpa1-PTH is in the sixth transmembrane domain and part of the third extracellular loop, spanning residues Ser364-Met395 of the receptor. This domain was further confirmed and refined by cross-linking 125I-Bpa1-PTH to two receptor mutants, PTH2R[V380M]- and PTH2R[V380M,M395L]-receptors. Treatment of the cross-linked conjugates with cyanogen bromide identified a single amino acid (position 380) as the putative contact point. The contact domain for 125I-K13 is located in the N-terminal extracellular tail of the receptor (in the C-terminal portion) and spans Gln138-Met147. Further validation of this contact domain was accomplished by photocross-linking to point-mutated PTH2R[K137R] receptor. Previous studies in which PTH analogs were cross-linked to human PTH/PTHrP receptor (PTH1R) identified Met425 and Phe173-Met189 as the contact sites for Bpa1-PTH and K13, respectively. These studies demonstrate that both receptor subtypes, PTH1- and PTH2-receptors, use analogous sites for interaction with positions 1 and 13 in PTH.

Lossy (15:1) JPEG compression of digital coronary angiograms does not limit detection of subtle morphological features.

BACKGROUND: Development of the "all-digital" cardiac catheterization laboratory has been slowed by substantial computer archival and transfer requirements. Lossy data compression reduces this burden but creates irreversible changes in images, potentially impairing detection of clinically important angiographic features. METHODS AND RESULTS: Fifty image sequences from 31 interventional procedures were viewed both in the original (uncompressed) state and after 15:1 lossy Joint Photographic Expert's Group (JPEG) compression. Experienced angiographers identified dissections, suspected thrombi, and coronary stents, and their results were compared with those from a consensus panel that served as a "gold standard." The panel and the individual observers reviewed the same image sequences 4 months after the first session to determine intraobserver variability. Intraobserver agreement for original images was not significantly different from that for compressed images (89.8% versus 89.5% for 600 pairs of observations in each group). Agreement of individual observers with the consensus panel was not significantly different for original images from that for compressed images (87.6% versus 87.3%; CIs for the difference, -4.0%, 4.0%). Subgroup analysis for each observer and for each detection task (dissection, suspected thrombus, and stent) revealed no significant difference in agreement. CONCLUSIONS: The identification of dissections, thrombi, and coronary stents is not substantially impaired by the application of 15:1 lossy JPEG compression to digital coronary angiograms. These data suggest that digital angiographic images compressed in this manner are acceptable for clinical decision-making.

Serial prognostic capabilities of electrocardiographic indices of infarcted and hibernating myocardium in predicting short- and long-term outcome following coronary artery bypass surgery.

The present study was performed to test the hypothesis that patients with a large amount of ischemic (hibernating) myocardium are most likely to develop a perioperative myocardial infarction undergoing coronary artery bypass grafting (CABG). Furthermore, we evaluated the Selvester QRS scoring system as a postoperative prognostic tool. A relationship between a high amount of hibernating myocardium determined by ventriculographic and electrocardiographic investigations and an increased risk of perioperative myocardial infarction was found. The Selvester QRS scoring system used in diagnosing and prognosing after acute myocardial infarction was proven valid in predicting prognosis after CABG as well.

Role of glycosylation in expression and function of the human parathyroid hormone/parathyroid hormone-related protein receptor.

Parathyroid hormone (PTH) regulates calcium metabolism through a specific G protein-coupled, seven-transmembrane helix-containing receptor. This receptor also binds and is activated by PTH-related protein (PTHrP). The human (h) PTH/PTHrP receptor is a membrane glycoprotein with an apparent molecular weight of approximately 85000 which contains four putative N-glycosylation sites. To elucidate the functional role of receptor glycosylation, if any, we studied hormone binding and signal transduction in human embryonic kidney cells transfected with hPTH/PTHrP receptor (HEK-293/C-21). These cells stably express 300000-400000 receptors per cell. Inhibition of N-glycosylation with an optimized concentration of tunicamycin yielded completely nonglycosylated hPTH/PTHrP receptor (approximately 60 kDa). This receptor form is fully functional; it maintains nanomolar binding affinity for PTH- and PTHrP-derived agonists and antagonists. PTH and PTHrP agonists stimulate cyclic AMP accumulation and increases in cytosolic calcium levels. In addition, the highly potent benzophenone (pBz2)-containing PTH-derived radioligand [Nle8,18,Lys13(epsilon-pBz2),L-2-Nal23,Tyr34 3-125I)]bPTH(1-34)NH2 can photoaffinity cross-link specifically to the nonglycosylated receptor. The molecular weight (approximately 60000) of the band representing the photo-cross-linked, nonglycosylated receptor (obtained from the tunicamycin-treated HEK-293/C-21 cells) was similar to that of the deglycosylated photo-cross-linked receptor (obtained by enzymatic treatment with Endoglycosidase-F/N-glycosidase-F). Our findings indicate that glycosylation of the hPTH/PTHrP receptor is not essential for its effective expression on the plasma membrane or for the binding of ligands known to interact with the native receptor. The nonglycosylated hPTH/PTHrP receptor remains fully functional with regard to both of its known signal transduction pathways: cAMP-protein kinase A and phospholipase C-cytosolic calcium.

Histidine at position 5 is the specificity "switch" between two parathyroid hormone receptor subtypes.

The PTH and PTH-related protein (PTHrP) system consists of two hormones, at least two G protein-coupled seven-transmembrane domain receptors, and at least two intracellular signal transduction pathways for each receptor. The PTH/PTHrP receptor is present in the conventional target tissues of PTH action, namely kidney and bone. Both PTH and PTHrP bind to and activate the PTH/PTHrP receptor with equal affinity and efficacy. The newly discovered receptor, termed the human (h) PTH2 receptor, has 70% homology with the PTH/PTHrP receptor, but is found predominantly in brain and pancreas. It interacts selectively with PTH and not with PTHrP. PTH and PTHrP differ in several positions, including position 5 (Ile in PTH; His in PTHrP). To define the role of position 5 in receptor selectivity, we designed and synthesized a series of hybrid analogs containing specific elements of both the PTH and PTHrP sequences. Using human cell lines stably expressing either human receptor subtype, we evaluated the biological profile of the hybrids in assays of receptor binding and action. Both point-mutated hybrids, [Ile5]PTH-(1-34) and [His5]PTH-(1-34), bind to and stimulate cAMP accumulation and the release of cytosolic free calcium in HEK293/C-21, a clonal human embryonic kidney cell line stably expressing the recombinant hPTH/PTHrP receptor. However, only [Ile5]PTHrP-(1-34), and not [His5]PTH-(1-34), binds to and stimulates cAMP accumulation and the release of cytosolic free calcium in HEK293/BP-16, a clonal human embryonic kidney cell line stably expressing the recombinant hPTH2 receptor. The segmental hybrid PTHrP-(1-14)-PTH-(15-34) binds to and activates the hPTH/PTHrP receptor, but not the hPTH2 receptor, similar to the biological profile of His5-containing ligands: PTHrP-(1-34) and [His5]PTH-(1-34). Exchanging Ile5 for His5 in the segmental hybrid produces the analog [Ile5]PTHrP-(1-14)-PTH-(15-34), which interacts with both receptor subtypes. We conclude that His5 in PTHrP is the major structural determinant of receptor subtype specificity in the hPTH/PTHrP and hPTH2 two-receptor system. The mechanism of the specificity "switch" remains to be elucidated, but may result from a subtle perturbation of the bioactive conformation and/or from a direct steric hindrance at the hPTH2 receptor-ligand interface created by histidine at position 5. The hPTH2, but not the hPTH/PTHrP, receptor can discriminate between the two hormones based on the structural differences generated at position 5.

Evaluation in vivo of a potent parathyroid hormone antagonist: [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2.

In an effort to design and select potent parathyroid hormone (PTH) antagonists suitable for clinical utility, a PTH analog was evaluated in vivo in an animal model to assess its properties in preparation for human studies. The previously described PTH antagonist, [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2, which is highly active in vitro, was documented in these studies to be an effective antagonist of the PTH-stimulated calcemic response in vivo. In thyroparathyroidectomized (TPTX) rats, the efficacy of the antagonist was demonstrated to be dose-dependent. Inhibition was demonstrated when intravenous administration of antagonist started 1 h prior to coinfusion with the PTH agonist [Nle8,18,Tyr34]bPTH(1-34)NH2. Maximal inhibition by antagonist (an 84% decline in serum calcium levels compared with agonist alone) of the calcemic response was observed when a 200-fold molar excess of antagonist (12 nmol/h) was administered. At dose ratios of antagonist:agonist as low as 10:1, a 40-50% inhibition of PTH-stimulated calcemic response is evident, provided a longer (2 h) lead time for antagonist infusion is allowed. Based on these and related studies, the antagonist [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2 has displayed sufficient potency to obtain approval from the appropriate institutional and regulatory agencies for clinical trials in hypercalcemic states of parathyroid and tumor origin.

The human PTH2 receptor: binding and signal transduction properties of the stably expressed recombinant receptor.

We have generated a series of stably transfected HEK-293 cell lines expressing the newly identified alternate human PTH receptor (hPTH2 receptor). This receptor subtype is selectively activated by N-terminal PTH-(1-34) and not the corresponding N-terminal (1-34) region of the functionally and structurally related hormone, PTH-related protein (PTHrP). A total of 20 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed. None responded to PTHrP-(1-34). One of these clones (BP-16), displaying maximal PTH responsiveness, was chosen for more detailed evaluation. The BP-16 clone (and the parental HEK-293 cell line lacking both the hPTH/PTHrP receptor and the hPTH2 receptor) were examined for PTH binding, PTH-stimulated cAMP accumulation, PTH-stimulated changes in intracellular calcium ([Ca2+]i) levels, and hPTH2 receptor messenger RNA expression. In addition, we studied the photomediated cross-linking of a potent PTH agonist, namely [Nle8,18,Lys13 (epsilon-pBz2), 2-L-Nal23,Tyr34]bPTH(1-34)NH2 (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an approximately 90-kDa cell membrane component that was specifically competed by PTH-(1-34) and other receptor-interacting ligands. PTH-(1-34) and K13 are potent stimulators of both cAMP accumulation and increases in (Ca2+]i levels, and both bind to the hPTH2 receptor with high affinity (apparent Kd, 2.8 +/- 0.9 x 10(-8) and 8.5 +/- 1.7 x 10(-8) M, respectively). There was no apparent binding, cAMP-stimulating activity, or [Ca 2+]i signaling observed, nor was specific competition vs. binding of a PTH-(1-34) radioligand ([125I]PTH) with PTHrP-(1-34)NH2 found. PTHrP-(1-34) failed to inhibit cross-linking of the hPTH2 receptor by radiolabeled K13 ([125I]K13). However, effective competition vs. [125I]PTH and [125I]K13 binding and [125I]K13 cross-linking were observed with the potent PTH/PTHrP receptor antagonists, PTHrP-(7-34)NH2 and PTH-(7-34)NH2. PTHrP-(7-34)NH2 was shown to be a partial agonist that weakly stimulates both cAMP accumulation and increases in [Ca 2+]i levels in BP-16 cells. These data suggest that the hPTH2 receptor is distinct from the hPTH/PTHrP receptor in the structural features it requires for ligand binding in the family of PTH and PTHrP peptides.

Inositol 1-,4-,5-trisphosphate-dependent Ca2+ signaling by the recombinant human PTH/PTHrP receptor stably expressed in a human kidney cell line.

We previously reported the preparation and partial characterization of a series of human embryonic kidney cell lines (HEK-293) stably expressing various numbers of the recombinant human (h) parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (Rc). Using this expression system we examined ligand (PTH or PTHrP) binding characteristics and cyclic AMP responsiveness. We have now extended these studies to investigate the calcium signal transduction pathways activated by the hPTH/PTHrP Rc. In parental HEK-293 cells, which lack endogenous PTH/PTHrP Rc, incubation with hPTH(1-34) had no effect on cytosolic free Ca2+ concentration [Ca2+]i. In HEK-293 clone C-21, stably expressing approximately 400,000 Rc/cell, PTH stimulated an increase in [Ca2+]i by Ca2+ release from intracellular stores; PTH released Ca2+ exclusively from the IP3 sensitive Ca2+ pool. Unlike previous studies, the ability of PTH to elicit both cAMP responses and [Ca2+]i transients occurred over a wide range of Rc numbers (between 400,000 and 3000 Rc/cell); both responses were always observed at PTH concentrations in the same dose range although the magnitude of the responses decrease with Rc number. Pretreatment of C-21 cells with pertussis toxin for 24 h, which significantly enhanced PTH-stimulated cAMP accumulation, did not modulate PTH-stimulated [Ca2+]i transients. At each PTH concentration tested which resulted in increased cAMP levels, there was also an increase in [Ca2+]i transients. Treatment of C-21 cells with a battery of midregion and C-terminal PTH or PTHrP peptides showed no effect on either [Ca2+]i transients or cAMP accumulation, indicating a lack of functional interactions between these peptides and the form of the hPTH/PTHrP Rc stably expressed in these cells. Immunological analysis of G-protein expression demonstrated the presence of Gs, Gi, and Gq in all parental and transfected cells lines examined. Taken together, these data demonstrate that the hPTH/PTHrP Rc, stably expressed in HEK-293 cells, elicits responses in both the cAMP and IP3-dependent [Ca2+]i pathways and is responsive only to N-terminal PTH/PTHrP peptides.

Conformation and interactions of bombolitin I analogues with SDS micelles and phospholipid vesicles: CD, fluorescence, two-dimensional NMR and computer simulations.

Bombolitins are five structurally related heptadecapeptides acting at the membrane level able to lyse erythrocytes and liposomes and to enhance the activity of phospholipase A2 (PLA2). In the presence of SDS or phospholipid vesicles bombolitins are able to form amphiphilic alpha-helical structures and this property seems to be the major determinant of bioactivity. In order to test the model of interaction between bombolitin I and membranes, an analogue was synthesized in which all the lysines were replaced by arginines: Ile-Arg-Ile-Thr-Thr-Met-Leu-Ala-Arg-Ile-Gly-Arg-Val-Leu-Ala-His-Val-NH2 ([Arg2,9,12, Ile10]bombolitin I). The design of this sequence allowed the synthesis of a second analogue through a specific postsynthetic dansylation at the epsilon-amino group of a lysine residue replacing the original leucine residue at position 7. The first analogue was fully characterized by CD and two-dimensional nmr in the presence of SDS or phospholipid vesicles. The peptide folds into an amphiphilic alpha-helical conformation with the helical segment spanning the central part of the sequence from Ile3 to His16. This behavior is identical to that observed for the native sequence. The replacement of lysine residues by arginine has no detectable effect on the conformational preference of the peptide chain. By CD and fluorescence spectroscopy measurements, the fluorophore-containing analogue [Arg2,9,12, Lys7 (epsilon-dansyl)] bombolitin I also folded into the alpha-helical conformation in the presence of SDS micelles or phospholipid vesicles. In particular, the dansyl fluorophore, which is located approximately in the middle of the apolar surface of the amphiphilic helix, is clearly buried in a hydrophobic environment when the peptide is bound to phospholipid vesicles. These findings support the hypothesis that the peptide helices are oriented parallel to the vesicle surface.

Conformation of parathyroid hormone antagonists by CD, NMR, and molecular dynamics simulations.

The conformation of two highly potent parathyroid hormone (PTH) antagonists was investigated in water/2,2,2-trifluoroethanol mixtures. The two peptides are derived from the sequence (7-34) of PTH and of PTH-related protein (PTHrP) and have a D-Trp replacing Gly in position 12. In the analogue derived from PTHrP, Lys11 was replaced by Leu to remove the residual agonist activity. The study was conducted by CD and two-dimensional proton magnetic resonance spectroscopy, and the nuclear Overhauser effects found were utilized in restrained distance geometry and molecular dynamics simulations. Both peptides adopt a helical C-terminal conformation, which seems more stable in the case of the PTHrP analogue. A type II' beta-turn centered around D-Trp12 and Lys13 is present in both structures.

Probing the bimolecular interactions of parathyroid hormone with the human parathyroid hormone/parathyroid hormone-related protein receptor. 1. Design, synthesis and characterization of photoreactive benzophenone-containing analogs of parathyroid hormone.

Parathyroid hormone (PTH) regulates calcium and phosphate metabolism through a G-protein-coupled receptor which is shared with PTH-related protein (PTHrP). Therefore, structure-activity studies of PTH and PTHrP with their common receptor provide an unusual opportunity to examine the structural elements in the two hormones and their common receptor which are involved in the expression of biological activity. Our approach to studying the nature of the bimolecular interface between hormone and receptor is to use a series of specially designed photoreactive benzophenone- (BP-) containing PTH analogs in "photoaffinity scanning" of the PTH/PTHrP receptor. In this report we describe a series of BP-containing agonists and antagonists which have been synthesized by solid-phase methodology and characterized physiocochemically and biologically. Each of the 12 analogs contains a single BP moiety at a different defined position. Examples of BP-containing agonists prepared and studied in human osteogenic sarcoma Saos-2/B-10 cells are [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13)(Kb = 13 nM; Km = 2.7 nM) and [Nle8,18,L-Bpa23,Tyr34[bPTH(1-34)NH2(L-Bpa23) (Kb = 42 nM; Km = 8.5 nM). Another BP-containing analog, [Nle8,18,D-2-Nal12,Lys13(epsilon-pBZ2),L-2-Nal23 ,Tyr34]bPTH(7-34)NH2, was a potent antagonist (Kb = 95 nM; Ki = 72 nM). The amino acids substituted by residues carrying the BP moiety span the biologically active domain of the hormone (Phe7, Gly12, Lys13, Trp23, and Lys26). Analysis of photo-cross-linked conjugates of PTH/PTHrP receptor with BP-containing PTH analogs should help to identify the "contact points" between ligand and receptor.

Probing the bimolecular interactions of parathyroid hormone and the human parathyroid hormone/parathyroid hormone-related protein receptor. 2. Cloning, characterization, and photoaffinity labeling of the recombinant human receptor.

Parathyroid hormone (PTH) acts to regulate calcium homeostasis by interacting with a G-protein-coupled receptor that also binds PTH-related protein (PTHrP). In this report we describe the cloning, characterization, and biological activity of the cloned human (h) PTH/PTHrP receptor (Rc) and cross-linking of a benzophenone-substituted PTH analog, [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13), to cells endogenously expressing the Rc and cells transiently or stably transfected with the human Rc. A full-length cDNA clone was isolated and fully sequenced from a human kidney cDNA library. Northern blot analysis of normal human tissues revealed a limited tissue distribution: a single transcript of approximately 2.3 kb was detected in kidney, lung, placenta, and liver. In human embryonic kidney cells (HEK-293, clone C-21) stably transfected with hPTH/PTHrP Rc, a single 85-90 kDa Rc-hormone complex was formed after photolysis in the presence of K13. This covalent cross-linking reaction was specifically inhibited by excess quantities of biologically active 1-34 analogs of bovine (b) PTH or hPTHrP but not by C-terminal and midregion PTH peptides. Photoincorporation of 125I-labeled K13 into the Rc occurred with high efficiency (60-70%), approximately an order of magnitude greater than that achieved with conventional aryl azide cross-linking reagents. These results support the feasibility of our approach for specifically cross-linking a tagged PTH analog to the Rc, as a first step in the effort to identify directly the amino acid residues that constitute the Rc binding site.