Structure and Dynamics of Adrenomedullin Receptors AM1 and AM2 Reveal Key Mechanisms in the Control of Receptor Phenotype by Receptor Activity-Modifying Proteins

Abstract

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors are critically important for metabolism, vascular tone, and inflammatory response. AM receptors are also required for normal lymphatic and blood vascular development and angiogenesis. They play a pivotal role in embryo implantation and fertility and can provide protection against hypoxic and oxidative stress. CGRP and AM receptors are heterodimers of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) (CGRPR), as well as RAMP2 or RAMP3 (AM₁R and AM₂R, respectively). However, the mechanistic basis for RAMP modulation of CLR phenotype is unclear. In this study, we report the cryo-EM structure of the AM₁R in complex with AM and Gs at a global resolution of 3.0 Å, and structures of the AM₂R in complex with either AM or intermedin/adrenomedullin 2 (AM2) and Gs at 2.4 and 2.3 Å, respectively. The structures reveal distinctions in the primary orientation of the extracellular domains (ECDs) relative to the receptor core and distinct positioning of extracellular loop 3 (ECL3) that are receptor-dependent. Analysis of dynamic data present in the cryo-EM micrographs revealed additional distinctions in the extent of mobility of the ECDs. Chimeric exchange of the linker region of the RAMPs connecting the TM helix and the ECD supports a role for this segment in controlling receptor phenotype. Moreover, a subset of the motions of the ECD appeared coordinated with motions of the G protein relative to the receptor core, suggesting that receptor ECD dynamics could influence G protein interactions. This work provides fundamental advances in our understanding of GPCR function and how this can be allosterically modulated by accessory proteins.

Figure 1

Refined EM maps of the AM receptor complexes. (A–C) AM:CLR:RAMP2:GsDN:Nb35 complex. (D–F) AM:CLR:RAMP3:GsDN:Nb35 complex. (G–I) AM2:CLR:RAMP3:GsDN:Nb35 complex. (A, D, G) Full maps and receptor only maps (D, G) containing the backbone model of the complexes in ribbon format; CLR (blue), RAMP2 (green), RAMP3 (coral), AM (red), AM2 (dark pink), G protein α-subunit (gold), β-subunit (cyan), γ-subunit (dark purple), and Nb35 (white). (B, E, H) Local-resolution-filtered EM maps displaying local resolution (Å) colored from highest (dark blue) to lowest resolution (red). (C, F, I) Gold standard Fourier shell correlation (FSC) curves for the final maps and map validation from half maps, showing overall nominal resolutions of 3.0, 2.4, and 2.3 Å for the AM:AM₁R (C), AM:AM₂R (F), and AM2:AM₂R (I), respectively, and 2.6 Å for the receptor only maps (F, I). (J–L) 3D histogram representations of the Euler angle distribution of all the particles used in the reconstruction overlaid on the density map drawn on the same coordinate axis for complexes of the AM:AM₁R (J), AM:AM₂R (K), and AM2:AM₂R (L), respectively.

Figure 2

Overlay of the backbone structures in protein worm format of (A) the AM:AM₁R (CLR:R2:AM) and AM:AM₂R (CLR:R3:AM) complexes and (B) the AM:AM₂R and AM2:AM₂R (CLR:R3:AM2) complexes. The peptide and G proteins have been omitted for clarity. (A) RAMP2 and RAMP3 ECDs have different orientations relative to the CLR ECD, whereas (B) the difference in positioning of the ECD between the two AM₂Rs is due to a rigid body lateral movement. Distances in the TM domain in (A) are between the Cα of TM6 L351^6.55 (∼5 Å) and ICL2 F246^ICL2 (∼3 Å) and in (B) are ECL1 V205^ECL1 (2.6 Å) and TM7 V364^7.37 (2.6 Å). The CLR in the RAMP3 (R3) complexes is colored blue, and it is gray in the RAMP2 complex. RAMP2 is green, and RAMP3 is coral.

Figure 3

(A) Overlay of the backbone structures in ribbon format of the CGRP bound CGRPR (CLR:R1:CGRP), AM-bound AM₁R (CLR:R2:AM), and AM-bound AM₂R (CLR:R3:AM) complexes. CLR is colored as follows: in the CGRPR, dark pink, in the AM₁R, gray, and in the AM₂R, blue. RAMP1 is colored dark red, and RAMP2 is shown as green. RAMP3 is shown as coral. (B) Backbone (ribbon format) overlay of the AM:CLR:RAMP2 structure with the X-ray crystal structure of the ECDs of AM:CLR:RAMP2 (4RWF). Colors are as follows: cryo-EM structure: AM (red), CLR (blue), RAMP2 (green); 4RWF: AM (orange), CLR (light blue), RAMP2 (aquamarine).

Figure 4

CLR-RAMP interface for the AM_AM₁R (A), AM_AM₂R (B), and AM2_AM₂R (C). Map to model figures illustrating tightness of TM packing (left panels), and extent of engagement of the proximal RAMP linker with ECL2 (right panel). Map density for the RAMP is shown as mesh. Map density for CLR is shown as a transparent surface. RAMP2 (green), RAMP3 (coral), and CLR (blue).

Figure 5

AM and AM2 peptide binding to AM receptors. (A) Electrostatic surface potential for CLR for each of the receptor complexes (AM₁R ECD is from PDB: 4WRF), with the peptides and RAMPs shown as ribbon representation. Colors are as follows: in the AM₁R: AM (red) and RAMP2 (green); in the AM₂R: AM (dark red) and RAMP3 (light red); in the AM₂R: AM2 (dark pink) and RAMP3 (light pink). The electrostatic potential ranges from −5 (red) to +5 (blue) kT e^–1. (B) CLR residues selectively engaged (left panels) or differentially engaged (right panels) by equivalent amino acids of AM with the AM₁R and AM₂R, with common residues shown as gray surface representation and distinct interactions mapped in red (AM_AM₂R) or dark red (AM_AM₁R); specific interacting residues are detailed below. (C) CLR residues selectively engaged (left panels) or differentially engaged by (right panels) positionally equivalent amino acids of AM or AM2 with the AM₂R, with common residues shown as gray surface representation and distinct interactions mapped in red (AM_AM₂R) or dark pink (AM2_AM₂R); specific interacting residues are detailed below. The location of the deepest peptide residue in the binding pocket is highlighted (G19^AM; G13^AM2). For clarity in (B) and (C), CLR is colored differently; other colors are as follows: light gray, AM_AM₁R; blue, AM_AM₂R; light blue, AM2_AM₂R.

Figure 6

N-terminal peptide interactions with the TM core of CLR for the AM-bound AM₁R (A), AM-bound AM₂R (B), and AM2-bound AM₂R (C) calculated with using LigPlot+. Peptide residues are colored dark red (AM at AM₁R), red (AM at AM₂R), and dark pink (AM2 at AM₂R), and receptor residues are colored blue. Hydrophobic interactions are illustrated by red (AM) or pink (AM2) arcs with CLR in the reverse color, and interacting residues are joined by a red line. Amino acids involved in H-bonds are shown in atomic detail and H-bonds are shown as dashed green lines. (D) Structure models of N-terminal peptide binding to the AM₁R (left panel; AM, dark red; RAMP2, green; CLR, gray) or the AM₂R (middle panel: AM, red; RAMP3, coral; CLR, blue; right panel: AM2, dark pink; RAMP3, coral; CLR, light blue). The protein backbone is shown in a ribbon format, and side chains are shown in the x-stick format.

Figure 7

Comparison of the receptor:G protein interface across CLR:RAMP receptor heterodimers. (A) Overlay of the intracellular face of the CGRP and AM₁ receptors, highlighting the common positioning of most side chains. The largest exception was in the position of F246 in ICL2 that occupied a common position between the CGRPR and AM₁R (upper panel), as well as between the different peptide-bound AM₂R (middle panel), but was distinct between AM₂R and the other receptors (lower panel). (B) The G protein occupies a common global interaction position that is highly similar in conformation across receptors. (C) Close up of panel B, boxed area, focusing on the interaction with ICL2. (D) The bound Gs heterotrimer has a similar surface electrostatic potential when binding to AM receptors. The protein backbone is shown in a ribbon format, and side chains are shown in the x-stick format. RAMP1 is colored dark red, RAMP2, green, RAMP3, coral. CLR in the CGRPR is colored dark pink, in the AM-bound AM₁R, gray, in the AM-bound AM₂R, blue, in the AM2-bound AM₂R, light blue. G proteins are colored equivalent to CLR for each receptor.

Figure 8

CLR–Gαs interface for AM_AM₁R (A), AM_AM₂R (B), and AM2_AM₂R (C). Interactions were determined using LigPlot+. Gαs residues are gold, and receptor residues are blue. Hydrophobic interactions are illustrated by red or pink arcs adjacent to residue labels, and interacting residues are joined by a red line. Amino acids involved in H-bonds are shown in atomic detail with H-bonds shown as dashed green lines. (D) Summary of the specific interactions. Interactions common to all 3 receptor complexes are shown in bold and are shaded light green. Residues involved in H-bond interactions are shown in green type. Interactions common to AM bound to AM₁R and AM₂R only are shaded light orange. Interactions common to complexes of the AM₂R only are shaded yellow. Interactions common to AM bound to AM₁R and AM2 bound to AM₂R only are shaded blue.

Figure 9

cryoSPARC multivariate analysis of the AM-bound AM₁R (A), AM-bound AM₂R (B), and AM2-bound AM₂R (C) focusing on the interface of the RAMP and ECL2. Three normal modes were captured as 20 map files for each receptor complex and snapshots of the first and last frame (F1 and F19, respectively) are displayed for the AM₂R complexes (B, C). For AM₁R, due to the greater noise in the data, the early and late frames primarily reflected loss of signal; as such, F6 and F16, which represent the ends of the motion, are displayed (A). Map density is displayed as a gray surface.

Figure 10

cryoSPARC multivariate analysis of the AM-bound AM₁R (A), AM-bound AM₂R (B), and AM2-bound AM₂R (C) focusing on the interface of Gαs and the receptors, particularly the RAMP C-terminus and ICL2. Three normal modes were captured as 20 map files for each receptor complex and snapshots of the first and last frame (F1 and F19, respectively) are displayed for the AM₂R complexes (B, C). For the AM₁R, due to the greater noise in the data, the early and late frames primarily reflected loss of signal; as such, F6 and F16, which represent the ends of the motion, are displayed (A). Map density is displayed as a gray surface. Color key: CLR, blue; RAMP2, green; RAMP3, coral; Gαs, gold. The end of the resolved density for each of the RAMPs is illustrated with a dashed line labeled with R2b (RAMP2) or R3b (RAMP3).

Figure 11

Pharmacological analysis of RAMP1 linker chimeras with RAMP2 or RAMP3. (A) Amino acid sequence of the linker regions (numbered using the RAMP1 sequence for simplicity), with the different length chimeras denoted by colored boxes: red, whole linker (102–118); blue, N-terminal linker region (102–112); green, mid-linker region (108–112); purple, C-terminal linker region (116–118). Conserved residues are in gray, and divergent residues are in black. (B, C) Peptide concentration–response was measured in cAMP accumulation assay, following transient expression of constructs into COS-7 cells, for full linker exchange (B, upper panel) or exchange of the 108–112 segment (B, lower panel), the 116–118 segment (C, upper panel), or the 102–112 segment (C, lower panel).

Figure 12

Pharmacological analysis of RAMP2 linker chimeras with RAMP1 or RAMP3 (A) or RAMP3 chimeras with RAMP1 or RAMP2 (B). Peptide concentration–response was measured in cAMP accumulation assay, following transient expression of constructs into COS-7 cells, for full linker exchange (A, B, upper panels) or exchange of the RAMP2 108–112 segment, the 116–118 segment, or the 102–112 segment with that of RAMP3 (A, lower panel), or exchange of the RAMP3 108–112 segment, the 116–118 segment, or the 102–112 segment with that of RAMP2 (B, lower panel).