Abstract
The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor-like class B G protein-coupled receptor (CLR) and a variable receptor activity-modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM1 (RAMP2:CLR) and AM2 (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP-CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM2 receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment-bound RAMP1-CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple β-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM1 and AM2 receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.
Highlights
AM2/IMD turn 1 is a type VIII -turn, and turns 2 and 3 are type I -turns [40]
47), and AM2/IMD[32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47] bound MBP-RAMP3–CLR ECD with similar affinities that were slightly weaker than the traditional antagonist fragment, whereas the AM2/IMD[35,36,37,38,39,40,41,42,43,44,45,46,47] and AM2/IMD(38 – 47) fragments exhibited more pronounced reductions in binding affinity (Table S2)
We used MBP-RAMP1–CLR ECD for crystallization because it strongly bound AM2/IMD and we could costeffectively produce it in E. coli
Summary
Expression and purification of soluble tethered RAMP–CLR ECD complexes of the human CGRP, AM1, and AM2 receptors in their native N-glycosylated states. We used a competition assay format to determine the equilibrium dissociation constants (pKI) for unlabeled AM2/IMD(16 – 47), AM[22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52], and CGRP(8 –37) (Fig. 2 (D–F) and Table 1) These traditional antagonist fragments include the C-terminal ECD-binding region and the central ␣-helical region that contacts the CLR 7TM domain, but they lack the N-terminal disulfide-bonded portion required for 7TM domain activation (Fig. 1C). Ala-37 was substituted with Gly. Binding of the mutant peptides to the three N-glycosylated MBP-RAMP–CLR ECD fusion proteins was assessed in a single point FP competition assay (Fig. 3, G–I). Effects of alanine substitutions in RAMP1:CLR on AM2/IMD activation of cAMP signaling *, p Ͻ 0.05
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call