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Structure–activity relationships of the N ‐terminus of calcitonin gene‐related peptide: key roles of alanine‐5 and threonine‐6 in receptor activation
Author(s) -
Hay Debbie L,
Harris Paul W R,
Kowalczyk Renata,
Brimble Margaret A,
Rathbone Dan L,
Barwell James,
Conner Alex C,
Poyner David R
Publication year - 2014
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.12464
Subject(s) - calcitonin gene related peptide , agonist , receptor , alanine , chemistry , serine , biochemistry , cysteine , threonine , peptide , amino acid , phosphorylation , neuropeptide , enzyme
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 C ys‐2 and C ys‐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 β‐arrestin 2 translocation in CHO ‐ K1 cells at the CGRP receptor. Key Results Substitution of A la‐5 by cysteine reduced affinity 270‐fold and reduced efficacy for production of cAMP in SK ‐ N ‐ MCs . Potency at β‐arrestin translocation was reduced by ninefold. Substitution of T hr‐6 by cysteine destroyed all measurable efficacy of both cAMP and β‐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 AMY 1(a) receptor. Conclusions and Implications A la‐5 and T hr‐6 are key determinants of agonist activity for CGRP . A la‐5 is also very important for receptor binding. Residues outside of the 1–7 ring also contribute to agonist activity.