Insights into the structure of class B GPCRs

Abstract

The secretin-like (class B) family of G protein-coupled receptors (GPCRs) are key players in hormonal homeostasis and are interesting drug targets for the treatment of several metabolic disorders (such as type 2 diabetes, osteoporosis, and obesity) and nervous system diseases (such as migraine, anxiety, and depression). The recently solved crystal structures of the transmembrane domains of the human glucagon receptor and human corticotropin-releasing factor receptor 1 have opened up new opportunities to study the structure and function of class B GPCRs. The current review shows how these structures offer more detailed explanations to previous biochemical and pharmacological studies of class B GPCRs, and provides new insights into their interactions with ligands.

Keywords: class B G protein-coupled receptor (GPCR); corticotropin-releasing factor receptor 1 (CRF(1)); crystal structure; glucagon receptor (GCGR); ligand binding.

Figure 1

(a, b) Crystal structures of class B GPCRs CRF₁ (PDB ID 4K5Y) and GCGR (PDB ID 4L6R) are shown in blue and orange ribbons, respectively, in two different views from within the membrane. TM helices and helix 8 are labeled. The disulfide bond tethering ECL2 to the tip of TM3 is shown as purple sticks. In CRF₁ the small-molecule antagonist CP-376395 is shown in stick representation with carbon, nitrogen and oxygen atoms colored magenta, blue and red, respectively, and as skeletal formula in an inset. (c) Superposition of the two structures, with insets highlighting regions of particular interest. To highlight the structural differences in the extracellular halves of CRF₁ and GCGR, the distance of approximately 11 Å between the Cα-atoms of residues 7.33b at the N-terminal end of TM7 is indicated with a red arrow. The small-molecule binding pocket is shown as a superposition of the two receptors around CP-376395, illustrating the antagonist binding mode and the substantial structural differences observed for TM6 of the two receptors.

Figure 2

(a) Overview of NMR and crystal structures of class B GPCR ECDs (magenta) and their complexes with peptide ligands (different colors) [,,,,,,–68]. A complete overview of corresponding PDB IDs is presented in Table 1. (b) Structure-based sequence alignment of representative peptide ligands of class B GPCR, adopted from Parthier et al. [6]. The residues of the peptide ligands solved in ECD-ligand complex crystal structures [,,,,,,–68] are marked using the same color as in Figure 2. The residues that are boxed black are found in an alpha helical conformation in the complex. Peptide ligand residues that covalently bind receptors in photo cross-linking or cysteine trapping studies [22,25,47,50,54,69,70] are colored and boxed green, while peptide ligand residues that have been mutated and studied in combination with receptor mutants [29,30,45,46,48] are colored and boxed red. Note that the first residue of GLP-1 is His7. A complete overview of all ECD structures and important peptide ligands for all class B GPCRs is presented in Table 1. Putative helix-capping residues [6] are colored blue and cysteines involved in a disulfide-bridge (calcitonin) are colored orange. D-phenylalanine (f), and norleucine (m) residues are indicated in stressin and asstressin. The last 41 and 99 residues of PTH and PTHrP, respectively, are not displayed.

Figure 3

The spatial correspondence between residues in TM helices of class A and class B GPCRs makes it possible to align the most conserved residues in class A (designated X.50, Ballesteros-Weinstein numbering) and class B (designated X.50b, Wootten numbering) for comparisons between GPCR classes (Box 1). (b) Structural alignment of CRF₁ (blue) and GCGR (orange) to two representative class A GPCRs, H₁R (PDB ID 3RZE) and CXCR4 (PDB IDs 3ODU/3OE0) (in grey). Helices are depicted as cylinders, ligands glucagon (GCGR), CP-376395 (CRF₁), doxepin (H₁R), and IT1t and CVX15 (CXCR4) as sticks. The location of the Cα-atoms of the most conserved residues of TM1–3 and TM5 among class A and class B GPCRs (Box 1) are indicated by spheres (TM4 is not depicted for clarity).

Figure 4

(a) Model of full GCGR-glucagon complex [11]. GCGR with the ECD (PDB ID 4ERS, in magenta) and TMD (PDB ID 4L6R, transparent surface) bound to glucagon (green). The effects of mutation studies of individual GCGR residues on glucagon binding are mapped onto the GCGR binding surface using the color coding presented in Figure 4b. (b) Effects of mutation studies in GCGR, GLP-1, GIP, secretin, VPAC₁, PTH₁, and CRF₁ receptors [,,,,,,–,–53] are mapped on a structure-based (Figure 3b) sequence alignment between representative class A GPCR crystal structures (H₁R and CXCR4) and representative class B GPCRs. Only specific parts of TM1–3 and TM5–7 are shown, separated by grey dashed lines. Mutated residues that show <4 fold (blue), 4–10 fold (orange), and >10 fold (red) changes of K_i/IC₅₀ values for ligand binding (or ligand potency/EC₅₀ value if no K_i/IC₅₀ value has been reported) are marked (peptide ligands) and colored (non-peptide ligands). Mutants that show receptor expression <30% of wild-type are marked grey. Ligand contact residues in CRF₁ (Figure 1c), H₁R, and CXCR4 crystal structures are boxed red. The most conserved residues in TM1–7 of class A (x.50) and class B (x.50b) GPCRs (Box 1, Figure 3a) are shown in bold. Receptor residues that covalently bind peptide ligands in photo cross-linking or cysteine trapping studies [22,25,47,50,54,69,70] are boxed green. Note that several mutation and photo cross-linking studies annotated for secretin and PTH receptors are performed with rat orthologs. Residues that are mutated in the conformationally thermostabilized [10] CRF₁ crystal structure are underlined.

Figure 5

Comparison of druggable binding sites of (a) GCGR, (c) CRF₁, and the class A GPCRs (b) CXCR4 (PDB ID 3ODU/3EO0) and (d) H₁R (PDB ID 3RZE). The surfaces of binding sites that are considered druggable [59] are colored orange (orthosteric pocket) and red (CP-376395 binding pocket). Of eight structurally aligned residues (see Figure 4b), the residues that line the binding pocket shown in the figures are labeled black; the residues that are not part of the depicted pockets are labeled light grey. Note that A^6.56b and A^7.43b are thermostabilizing mutations [10] in the CRF₁ crystal structure (see also Figure 4b). The approximate position of the extracellular membrane boundary is shown as a dotted black line. Carbon atoms of peptide ligands (glucagon in GCGR and CVX15 in CXCR4) and non-peptide ligands (IT1t in CXCR4 and doxepin in H₁R) are colored green and magenta, respectively.