Enhancement of Thrombin Receptor Activation by Thrombin Receptor-Derived Heptapeptide with para-Fluorophenylalanine in Place of Phenylalanine

doi:10.1006/bbrc.1993.1680

Biochemical and Biophysical Research Communications

Volume 193, Issue 2, 15 June 1993, Pages 694-699

https://doi.org/10.1006/bbrc.1993.1680 Get rights and content

Abstract

Thrombin receptor-derived peptide SFLLRNP (one-letter amino acid code) which corresponds to the N-terminal heptapeptide of tethered ligand is able to activate thrombin receptor and to stimulate the phosphoinositide (PI) turnover. The replacement of Phe-2 by Ala eliminated this activity completely, showing the crucial role of the Phe-phenyl group in receptor activation. It was found that substitution of para-fluorophenylalanine ((p-F)Phe) for Phe-2 enhanced several times the PI-turnover activity of SFLLRNP. This is the first example to date of a substitution with one order of magnitude greater increase in receptor activation. The Phe-2/Tyr substitution diminished the activity drasticaliy (almost 2% of SFLLRNP), indicating the importance of hydrophobicity of Phe²-phenyl. The Phe-2/Leu substitution, however, diminished also the activity (less than 2% of SFLLRNP). These results suggested that highly specific hydrophobic interaction exists between Phe-2 of the tethered ligand and its binding site in thrombin receptor.

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Cited by (38)

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Citation Excerpt :
Under these circumstances, we have focused on the functional roles of Phe-phenyl benzene-hydrogens. We reported that para-fluorophenylalanine [(4-F1)Phe] at position 2 of S/Phe/LLRNP enhances activity levels several-fold in assays of phosphoinositide turnover in human epithelial-like SH-EP cells and of human platelet aggregation.22–23 The role of the para-fluorine (F) atom of the Phe-phenyl benzene ring was immediately established as an essential structural element to reinforce the acidity of neighboring benzene-hydrogen (H) atoms, which were suggested to participate directly in the interaction with the PAR-1 receptor.22
Heptapeptide SFLLRNP is a receptor–tethered ligand of protease-activated receptor 1 (PAR-1), and its Phe at position 2 is essential for the aggregation of human platelets. To validate the structural elements of the Phe-phenyl group in receptor activation, we have synthesized a complete set of S/Phe/LLRNP peptides comprising different series of fluorophenylalanine isomers (F_n)Phe, where n = 1, 2, 3, and 5. Phe-2-phenyl was strongly suggested to be involved in the edge-to-face CH/π interaction with the receptor aromatic group. In the present study, to prove this receptor interaction definitively, we synthesized another series of peptide analogs containing (F₄)Phe-isomers, with the phenyl group of each isomer possessing only one hydrogen atom at the ortho, meta, or para position. When the peptides were assayed for their platelet aggregation activity, S/(2,3,4,6-F₄)Phe/LLRNP and S/(2,3,4,5-F₄)Phe/LLRNP exhibited noticeable activity (34% and 6% intensities of the native peptide, respectively), whereas S/(2,3,5,6-F₄)Phe/LLRNP was completely inactive. The results indicated that, at the ortho and meta positions but not at the para position, benzene-hydrogen atoms are required for the CH/π interaction to activate the receptor. The results provided a decisive evidence of the molecular recognition property of Phe, the phenyl benzene-hydrogen atom of which participates directly in the interaction with the receptor aromatic π plane.
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Proteinase-activated receptors are a family of seven-transmembrane G-protein-coupled receptors. Activation of PARs is initiated through cleavage of the N-terminus, unmasking a tethered ligand that can then interact with the receptor and lead to its activation. PARs exhibit both anti- and pro-inflammatory properties, although recent evidence has pointed towards a detrimental role for PARs, particularly PAR-2, in arthritis. Initial research using PAR-2 knockout mice identified PAR-2 as a key mediator of chronic joint inflammation. Further research examined the role of PAR-2 in human articular cell types, demonstrating upregulation of PAR-2 in cells from an inflammatory background compared with non-inflammatory cells, with PAR-2 levels being further upregulated by pro-inflammatory cytokines and growth factors. To date, there is no clinical evidence of a role for PAR-2 in vivo in humans, although recent studies utilizing human joint tissue and articular cells are emerging.
Differential receptor binding characteristics of consecutive phenylalanines in μ-opioid specific peptide ligand endomorphin-2
2007, Bioorganic and Medicinal Chemistry
Endogenous opioid peptides consist of a conserved amino acid residue of Phe³ and Phe⁴, although their binding modes for opioid receptors have not been elucidated in detail. Endomorphin-2, which is highly selective and specific for the μ opioid receptor, possesses two Phe residues at the consecutive positions 3 and 4. In order to clarify the role of Phe³ and Phe⁴ in binding to the μ receptor, we synthesized a series of analogs in which Phe³ and Phe⁴ were replaced by various amino acids. It was found that the aromaticity of the Phe-β-phenyl groups of Phe³ and Phe⁴ is a principal determinant of how strongly it binds to the receptor, although better molecular hydrophobicity reinforces the activity. The receptor binding subsites of Phe³ and Phe⁴ of endomorphin-2 were found to exhibit different structural requirements. The results suggest that [Trp³]endomorphin-2 (native endomorphin-1) and endomorphin-2 bind to different receptor subclasses.
Protease-activated receptor-2 (PAR-2): Structure-function study of receptor activation by diverse peptides related to tethered-ligand epitopes
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Protease-activated receptor-2 (PAR-2) is a tethered-ligand, G-protein-coupled receptor that is activated by proteolytic cleavage or by small peptides derived from its cleaved N-terminal sequence, such as SLIGRL-NH₂. To assess specific PAR activity, we developed an immortalized murine PAR-1 (−/−) cell line transfected with either human PAR-2 or PAR-1. A “directed” library of more than 100 PAR agonist peptide analogues was synthesized and evaluated for PAR-2 and PAR-1 activity to establish an in-depth structure-function profile for specific action on PAR-2. The most potent agonist peptides (EC₅₀ = 2–4 μM) had Lys at position 6, Ala at position 4, and pFPhe at position 2; however, these also exhibited potent PAR-1 activity (EC₅₀ = 0.05–0.35 μM). We identified SLIARK-NH₂ and SL-Cha-ARL-NH₂ as relatively potent, highly selective PAR-2 agonists with EC₅₀ values of 4 μM. Position 1 did not tolerate basic, acidic, or large hydrophobic amino acids. N-Terminal capping by acetyl eliminated PAR-2 activity, although removal of the amino group reduced potency by just 4-fold. At position 2, substitution of Leu by Cha or Phe gave equivalent PAR-2 potency, but this modification also activated PAR-1, whereas Ala, Asp, Lys, or Gln abolished PAR-2 activity; at position 3, Ile and Cha were optimal, although various amino acids were tolerated; at position 4, Ala or Cha increased PAR-2 potency 2-fold, although Cha introduced PAR-1 activity; at position 5, Arg or Lys could be replaced successfully by large hydrophobic amino acids. These results with hexapeptide C-terminal amides that mimic the native PAR-2 ligand indicate structural modes for obtaining optimal PAR-2 activity, which could be useful for the design of PAR-2 antagonists.
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The thrombin receptor (PAR-1) is an unusual transmembrane G-protein coupled receptor in that it is activated by serine protease cleavage of its extracellular N-terminus to expose an agonist peptide ligand, which is tethered to the receptor itself. Synthetic peptides containing the agonist motif, such as SFLLRN for human PAR-1, are capable of causing full receptor activation. We have probed the possible bioactive conformations of thrombin receptor-activating peptides (TRAPs) by systematic introduction of certain conformational perturbations, involving α-methyl, ester Ψ(COO), and reduced-amide Ψ(CH₂N) scans, into the minimum-essential agonist sequence (SFLLR) to probe the importance of the backbone conformation and amide NH hydrogen bonding. We performed extensive conformational searches of representative pentapeptides to derive families of putative bioactive structures. In addition, we employed ¹H NMR and circular dichroism (CD) to characterize the conformational disposition of certain pentapeptide analogues experimentally. Activation of platelet aggregation by our pentapeptide analogues afforded a structure-function correlation for PAR-1 agonist activity. This correlation was assisted by PAR-1 receptor binding data, which gauged the affinity of peptide ligands for the thrombin receptor independent of a functional cellular response derived from receptor activation (i.e. a pure molecular recognition event). Series of alanine-, proline-, and N-methyl-scan peptides were also evaluated for comparison. Along with the known structural features for PAR-1 agonist peptides, our work adds to the understanding of peptide topography relative to platelet functional activity and PAR-1 binding. The absolute requirement of a positively charged N-terminus for strong agonist activity was contradicted by the N-terminal hydroxyl peptide Ψ(HO)S-FLLR-NH₂. The amide nitrogen between residues 1 and 2 was found to be a determinant of receptor recognition and the carbonyl groups along the backbone may be involved in hydrogen bonding with the receptor. Position 3 (P3) of TRAP-5 is known to tolerate a wide variety of side chains, but we also found that the amide nitrogen at this position can be substituted by an oxygen, as in SF-Ψ(COO)-LLR-NH₂, without diminishing activity. However, this peptide bond is sensitive to conformational changes in that SFPLR-NH₂ was active, whereas SF-NMeL-LR-NH₂ was not. Additionally, we found that position 3 does not tolerate rigid spacers, such as 3-aminocyclohexane-1-carboxylic acid and 2-aminocycloalkane-1-carboxylic acid, as analogues 1A, 1B, 2A, 2B, 3, 4, 5A and 5B lack agonist activity. On the basis of our results, we suggest that an extended structure of the agonist peptide is principally responsible for receptor recognition (i.e. binding) and that hydrophobic contact may occur between the side chains of the second (Phe) and fourth (Leu) residues (i.e. P2-P4 interaction).

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Regular ArticleEnhancement of Thrombin Receptor Activation by Thrombin Receptor-Derived Heptapeptide with para-Fluorophenylalanine in Place of Phenylalanine

Abstract

Regular Article
Enhancement of Thrombin Receptor Activation by Thrombin Receptor-Derived Heptapeptide with para-Fluorophenylalanine in Place of Phenylalanine