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

Abstract

We determined the AM22-52 binding epitope for the AM1 receptor extracellular domain using biophysical techniques, heteronuclear magnetic resonance spectroscopy and alanine scanning. KEY RESULTSChemical 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 Ն100-fold reduction in antagonist potency compared with AM22-52. R44A, K46A, S48A and P49A AM22-52 decreased antagonist potency by approximately 10-fold. CONCLUSIONS AND IMPLICATIONSThis 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.

Selective CGRP and adrenomedullin peptide binding by tethered RAMP‐calcitonin receptor‐like receptor extracellular domain fusion proteins

“…Our DsbC-assisted disulfide shuffling methodology is distinct from the traditional denaturant-based refolding protocols employed by other groups to produce CLR-RAMP ECD complexes. 17,27,37 We previously employed our methodology to produce several other class B GPCR ECDs with 3 disulfide bonds. [30][31][32][33][34] Here, the methodology was successful for proteins with up to 6 disulfide bonds, which suggests that it may be more broadly applicable to various disulfide bond-containing proteins.…”

“…22,26,36,37 It was recently demonstrated that the C-terminal eight amino acids of AM, in the context of the 22-52 fragment, constituted the primary AM receptor ECD binding epitope. 27 We examined the ability of N-terminally truncated CGRP and AM peptides of varying lengths to bind their respective tethered receptor ECD fusion proteins in the competition AlphaScreen assay. In agreement with previous studies, we observed that CGRP peptides as short as (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) retained the ability to bind the tethered CGRP receptor ECD fusion protein and that the affinity of the (27-37) fragment was only slightly reduced compared to the (8-37) fragment (Table I).…”

“…27 We examined the ability of N-terminally truncated CGRP and AM peptides of varying lengths to bind their respective tethered receptor ECD fusion proteins in the competition AlphaScreen assay. In agreement with previous studies, we observed that CGRP peptides as short as (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) retained the ability to bind the tethered CGRP receptor ECD fusion protein and that the affinity of the (27-37) fragment was only slightly reduced compared to the (8-37) fragment (Table I). The AM(37-52) fragment retained the ability to bind the tethered AM receptor ECD fusion with similar affinity as the (22-52) fragment, but the (39-52) fragment exhibited significantly diminished binding and the (41-52) fragment did not exhibit any detectable binding (Table I).…”

“…21 It is unclear if CGRP and AM bind to their receptor ECDs as continuous a-helices as observed for other class B GPCR ECD-peptide pairs. Chimeric receptor and mutagenesis studies indicated that the RAMP ECDs are sufficient to determine peptide selectivity and identified receptor and CGRP and AM peptide residues that are important for their interactions, [22][23][24][25][26][27] but precisely how RAMPs determine peptide selectivity remains uncertain. The CGRP and AM receptor stoichiometries are a topic of debate.…”

“…17 Watkins et al reported the production and biochemical characterization of recombinant CLR:RAMP2 ECD complex with evidence for a 2:1 CLR:RAMP2 stoichiometry. 27 We previously described novel methodology for bacterial production of the AM receptor ECD complex as a maltose binding protein (MBP)-CLR ECD fusion protein in association with the RAMP2 ECD. 29 Fusion of the CLR ECD to MBP was designed to facilitate crystallization as for other class B GPCR ECDs.…”