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


METHODS
We determined the AM22-52 binding epitope for the AM1 receptor extracellular domain using biophysical techniques, heteronuclear magnetic resonance spectroscopy and alanine scanning.


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. 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 AM22-52 . R44A, K46A, S48A and P49A AM22-52 decreased antagonist potency by approximately 10-fold.


CONCLUSIONS
This study localizes 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.
School of Biological Sciences, University of Auckland, Auckland, New Zealand.
H A
HA
Watkins
M
M
Au
R
R
Bobby
J K
JK
Archbold
N
N
Abdul-Manan
J M
JM
Moore
M J
MJ
Middleditch
G M
GM
Williams
M A
MA
Brimble
A J
AJ
Dingley
D L
DL
Hay
D053607:Adrenomedullin / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002151:Calorimetry / Q000379:methods; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism*; D008279:Magnetic Resonance Imaging / Q000379:methods; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D058265:Receptors, Adrenomedullin / Q000378:metabolism*
D000939:Epitopes; D010446:Peptide Fragments; D058265:Receptors, Adrenomedullin; C104782:adrenomedullin (22-52); D053607:Adrenomedullin; D000242:Cyclic AMP