title: Structure-activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  Substitution of Ala-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 Thr-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
  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.
authors.affiliation: School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand.
authors.forename: Debbie L
authors.initials: DL
authors.lastname: Hay
authors.forename: Paul W R
authors.initials: PW
authors.lastname: Harris
authors.forename: Renata
authors.initials: R
authors.lastname: Kowalczyk
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Brimble
authors.forename: Dan L
authors.initials: DL
authors.lastname: Rathbone
authors.forename: James
authors.initials: J
authors.lastname: Barwell
authors.forename: Alex C
authors.initials: AC
authors.lastname: Conner
authors.forename: David R
authors.initials: DR
authors.lastname: Poyner
mesh_terms: D000409:Alanine / Q000502:physiology*; D000818:Animals; D019390:Arrestins / Q000096:biosynthesis; D016466:CHO Cells; D015740:Calcitonin Gene-Related Peptide / Q000737:chemistry* / Q000235:genetics; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D007553:Isotope Labeling; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D021381:Protein Transport; D011869:Radioligand Assay; D019275:Radiopharmaceuticals; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000819:agonists*; D055291:Salivary alpha-Amylases / Q000187:drug effects; D013329:Structure-Activity Relationship; D013912:Threonine / Q000502:physiology*; D014162:Transfection
chemical_list: D019390:Arrestins; D010455:Peptides; D019275:Radiopharmaceuticals; D018015:Receptors, Calcitonin Gene-Related Peptide; D013912:Threonine; D000242:Cyclic AMP; C524788:AMY1A protein, human; D055291:Salivary alpha-Amylases; D015740:Calcitonin Gene-Related Peptide; D000409:Alanine
keywords: GPCR; amylin; cAMP; calcitonin gene-related peptide; efficacy; ligand binding; structure-activity relationship; β-arrestin