Structure-activity study of hCGRP8-37, a calcitonin gene-related peptide receptor antagonist

Mimeault, Murielle; Quirion, Rémi; Dumont, Yvan; St‐Pierre, Serge; Fournier, Alain

doi:10.1021/jm00090a003

Cited by 44 publications

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“…Replacement of residues 44 to 52 of AM with the corresponding residues of AM2 (C8) resulted in a somewhat reduced ␣-helical content and a substantial increase in ␤-sheet, suggesting that the correct sequence of the C-terminal residues of AM may be required for the formation or stabilization of an ␣-helical structure. It has been reported previously that ␣-helical content correlates with the affinity of CGRP antagonist fragments (Mimeault et al, 1992). However, for the peptides in this study, differences in affinity or selectivity of these peptides did not necessarily correlate with differences in secondary structural composition.…”

Section: Discussioncontrasting

confidence: 67%

“…Progressive deletion of residues 22 to 30 from AM reduced affinity at both AM receptors. The corresponding region in CGRP (i.e., the residues directly after the ring structure) is also required for high-affinity binding (Mimeault et al, 1992). Residues 22 to 25 of AM seem to be more important than 26 to 29 of AM because their removal resulted in a similar loss of affinity to removal of the residues 22 to 29.…”

Section: Discussionmentioning

confidence: 99%

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Novel Peptide Antagonists of Adrenomedullin and Calcitonin Gene-Related Peptide Receptors: Identification, Pharmacological Characterization, and Interactions with Position 74 in Receptor Activity-Modifying Protein 1/3

Robinson

Aitken²,

Bailey³

et al. 2009

J Pharmacol Exp Ther

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Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin peptide family, which also includes calcitonin gene-related peptide (CGRP) and AM2. The two AM receptors, AM 1 and AM 2 , are calcitonin receptor-like receptor (CL)/receptor activity-modifying protein (RAMP) (RAMP2 and RAMP3, respectively) heterodimers. CGRP receptors comprise CL/RAMP1. The only human AM receptor antagonist (AM ) is a truncated form of AM; it has low affinity and is only weakly selective for AM 1 over AM 2 receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM 1 , AM 2 , and CGRP receptors. AM 22-52 was the framework for generating further AM fragments (AM 26 -52 and AM 30 -52 ), novel AM/␣CGRP chimeras (C1-C5 and C9), and AM/AM 2 chimeras (C6 -C8). cAMP assays were used to screen the antagonists at all receptors to determine their affinity and selectivity. Circular dichroism spectroscopy was used to investigate the secondary structures of AM and its related peptides. The data indicate that the structures of AM, AM2, and ␣CGRP differ from one another. Our chimeric approach enabled the identification of two nonselective highaffinity antagonists of AM 1 , AM 2 , and CGRP receptors (C2 and C6), one high-affinity antagonist of AM 2 receptors (C7), and a weak antagonist selective for the CGRP receptor (C5). By use of receptor mutagenesis, we also determined that the C-terminal nine amino acids of AM seem to be responsible for its interaction with Glu74 of RAMP3. We provide new information on the structure-activity relationship of AM, ␣CGRP, and AM2 and how AM interacts with CGRP and AM 2 receptors.Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin family of peptides, which also includes calcitonin gene-related peptide (CGRP) and AM2 (also known as intermedin). The calcitonin peptide family shows weak homology at the level of the primary sequence, but there are stronger relationships at the secondary structure level. Each member of the family has an N-terminal ring structure and an amidated carboxyl terminus. Both of these structures are critical for receptor binding and subsequent signaling (Eguchi et al., 1994;Conner et al., 2002). Truncation of the N-terminal ring structure produces antagonist peptides in all family members. The ring structure of AM is created by the formation of a disulfide bond between two cysteine residues at positions 16 and 21 of the peptide (Eguchi et al., 1994). Truncation of AM yields the AM antagonist AM . Interfering with the C-terminal amidated tyrosine residue of AM results in a peptide with reduced receptor affinity (Eguchi et al., 1994). AM 13-52 and AM 15-52 are both agonists with similar affinity to full-length AM, however, little is known structurally about AM.The combination of the calcitonin receptor-like receptor (CL) with a receptor activity-modifying protein (RAMP) constitutes receptors that bind AM . CL is unusual for a G protein-coupled receptor in that it cannot f...

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Section: Discussioncontrasting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Novel Peptide Antagonists of Adrenomedullin and Calcitonin Gene-Related Peptide Receptors: Identification, Pharmacological Characterization, and Interactions with Position 74 in Receptor Activity-Modifying Protein 1/3

Robinson

Aitken²,

Bailey³

et al. 2009

J Pharmacol Exp Ther

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“…Another ␣-helix structure is predicted at the C terminus. In the case of CGRP, deletion of the disulfide ring structure resulted in an antagonist (11,12). This result suggested that the disulfide ring might be a transduction domain or might be important in the maintenance of the proper structure for signal transduction.…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of Structural Alterations in the Sequence of the Vasodilatory Peptide Maxadilan Yields a Pituitary Adenylate Cyclase-activating Peptide Type 1 Receptor-specific Antagonist

Moro

Wakita

Ohnuma

et al. 1999

Journal of Biological Chemistry

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Maxadilan is a vasodilatory peptide derived from sand flies that is an agonist at the pituitary adenylate cyclase-activating peptide (PACAP) type 1 receptor. Surprisingly, maxadilan does not share significant sequence homology with PACAP. To examine the relationship between structure and activity of maxadilan, several amino acid substitutions and deletions were made in the peptide. These peptides were examined in vitro for binding to crude membranes derived from rabbit brain, a tissue that expresses PACAP type 1 receptors; and induction of cAMP was determined in PC12 cells, a line that expresses these receptors. The peptides were examined in vivo for their ability to induce erythema in rabbit skin. Substitution of the individual cysteines at positions 1 and 5 or deletion of this ring structure had little effect on activity. Substitution of either cysteine at position 14 or 51 eliminated activity. Deletion of the 19 amino acids between positions 24 and 42 resulted in a peptide with binding, but no functional activity. The capacity of this deletion mutant to interact with COS cells transfected with the PACAP type 1 receptor revealed that this peptide was a specific antagonist to the PACAP type 1 receptor.

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“…Briefly, the syntheses were performed with a homemade manual multireactor synthesizer, using a benzhydrylamine resin as solid support and benzotriazol-1 -yloxy-tris-(dimethy1amino)phosphonium hexafluorophosphate (BOP) as coupling agent. Side-chain protection of a-tert-butoxycarbonyl-amino-acids was as previously reported (Mimeault et al, 1992). Deprotection of side chains and cleavage of peptides from the resin were achieved by treatment with liquid hydrofluoric acid in the presence of m-cresol.…”

Section: Materials and Methods Peptide Synthesis And Purificationmentioning

confidence: 98%

“…All the peptides were synthesized by the solid-phase peptide synthesis methodology, using experimental protocols described in previous reports (Forest et al, 1990;Mimeault et al, 1992). Briefly, the syntheses were performed with a homemade manual multireactor synthesizer, using a benzhydrylamine resin as solid support and benzotriazol-1 -yloxy-tris-(dimethy1amino)phosphonium hexafluorophosphate (BOP) as coupling agent.…”

Section: Materials and Methods Peptide Synthesis And Purificationmentioning

confidence: 99%

Conformational characterization by circular‐dichroism spectroscopy of various fragments and analogs of calcitonin‐gene‐related peptide

Mimeault

St‐Pierre

Fournier

1993

European Journal of Biochemistry

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A conformational study by circular‐dichroism spectroscopy of calcitonin‐gene‐related peptide (CGRP) and related fragments and analogs was carried out in structure‐promoting solvent mixtures. The structural characterization of rat CGRPα and the two isoforms of human CGRP, α and β, revealed that these peptides possess very similar conformational features. The far‐ultraviolet circular‐dichroism spectra, in pure water, of human CGRPα, (hCGRPα), [Acm‐Cys2,7]hCGRPα, various fragments and analogs indicated that these peptides exhibited predominantly a random‐coil conformation. The addition of increasing concentrations of 1,1,1,3,3,3‐hexafluoro‐2‐propanol to the peptide solutions resulted in a transition from a random‐coil conformation to a stabilized α‐helical structure. The substantial loss of helical content measured with [Acm‐Cys2,7]hCGRPα, [Acm‐Cys2,7]hCGRP‐(1–24)‐CONH2 and hCGRP‐(8–37), compared to hCGRPα, suggested that the N‐terminal disulfide bridge of hCGRPα is essential for adopting a highly stabilized α‐helical conformation. Moreover, the lower helical content of hCGRP‐(8–37), as compared to [Acm‐Cys2,7]hCGRPα, as well as spectroscopic results measured with various fragments and analogs of hCGRP‐(8–37) revealed that N‐terminal residues found in the peptide segment 1–12 are important for the full conservation of the amphiphilic α‐helix. In addition, the similar α‐helical content of hCGRP‐(8–37) and hCGRP‐(8–18) indicated that the C‐terminal segment 19–37 is not essential for the stabilization of the α‐helix structure.

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Structure-activity study of hCGRP8-37, a calcitonin gene-related peptide receptor antagonist

Cited by 44 publications

References 0 publications

Novel Peptide Antagonists of Adrenomedullin and Calcitonin Gene-Related Peptide Receptors: Identification, Pharmacological Characterization, and Interactions with Position 74 in Receptor Activity-Modifying Protein 1/3

Novel Peptide Antagonists of Adrenomedullin and Calcitonin Gene-Related Peptide Receptors: Identification, Pharmacological Characterization, and Interactions with Position 74 in Receptor Activity-Modifying Protein 1/3

Functional Characterization of Structural Alterations in the Sequence of the Vasodilatory Peptide Maxadilan Yields a Pituitary Adenylate Cyclase-activating Peptide Type 1 Receptor-specific Antagonist

Conformational characterization by circular‐dichroism spectroscopy of various fragments and analogs of calcitonin‐gene‐related peptide

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