Design of analogues of parathyroid hormone: A conformational approach

Nussbaum, Samuel R.; Beaudette, Norman V.; Fasman, Gerald D.; Potts, John T.; Rosenblatt, Michael

doi:10.1007/bf01025179

Cited by 16 publications

(42 citation statements)

References 54 publications

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“…As described herein, we show that this can be accomplished by tethering the N-terminal residues of PTH directly to a truncated receptor lacking the N-terminal extracellular domain. The resulting tethered ligand/receptor constructs are active and exhibit a similar, yet not identical, mutational profile to that seen previously with exogenous PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and PTH(1-34) peptide ligands. This system provides a new approach for analyzing how the Nterminal residues of PTH contribute to interactions with the PTH-1 receptor.…”

supporting

confidence: 61%

“…1). The N-terminal tyrosine was not expected to be a major detriment to the potential signaling activity induced by the tethered PTH segment because [Tyr Ϫ1 ]rPTH(1-14)NH 2 was 50% as active as native PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) in stimulating a cAMP response in cells expressing the P1Rc. 3 The basal and ligand-stimulated signaling properties of the tethered PTH-1 receptors in transiently transfected COS-7 cells are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In further support of the latter component of this hypothesis, we recently showed that a peptide as small as PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) could stimulate cAMP formation, albeit weakly, with both the wild type PTH receptor and a truncated PTH receptor (P1Rc-delNt) that lacked most of the N-terminal domain (11). The potency of PTH(1-34) was severely diminished with P1Rc-delNt, as compared with its potency with the wild type receptor (EC 50 values ϭ ϳ500 nM and ϳ2 nM, respectively).…”

mentioning

confidence: 76%

“…The potency of PTH(1-34) was severely diminished with P1Rc-delNt, as compared with its potency with the wild type receptor (EC 50 values ϭ ϳ500 nM and ϳ2 nM, respectively). In contrast, the potency of PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) was equivalent with both the intact receptor and with P1Rc-delNt (EC 50 values ϭ ϳ200 M). An alanine-scanning analysis performed on PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) revealed that residues (1-9) were critical for interacting with the heptahelical and extracellular loop region of the receptor (11).…”

mentioning

confidence: 95%

“…Structure-activity analyses of PTH have shown that the 1-34 fragment is sufficient for full biological activity (4). Within the PTH(1-34) peptide, the N-terminal residues are the most critical for receptor activation, whereas the C-terminal residues are more important for determining receptor binding affinity (4,5).…”

mentioning

confidence: 99%

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Autoactivation of Type-1 Parathyroid Hormone Receptors Containing a Tethered Ligand

Shimizu

Carter

Gardella

2000

Journal of Biological Chemistry

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Interactions between the N-terminal residues of parathyroid hormone (PTH) and the region of the PTH receptor containing the extracellular loops and transmembrane domains are thought to be critical for receptor activation. We evaluated this hypothesis by replacing the large N-terminal extracellular domain of the human type 1 PTH receptor (hP1Rc-WT) with residues 1-9 of PTH (AVSEIQLMH) using a tetraglycine linker between His-9 of the ligand and Glu-182 of the receptor near the extracellular terminus of transmembrane domain-1. Expression of this construct, hP1Rc-Tether(1-9), in COS-7 cells resulted in basal cAMP levels that were 10-fold higher than those seen in control cells transfected with hP1Rc-WT. Extending the ligand sequence to include Asn-10 and the activity-enhancing substitution of Leu-11 3 Arg yielded hP1Rc-[Arg 11 ]Tether(1-11), for which we observed basal cAMP levels that were 50-fold higher than those seen with P1Rc-WT. An alaninescan analysis of hP1Rc-[Arg 11 ]Tether(1-11) revealed that Gln-6 and His-9 were not critical for autoactivation, whereas Val-2, Ile-5, and Met-8 were. The data show that tethered PTH/PTH receptors can autoactivate. Analysis of the structure-activity relationships in these tethered receptor constructs can provide new information concerning how the N-terminal residues of PTH interact with the extracellular loops and transmembrane regions of the PTH-1 receptor, particularly in regard to receptor activation. Human parathyroid hormone (hPTH)1 is an 84-amino acid polypeptide that binds to a class B G protein-coupled receptor, the PTH-1 receptor, and thereby plays a vital role in regulating the extracellular concentrations of ionized calcium (1). The PTH-1 receptor also mediates the actions of PTH-related peptide, a large (ϳ140 amino acids) polypeptide ligand that plays a critical role in the developmental of several organs, particularly the skeleton (1). PTH has potent anabolic effects on bone in humans (2) and, thus, is a potential therapy for metabolic bone diseases, such as osteoporosis (3). Structure-activity analyses of PTH have shown that the 1-34 fragment is sufficient for full biological activity (4). Within the PTH(1-34) peptide, the N-terminal residues are the most critical for receptor activation, whereas the C-terminal residues are more important for determining receptor binding affinity (4, 5).Recent receptor mutagenesis and photochemical cross-linking approaches suggest that residues within the C-terminal 15-34 region of PTH(1-34) interact with the relatively large (ϳ170 amino acid) N-terminal extracellular domain of the PTH-1 receptor and that the N-terminal residues of PTH interact with the 7 transmembrane domains and extracellular loops of the receptor (6 -10). In further support of the latter component of this hypothesis, we recently showed that a peptide as small as PTH(1-14) could stimulate cAMP formation, albeit weakly, with both the wild type PTH receptor and a truncated PTH receptor (P1Rc-delNt) that lacked most of the N-terminal domain (11). The potency of P...

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supporting

confidence: 61%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 76%

mentioning

confidence: 95%

mentioning

confidence: 99%

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Autoactivation of Type-1 Parathyroid Hormone Receptors Containing a Tethered Ligand

Shimizu

Carter

Gardella

2000

Journal of Biological Chemistry

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The road from poly(α-amino acids) to the prediction of protein conformation

Fasman¹

1987

Biopolymers

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SynopsisThe physicochemical properties and the conformational transitions of various poly(a-amino acids) are described. Rotatory dispersion and CD techniques to determine the secondary structure were developed. The similarity between the helix-coil Zimm-Bragg parameters of the poly(aamino acids) and the frequencies of the amino acids found in the various secondary structures in proteins is utilized to develop an algorithm to predict the secondary structure of proteins from the primary sequence. Application of the predictive scheme to various biological/biochemical problems is described.The scientific community was introduced to the field of poly( a-amino acids) in an excellent review by Katchalski and Sela' in 1958, which was followed by a view of poly(a-amino acid) biological properties by these same authors.2 These biopolymem were quickly adopted as excellent models for studies of conformational transitions of proteins, synthetic antigens, enzyme substrates, models for protein-DNA interactions, interaction with viruses, antibacterial properties, and interactions with blood components (e.g., red blood cells); they were also tested as possible blood volume expanders. These fasicle polymers can be produced of any desired length-from lo3 to 10' daltons. The polymer chemist as well as the biochemist found these models fascinating. They became models par excellence for the study of the secondary and tertiary structure of proteins, and as models of the conformational changes these important biological systems were capable of undergoing. Using the techniques of light scattering, viscosity, and optical rotational dispersion, researchers demonstrated the various conformations these models proteins could undergo. These protein models were excellent substrates for physicochemical and biological studies, as well as for the study of the polymerization mechanism of the Leuchs anhydride, used predominantly to produce these semiproteins. Without a doubt, the father of this huge area of research was Ephraim Katchalski-Katzir.Ephraim Katchalski first summarized this literature up to 1951,3 which brought worldwide attention to their synthesis, and their physicochemical and biological properties. Attempts to synthesize these by polymerization of amino acids were made by Hofmeister4 and Fischer5 before the existence of the peptide bond was clearly established. Schaal,' as early as 1871, began experiments along these lines. Excellent monomers used for their synthesis were the N-carboxyamino acid anhydrides, first developed by Leuchs in 19CML7 Ephraim Katchalski and collaborators, including his mentor, Frankel,' had researched various procedures to produce high molecular-weight poly( a-amino

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