Structure-activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation

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Abstract

Background and purpose - The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7.
Experimental approach - CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and ß-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor.
Key results - Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at ß-arrestin translocation was reduced by 9-fold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and ß-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor.
Conclusions and implications - Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity.

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  • Structure-activity relationships of the N-terminus of calcitonin gene-related peptide

    Rights statement: © 2013 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Original languageEnglish
Pages (from-to)415-426
Number of pages12
JournalBritish Journal of Pharmacology
Volume171
Issue number2
DOIs
Publication statusPublished - 2014

Bibliographic note

© 2013 The Authors. British Journal of Pharmacology published by John Wiley &. Sons Ltd on behalf of The British Pharmacological Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

    Keywords

  • calcitonin gene-related peptide, efficacy, ligand binding, β-arrestin, cAMP, structure-activity relationship, GPCR, amylin

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