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Identification of Key Residues Involved in Adrenomedullin Binding to the AM1 Receptor.
MedLine Citation:
PMID:  23351143     Owner:  NLM     Status:  Publisher    
BACKGROUND AND PURPOSE: Adrenomedullin (AM) is a peptide hormone whose receptors are members of the class B G protein-coupled receptor (GPCR) family. They comprise a heteromer between the GPCR, the calcitonin receptor-like receptor (CLR) and one of the receptor activity-modifying proteins (RAMPs) 1-3. AM plays a significant role in angiogenesis and its antagonist fragment AM22-52 can inhibit blood vessel and tumour growth. The mechanism by which AM interacts with its receptors is unknown. EXPERIMENTAL APPROACH: We determined the AM22-52 binding epitope for the AM1 receptor extracellular domain using biophysical techniques, heteronuclear magnetic resonance spectroscopy and alanine scanning. KEY RESULTS: Chemical shift perturbation experiments located the main binding epitope for AM22-52 at the AM1 receptor to the C-terminal 8 amino acids. Isothermal titration calorimetry of AM22-52 alanine substituted peptides indicated that Y52, G51 and I47 are essential for AM1 receptor binding and that K46 and P49 and R44 have a smaller role to play. Characterisation of these peptides at the full length AM receptors was assessed in Cos7 cells by cAMP assay. This confirmed the essential role of Y52, G51 and I47 in binding to the AM1 receptor, with their substitution resulting in ≥100-fold reduction in antagonist potency compared to AM22-52. R44A, K46A, S48A and P49A AM22-52 decreased antagonist potency by approximately 10-fold. CONCLUSIONS AND IMPLICATIONS: This study localises the main binding epitope of AM22-52 to its C-terminal amino acids and distinguishes essential residues involved in this binding. This will inform the development of improved AM receptor antagonists.
H A Watkins; M Au; R Bobby; J K Archbold; N Abdul-Manan; J M Moore; M J Middleditch; G M Williams; M A Brimble; A J Dingley; D L Hay
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-28
Journal Detail:
Title:  British journal of pharmacology     Volume:  -     ISSN:  1476-5381     ISO Abbreviation:  Br. J. Pharmacol.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-28     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  7502536     Medline TA:  Br J Pharmacol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
© 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.
School of Biological Sciences, University of Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, New Zealand.
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