Identification of key residues involved in adrenomedullin binding to the AM1 receptor

Background and Purpose Adrenomedullin (AM) is a peptide hormone whose receptors are members of the class B GPCR family. They comprise a heteromer between the GPCR, the calcitonin receptor-like receptor and one of the receptor activity-modifying proteins 13. AM plays a significant role in angiogenesis and its antagonist fragment AM2252 can inhibit blood vessel and tumour growth. The mechanism by which AM interacts with its receptors is unknown. Experimental Approach We determined the AM2252 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 AM2252 at the AM1 receptor to the C-terminal 8 amino acids. Isothermal titration calorimetry of AM2252 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. Characterization 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 with AM2252. R44A, K46A, S48A and P49A AM2252 decreased antagonist potency by approximately 10-fold. Conclusions and Implications This study localizes the main binding epitope of AM2252 to its C-terminal amino acids and distinguishes essential residues involved in this binding. This will inform the development of improved AM receptor antagonists.