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. 2023 Mar 30;186(7):1465-1477.e18.
doi: 10.1016/j.cell.2023.02.028.

Negative allosteric modulation of the glucagon receptor by RAMP2

Kaavya Krishna Kumar  1 , Evan S O'Brien  1 , Chris H Habrian  1 , Naomi R Latorraca  2 , Haoqing Wang  1 , Inga Tuneew  3 , Elizabeth Montabana  1 , Susan Marqusee  4 , Daniel Hilger  5 , Ehud Y Isacoff  6 , Jesper Mosolff Mathiesen  3 , Brian K Kobilka  7
Affiliations

Negative allosteric modulation of the glucagon receptor by RAMP2

Kaavya Krishna Kumar et al. Cell. .

Abstract

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

Keywords: G-protein; G-protein coupled receptor; GPCR; HDX-MS; allostery; cell signaling; cryo-EM; cryo-electron microscopy; glucagon receptor; hydrogen-deuterium exchange monitored by mass spectrometry; protein dynamics; receptor activity-modifying protein RAMP; single molecule fluorescence resonance energy transfer; smFRET.

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Conflict of interest statement

Declaration of interests B.K.K. is a cofounder of and consultant for ConfometRx. J.M.M. and I.T. are employees of Zealand Pharma A/S.

Figures

Figure 1.
Figure 1.. Biochemical and in-cellulo consequences of RAMP2 interaction with GCGR.
(A) The GTP turnover assay shows that GCGR activation of Gs is largely independent of GDP concentration, but upon pre-coupling of GCGR with RAMP2, Gs activation is potently inhibited in a [GDP] dependent manner and appears to inhibit even intrinsic Gs activity. (B) The rate of 3H-GDP release from Gs is significantly reduced when GCGR is pre-incubated with RAMP2. (C) Increasing the cell-surface expression of the glucagon receptor, but not β2AR, results in an increase in surface expression of RAMP2. (D) Cells transfected with GCGR display increases in cAMP levels upon stimulation with glucagon with an observed EC50 of 0.28 nM (pEC50=9.6 [95% confidence limits 9.8–9.3]); cells expressing the same levels of GCGR in the presence of excess transfected RAMP2 display an EC50 of 1.10 nM (pEC50=9.0 [95% confidence limits 9.1–8.8]), a 4-fold right-shift in potency.
Figure 2.
Figure 2.. Design and characterization of a GCGR antagonist and widespread RAMP2-induced GCGR conformational changes.
(A) Sequences and estimated affinities of previously described GCGR antagonist des-His1[Glu9]-Glucagon-amide (KB =80 nM) based on the glucagon scaffold, and the high affinity GCGR antagonist ZP7680 (KB =2.6 nM) derived from the dasiglucagon scaffold. See Methods and Fig. S2 for details on the design and characterization of ZP7680 and TAMRA-labeled peptides. Nle; norleucine, Aib=2-aminoisobutyric acid. K-T; TAMRA-labeled lysine. (B) Observed affinities of various TAMRA-labeled peptides in the presence or absence of co-expressed RAMP2. GCGR antagonist (ZP7680-30K(TAMRA)) shows a slight (~1.5-fold, P<0.05) decrease in affinity in the presence of RAMP2, while both agonist peptides (Dasi-E21S-A17K(TAMRA) and ZP3780-E20K(TAMRA)) display 2.9 (P<0.0001) and 2.1-fold (P=0.0001) decreases in affinity, respectively (one way ANOVA, Šidák multiple comparisons test). (C) The antagonist ZP7680 has minimal intrinsic efficacy compared to des-His1[Glu9]-Glucagon-amide. Under conditions allowing for cAMP accumulation by addition of the PDE inhibitor IBMX, des-His1[Glu9]-Glucagon-amide have some agonistic activity whereas ZP7680 is largely silent. (D) In an ATP depletion assay, the presence of RAMP2 inhibits receptor phosphorylation regardless of what is bound in the orthosteric site, though the inhibitory effect is most significant for agonist-induced phosphorylation (P<0.001), where receptor phosphorylation returns to levels seen in the apo-state (P is not significant). Using agonist-bound GCGR site-specifically labeled at the intracellular end of TM6 (E) or on the N-terminal helix of the ECD (F) with the environmentally sensitive fluorophore NBD, addition of increasing amounts of RAMP2 results in titratable environmental changes in both regions of the receptor. P values in B and D are denoted as follows: ns (P>0.05), * (P≤0.05), ** (P≤0.01), *** (P≤0.001), and **** (P≤0.0001).
Figure 3.
Figure 3.. RAMP2 binding broadly increases the conformational heterogeneity in GCGR.
(A) Alphafold model of GCGR-RAMP2 complex onto which changes in HDX upon complex formation are plotted. (B) Example HDX-MS exchange curves in the presence and absence of excess RAMP2 (independent replicates in solid and dashed lines) show that several key regions of the RAMP2 (153–162) and receptor (33–55) are impacted by heterodimer complex formation. (C) HDX-MS plots showing bimodal distribution in GCGR TM1 (residues 128–137), an indication of conformational heterogeneity. This heterogeneity is increased in the presence of RAMP2. Though not present in the absence of RAMP2, a bimodal distribution is induced in TM6 (348–362) upon addition of RAMP2.
Figure 4.
Figure 4.. Enhanced conformational heterogeneity stabilizes an inactive intracellular conformation of TM6.
(A) smFRET experiments show that a dominant high-FRET state (~0.83) of GCGR labeled with donor and acceptor fluorophores at the intracellular ends of TM4 and TM6 is present in the absence of orthosteric agonist (A, black, N=162) with brief excursions to mid- (~0.63) FRET (~0.32) states (C, black), and the binding of agonist peptide results in a dominant mid-FRET state at the expense of the high-FRET state (A, blue, N=150; C). The mid-FRET intermediate and low-FRET active conformations of TM6 induced by agonist peptide are largely abrogated by the presence of RAMP2 (A, purple, N=117) as the distribution shifts towards and inactive-like conformation. Gs coupling to agonist-bound GCGR increases the population of the low-FRET, suggesting a full outward movement of TM6 at the expense of the mid-FRET agonist-specific intermediate state and high-FRET inactive state (B, cyan, N=158; C). However, pre-coupling of agonist-bound GCGR with RAMP2 potently inhibits a Gs-induced increase in the population of the fully outward TM6 conformation as well as the agonist-associated intermediate state (B, salmon, N=179; C). Histograms are shown with a 3-Gaussian fit to the data.
Figure 5.
Figure 5.. Structure of GCGR-Gs in the presence of RAMP2.
(A) Cryo-EM density map of the ZP3780 and Gs heterotrimer bound GCGR in the presence of RAMP2 colored by subunit. Salmon, GCGR; blue, ZP3780; Gαs, green; Gβ, grey; Gγ, cyan. Cryo-EM density for ZP3780-bound GCGR in the presence (B) and absence (C) of RAMP2 colored by local resolution demonstrates a significant disordering of the receptor ECD in the presence of RAMP2. The intracellular interface of GCGR with Gαs is perturbed by RAMP2 in several significant ways (D), including inducing disorder in ICL3 and the resulting loss of contacts with Gαs (black, top), a downward movement of ICL2 (purple), and a lack of stabilizing contacts across TM5/TM6 (black, bottom). Further, there are rearrangements in the backbone and side chains in the TCAT motif at the base of the α5 helix of Gαs. Comparing this motif to GDP-bound Gs (E) it is clear that this important loop in the G protein occupies a distinct conformation.
Figure 6.
Figure 6.. RAMP2-induced conformational dynamics in the GCGR ECD inhibits intracellular activation.
(A) The major principal component of the 3D variability analysis directly demonstrates that changes in the GCGR ECD and agonist conformation are linked to changes in the intracellular conformation including ICL3 and H8. (B) Proposed model for RAMP2-induced inhibition of GCGR activation. Glucagon binding to GCGR promotes population of a temporally distinct intermediate conformation of TM6, followed by full outward movement upon binding to Gs. RAMP2 binding to GCGR causes enhanced ECD conformational sampling as observed in both HDX-MS solution experiments as well as cryo-EM structural analysis. This increase in dynamics in the ECD inhibits formation of both intermediate and active states of TM6 observed by smFRET experiments, and any engagement with Gs results in a largely unproductive complex.
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