Structure of the human glucagon class B G-protein-coupled receptor

Abstract

Binding of the glucagon peptide to the glucagon receptor (GCGR) triggers the release of glucose from the liver during fasting; thus GCGR plays an important role in glucose homeostasis. Here we report the crystal structure of the seven transmembrane helical domain of human GCGR at 3.4 Å resolution, complemented by extensive site-specific mutagenesis, and a hybrid model of glucagon bound to GCGR to understand the molecular recognition of the receptor for its native ligand. Beyond the shared seven transmembrane fold, the GCGR transmembrane domain deviates from class A G-protein-coupled receptors with a large ligand-binding pocket and the first transmembrane helix having a 'stalk' region that extends three alpha-helical turns above the plane of the membrane. The stalk positions the extracellular domain (~12 kilodaltons) relative to the membrane to form the glucagon-binding site that captures the peptide and facilitates the insertion of glucagon's amino terminus into the seven transmembrane domain.

Figure 1. Structure of the 7TM domain of human GCGR and comparison to class A GPCR structures

a, Cartoon depiction of the 7TM domain structure of GCGR. The two views are rotated 180° relative to each other. The disulfide bond between helix III and extracellular loop 2 (ECL2) is shown as yellow sticks. b, Side view of structural superimposition of 7TM domains of GCGR (blue) and class A GPCRs (grey). Structures of class A GPCRs used (PDB): 1U19, 2RH1, 2YCW, 3RZE, 3PBL, 3UON, 4DAJ, 3EML, 3V2W, 3ODU, 4DJH, 4EA3, 4DKL, 4EJ4, and 3VW7. Extracellular (EC) and intracellular (IC) membrane boundaries are shown as brown and cyan ovals (a) or dotted lines (b), respectively.

Figure 2. Comparison of ligand binding pocket of GCGR with class A GPCRs

The binding cavity of GCGR compared with the binding cavities of human chemokine receptor CXCR4 (PDB 3ODU), human κ-opioid receptor (κ-OR) (PDB 4DJH), rat neurotensin receptor (NTSR1) (PDB 4GRV), human β₂ adrenergic receptor (β₂AR) (PDB 2RH1), and bovine rhodopsin (Rho) (PDB 1U19) for comparison purposes (Supplementary Table 4). The approximate position of the EC membrane boundary is shown as a red line, and bound ligands as magenta carbon atoms.

Figure 3. Structural features of class B GPCRs

Comparison of GCGR and class A GPCRs crystal structures indicates distinct and conserved features. a, d, and e, The homologous GCGR residues involved in helix I – II, III – IV, and III –VI interface interactions as discussed for class A receptors by Venkatakrishnan et al, and class B GPCR specific residues that mediate helix I – VII, II – VII, and III – V interface interactions. b, GCGR residues Glu406 of helix VIII, Arg173^2.46, and Arg346^6.37 form a class B receptor specific ionic network. Arg346^6.37 (grey) has a weak electron density. c, The disulfide bond between Cys224^3.29 and Cys294^ECL2 in the GCGR structure, a conserved feature between classes A and B receptors. Hydrogen bond interactions are indicated by black dashed lines. Electron density maps for residues in this figure are shown in Supplementary Fig. 10.

Figure 4. Effects of mutation studies in GCGR snake plot

a, Mutated residues that show < 4 fold (purple), 4–10 fold (orange), and > 10 fold (red) changes of IC₅₀ values for glucagon binding with receptor expression >30% of wild-type (Supplementary Table 5). Mutation studies to investigate peptide ligand binding have been previously reported for several class B GPCRs including GCGR^,,,,,,, GLP1R^,,,,,, GIPR^,, rSCTR^,,,,, and VPAC1(ref –41) (Supplementary Table 6). The most conserved residues in helices I to VII of class B GPCRs are boxed and bolded. b, c, and d, Representative binding curves of GCGR mutants with glucagon. Data are expressed as a percentage of specific ¹²⁵I-glucagon binding in the absence of unlabeled peptide. Each point (± S.E.M.) represents the mean value of three independent experiments done in triplicate (IC₅₀ are shown in Supplementary Table 5).

Figure 5. Model of GCGR bound to glucagon

a, and b, GCGR with the ECD (magenta) and 7TM domain (blue) bound to glucagon (green). Residues 122–126 and 199–218 (brown) are not defined in the GCGR ECD (GCGR-linker) (PDB 4ERS) and 7TM domain (ECL1) crystal structures, respectively. The GCGR ECD structure and the interactions between GCGR ECD and glucagon resemble those in the GCGR ECD (PDB 4ERS) and GLP1-GLP1R ECD complex (PDB 3IOL) structures, respectively. c, and d, The effects of mutation studies of individual GCGR residues on glucagon (green) binding mapped onto the GCGR binding surface using the color coding presented in Figure 4. Important glucagon residues are labeled black. GCGR residues proposed to be important in stabilizing extracellular loops are boxed. GCGR-glucagon residue pairs that are homologous to residue pairs identified in GLP1R-GLP1 cross-linking studies are underlined.