Opere di Dan Simmons; Canti di Hyperion: Hyperion · La caduta di Hyperion · Endymion · Il risveglio di Endymion · Gli orfani di Helix: Ilium: Ilium. helix binding to both the N-terminal segment and also to the looped-out . Adenylyl Cyclase Activity—AC activities were measured using a di- .. Oldenburg, K. R., Epand, R. F., D’Orfani, A., Vo, K., Selick, H., and Epand. This C-terminal α-helix, extending approximately from Ser17 to Val31 (5), Oldenburg, K. R., Epand, R. F., D’Orfani, A., Vo, K., Selick, H., and Epand, R. M. ( ) J. Biol. Dong, M., Pinon, D. I., and Miller, L. J. () Mol.
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We explored these hypotheses by introducing a variety of substitutions in region 17—31 of PTH- 1—31 and assessing, via competition assays, their effects on binding to the wild-type PTHR and to PTHR-delNt, which lacks most of the N domain. Glu substitutions at Arg 25Lys 26and Lys 27 which forms the hydrophilic face of the helix caused 4—fold reductions in affinity for both receptors. The data help define the roles that side chains in the binding domain of PTH play in the PTH-PTHR interaction process and provide new clues for understanding the overall topology of the bimolecular complex.
Parathyroid hormone PTH 2 plays a key role in calcium and phosphate homeostasis and has potent effects on the bone-remodeling process. PTH interacts with a Class 2 G protein-coupled receptor that is prominently expressed in bone osteoblasts and in cells located in the proximal and distal portions of the renal convoluted tubules.
Within region 1—34, the principal determinants of receptor-binding affinity and receptor-signaling activity map to the C- and N-terminal domains, respectively 23. The mechanism by which PTH interacts with its receptor has been investigated via the approaches of ligand analog design, receptor mutagenesis, and photochemical cross-linking reviewed in Ref.
The view that has emerged from these studies is that the overall mechanism consists of two principal and, to some extent, autonomous components: The N domain component of the interaction is thought to provide the major portion of binding energy and stability to the complex, and the J domain component is thought to mediate the conformational changes involved in receptor activation It now seems likely that most, if not all, of the 15 or so other Class 2 G protein-coupled receptors utilize a similar two-site binding mechanism for interacting with their cognate peptide ligands 14 — The potency of such N-terminal PTH fragments can be significantly enhanced by introducing substitutions that improve the affinity of the ligand for the PTHR J domain 18 — The cross-linking approach has indeed established spatial proximities between PTH residues 23, 27, and 28, when substituted with para -benzoyl- l -phenylalanine Bpaand the N domain of the receptor 21 This observation raised the possibility that the C-terminal binding domain of PTH can functionally interact with the receptor J domain.
Consistent with this possibility, we recently showed that methylation of several backbone nitrogen atoms in region 17—31 of PTH- 1—31 impairs, albeit modestly, the capacity of the ligand to stimulate cAMP formation in cells expressing PTHR-delNt 8. Taken together, these observations point to the uncertainty that exists in our understanding of the specific mechanisms by which the C-terminal domain of PTH contributes to the PTHR-binding process. This study was undertaken to examine further the mode of action used by the C-terminal binding domain of PTH.
We sought to address the roles that the side chains in this domain play in the PTHR-binding process, the general functional importance of amphiphilicity, and the potential for binding interactions with the receptor J domain.
The overall results indicate a dominant role for specific interactions between side chains on the hydrophobic face of the C-terminal helix and the receptor N-terminal domain.
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They also provide evidence for weak interactions between side chains projecting from the hydrophilic face of the helix and the PTHR J domain. The data also shed new light on the overall topology of the bimolecular complex, as they support folding of the bound ligand and proximity of the binding sites in the N and J domains of the receptor. Additional analogs of PTH- 1—31 -NH 2 with substitutions at position 20 were prepared as part of a previous study All peptides were verified by analytical HPLC, matrix-assisted laser desorption ionization mass spectrometry, and amino acid analysis, and peptide concentrations of stock solutions were established by amino acid analysis.
Four spectra were averaged, and the data were smoothed by the Jasco software. The cells were plated and assayed in well plates. For binding assays, cell membranes were prepared from the transfected COS-7 cells. Cells were harvested 3 days after transfection, and membranes were prepared as described Specifically bound radioactivity was calculated as a percentage of the radioactivity specifically bound in the absence of competing ligand.
The medium was then removed, and the cells were lysed by adding 50 m m HCl and freezing the plate on dry ice. The cAMP in the thawed lysate was quantified by radioimmunoassay. After 2 h on ice, the acid lysates were orfzni with ether and processed by ion-exchange chromatography 0. Data and Statistical Calculations —Binding and cAMP data were processed for curve fitting and derivation of IC 50 and EC 50 values using least-squares nonlinear regression analysis and the following equation: In cases in which incomplete inhibition of binding occurred, e.
Paired data sets were statistically compared using a two-tailed Student’s t test, assuming unequal variance for the two sets. The various alanine substitutions had a range of effects on this binding. The Ala 22 substitution was thus paired with Ala 19 as well as with Arg 19but neither pairing improved affinity further relative to the single substitutions alone Table 1.
Cit, citralline, Orn, ornithine; ND, not determined. This binding was nevertheless sufficient to assess the effects of the alanine substitutions on the capacity of the ligand to interact with PTHR-delNt.
SBspecific binding. None of the alanine substitutions had a major impact on the secondary structure of the peptide as revealed by CD spectroscopy analysis. To evaluate this further, we substituted each residue in region 17—31 of PTH with Cha, an amino acid analog that would preserve bulk hydrophobicity, yet alter the specific chemistry and topology of the side chain at the substituted site. The deleterious effects that the Cha substitutions at Trp 23 and Leu 24 had on PTHR-binding affinity indicate that hydrophobicity per se is not the main physicochemical property of these two side chains that underlies their contributions to PTHR-binding affinity.
Data were obtained as described in the legend to Table 1. The basis for these enhanced CD signals which were not accompanied by parallel changes in our receptor assays is not clear at present.
These results are in agreement with those obtained with the corresponding Ala substitutions in that they suggest that the detrimental effects that substitutions at positions 20, 23, and 24 have on binding to the intact PTHR are not due to altered interactions with the PTHR J domain, but rather involve interactions with the receptor N domain.
They also suggest that Arg 25 and Lys 26 can contribute to PTHR-binding affinity via mechanisms that are not dependent on interactions with the receptor N domain. The parental PTH- 1—31 -NH 2 peptide and derivatives thereof altered by a single alanine substitution in region 17—31 were analyzed by CD spectroscopy. The CD spectra obtained for other peptides used in this study are shown in supplemental Fig. Non-conservative Glu Substitutions —The data so far suggested that certain residues in the C-terminal domain of PTH- 1—31especially Trp 23Leu 24and Leu 28contribute significantly to PTHR-binding affinity by interacting predominantly with the receptor N domain.
To test this hypothesis further, we sought to introduce substitutions that would more strongly disrupt binding to the intact PTHR than did the alanine or Cha substitutions. If the hypothesis were correct, then such substitutions would have little or no effect on binding to PTHR-delNt.
We therefore introduced, as a non-conservative substitution, Glu at each position in the C-terminal segment of [Ala 1 ,Arg 19 ]PTH- 1—31 -NH 2 that was otherwise occupied by a hydrophobic or positively charged amino acid.
As predicted, several of the Glu substitutions caused severe reductions in binding affinity for the intact PTHR. Thus, the Glu substitutions at Arg 20 and Leu 24 abolished detectable binding to the intact PTHR, and those at Leu 28 and Trp 23 reduced apparent affinity by and fold, respectively Fig.
None of the Glu substitutions substantially altered the CD profile of the peptide Table 2 and supplemental Fig.
The overall data obtained for these Glu substitutions support the view that the C-terminal domain of PTH- 1—31 interacts predominantly with the PTHR N domain, but can also contribute modestly to binding affinity via mechanisms that are independent of the N domain. Analysis of Arg 20 —The arginine at position 20 is one of the most conserved residues in PTH and PTHrP ligands and has been shown to play a key role in the receptor interaction process 1011 Little has been revealed, however, about the mechanistic basis for this role.
To further dissect the functional role of Arg 20we examined the same position substituted PTH- 1—31 -NH 2 analogs from the study of Barbier et al.
Although potency was reduced, each substituted analog at the highest concentration produced approximately the same maximum cAMP response as did the parental peptide. Substitution analysis of Arg This study was designed to gain further information on the functional roles that the amino acid side chains in the principal receptor-binding domain of PTH play in the receptor interaction process.
Analysis of the binding of the analogs to PTHR-delNt expressed in COS-7 cell membranes was a key and novel aspect of our study, as it enabled us, for the first time, to assess the extent to which a targeted amino acid in the C-terminal domain of a relatively unmodified PTH ligand interacts with the receptor N domain absent in PTHR-delNt versus the receptor J domain containing the extracellular loops and transmembrane helices. Overall, our findings are largely consistent with the two-site model of the PTH-PTHR interaction mechanism outlined in the Introduction in that they suggest that the C-terminal domain of PTH- 1—31 interacts predominantly with the N domain of the receptor to contribute a large proportion of the overall binding energy to the complex.
The greatest impact on the binding of our PTH- 1—31 peptides to the intact PTHR occurred with the non-conservative Glu substitutions at Arg 20Trp 23Leu 24and Leu 28 ,as each reduced apparent affinity by fold or more. Such effects are consistent with previous PTH substitution studies showing important roles for these four residues in binding to the intact PTHR 69 — Our CD analyses indicated that the substitutions did not cause major perturbations in the helical structure of the peptide.
It thus seems clear from the data that the side chains of Arg 20Trp 23 ,Leu 24and Leu 28 contribute to the PTHR-binding process by mechanisms that are largely, if not completely, dependent on interactions with the receptor N-terminal domain.
Our data predict that this hydrophobic helical face contributes to the PTHR-binding process by interacting with the N-terminal domain of the receptor.
Our findings do not support a mechanism by which this hydrophobic face contributes to the PTHR-binding process by interacting nonspecifically with the lipid component of the cell membrane, as has been discussed for PTH 32 and for amphiphilic peptide ligands in general Thus, the mechanisms by which the side chains of Try 23 and Leu 24 contribute to the PTHR-binding process are not likely to be based simply on nonspecific hydrophobic interactions with the lipid bilayer, but instead involve additional spatial and chemical features of the side chains and their specific interactions with cognate functional groups in the receptor.
The molecular nature of these interactions is not clear at present, but likely involves multiple components of the arginine side chain, including the cationic and H-bonding nitrogen atoms of the guanidino group and the three methylenes of the linker Our data now imply that these functional groups of the Arg 20 side chain fit within a highly specific binding pocket within the N domain of the receptor.
These effects were accompanied by approximately proportional effects on interaction with the intact PTHR.
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Such findings suggest that the side chains of these residues, although not making major contributions to overall binding energy, can promote binding via mechanisms that involve interactions with the PTHR J domain.
If both the C-terminal region 20—31 and N-terminal region 1—19 domains of the ligand interact with the PTHR J domain, then a bend would most likely be required between the two domains of the receptor-bound ligand. The tertiary structure of receptor-bound PTH has been a matter of some debate, with linear and folded structures supported 4 A folded helix-turn-helix structure for PTH- 1—34 was indeed predicted early on based on modeling and structure-activity data 36and solution-phase NMR studies of PTH and PTHrP ligands generally reveal mid-region flexibility or a hinge between the N- and C-terminal domains 4537 In addition to the tertiary structure of the bound ligand, our data have implications for the topology of the occupied receptor and the spatial relationship of the N and J domains.
This possibility is again supported by cross-linking data: Because the spatial positioning of the Bp moieties in these two ligands is likely to be similar, a proximity of the two cross-linked sites in the receptor is also likely. One important goal that may be facilitated by our work is to identify sites in the receptor that are used by key ligand residues such as Arg 20Trp 23Leu 24and Leu Our results predict that such interaction sites will be located in the receptor N-terminal domain.
For the other ligand residues, few clues are available: The recently reported NMR-derived structure of the N-terminal domain of the related corticotropin-releasing factor receptor 15 may open paths for structure-based analyses of the ligand interaction sites in the N domains of the Class 2 G protein-coupled receptors Even with such an approach, additional functional studies will be needed.
The new PTH analogs presented here should be of value in this regard, as they can be used in conjunction with PTHR mutants altered at candidate sites in the N-terminal domain to probe for allele-specific rescue effects. This is a direction that we hope to pursue in future studies. The costs of publication of this article were defrayed in part by the payment of page charges. Section solely to indicate this fact. You’ll be in good company.
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This Article First Published on August 21, doi: Classifications Mechanisms of Signal Transduction.