Lifting the lid on GPCRs: the role of extracellular loops

Abstract

GPCRs exhibit a common architecture of seven transmembrane helices (TMs) linked by intracellular loops and extracellular loops (ECLs). Given their peripheral location to the site of G-protein interaction, it might be assumed that ECL segments merely link the important TMs within the helical bundle of the receptor. However, compelling evidence has emerged in recent years revealing a critical role for ECLs in many fundamental aspects of GPCR function, which supported by recent GPCR crystal structures has provided mechanistic insights. This review will present current understanding of the key roles of ECLs in ligand binding, activation and regulation of both family A and family B GPCRs.

Figure 1

ECL2 conformational diversity in Family A GPCRs. In Panels A–D, the ECL2 of the diffusible-ligand GPCR is compared with the ECL2 of rhodopsin (red; PDB accession 3CAP): A, β₂AR (blue; PDB accession 2RH1); B, D3R (yellow; PDB accession 3PBL); C, A_2AR (orange; PDB accession 2YDO); D, CXCR4 (green; PDB accession 3OE0). An overlay of ECL2 of all five GPCRs viewed from above (Panel E) or from within the plane of the membrane (Panel F). Only the TM bundle of rhodopsin is shown in Panels E and F for clarity.

Figure 2

ECL conformations in different receptor activation states. Panel (A) Rhodopsin: red – inactive rhodopsin (PDB accession 1U19), grey – metarhodopsin II (PDB accession 3P0X); green – constitutively active rhodopsin (PDB accession 2X72); yellow – opsin (PDB accession 3CAP); orange – opsin-GαCT (PDB accession 3DQB). In each case, the N-terminus has been omitted for greater clarity. Panel (B) β₂AR: red – carazolol (partial inverse agonist) bound (PDB accession 2RH1B2); orange – alprenolol (antagonist) bound (PDB accession 3NYA); yellow – compound 2 (inverse agonist) bound (PDB accession 3NY9); green – ICI 118551 (inverse agonist) bound (PDB accession 3NY8); blue – FAUC50 (irreversible agonist) bound (PDB accession 3PDS); grey – BI-167107 (full agonist) bound, nanobody stabilized (active conformation) (PDB accession 3P0G). Panel (C) β₁AR: grey – dobutamine (partial agonist) bound (PDB accession 2YO1); orange – carmeterol (full agonist) bound (PDB accession 2YO2); yellow -isoprenaline (full agonist) bound (PDB accession 2Y03); green – salbutumol (partial agonist) bound (PDB accession 2Y04); red – cyanopindolol (antagonist) bound (PDB accession 2VT4). Panel (D) A_2AR: red – antagonist ZM241385 bound (PDB accession 3EML); grey – agonist UK-423097 bound (PDB accession 3QAK); yellow – agonist NECA bound (PDB accession 2YDV); green – agonist adenosine bound (PDB accession 2YDO).

Figure 3

Alignment of the ECLs of Family B GPCRs. Human sequences of the entire receptors excluding the GLP2R were aligned with T-COFFEE Version_6.85 (http://www.tcoffee.org) after the putative signal peptides had been identified and manually removed. The GLP2R ECLs were subsequently aligned manually. Sequence conservation >50% is boxed in black.

Figure 4

Modes of natural peptide agonist binding at the extracellular surface of a family B GPCR. Possible binding modes for peptide agonists interacting with ECLs and associated TM bundle of family B GPCRs. Panel (A) N-terminus parallel to the TM bundle, facing TM1-TM2-TM7; panel (B) N-terminus parallel to the TM bundle, facing TM3-TM4-TM5-TM6; panel (C) N-terminus perpendicular to the TM bundle, interacting with TM1-TM2-TM7; panel (D) N-terminus perpendicular to the TM bundle, interacting with TM3-TM4-TM5-TM6.

Figure 5

The N-cap motif for peptide agonists at family B GPCRs. The structure of the PACAP 1–21 bound to the PACAP receptor (Inooka et al., 2001). Residues involved in the N-cap are indicated.