9.A.14 The G-protein-coupled receptor (GPCR) Family

G protein-coupled receptors (GPCRs) constitute a large family involved in various types of signal transduction pathways triggered by hormones, odorants, peptides, proteins, and other types of ligands. The family is so diverse that many members lack apparent sequence similarity, although they all span the cell membrane seven times with an extracellular N- and a cytosolic C-terminus. Wistrand et al. (2006) analyzed a divergent set of GPCRs and found distinct loop length patterns and differences in amino acid composition between cytosolic loops, extracellular loops, and membrane regions.

Menon et al. (2011) demonstrated that opsin is an ATP-independent phospholipid flippase in photoreceptor discs. They showed that reconstitution of opsin into large unilamellar vesicles promotes rapid flipping of phospholipid probes across the vesicle membrane. This is the first ATP-independent phospholipid flippase to be described in photoreceptor discs.

Crystal structures are available for rhodopsin, adrenergic receptors, and adenosine receptors in both inactive and activated forms, as well as for chemokine, dopamine, and histamine receptors in inactive conformations. Katritch et al. (2012) reviewed common structural features, outlined the scope of structural diversity of GPCRs at different levels of homology, and briefly discussed the impact of the structures on drug discovery. A distinct modularity is observed between the extracellular (ligand-binding) and intracellular (signaling) regions.

In the retinal binding pocket of rhodopsin, a Schiff base links the retinal ligand covalently to the Lys296 side chain. Light transforms the inverse agonist 11-cis-retinal into the agonist all-trans-retinal, leading to the active Meta II state. Crystal structures of Meta II and the active conformation of the opsin apoprotein revealed two openings of the 7-transmembrane (TM) bundle towards the hydrophobic core of the membrane, one between TM1/TM7 and one between TM5/TM6, respectively. Computational analysis revealed a putative ligand channel connecting the openings and traversing the binding pocket. Single amino acids lining the channel were replaced, and 11-cis-retinal uptake and all-trans-retinal release were measured (Piechnick et al., 2012). Most mutations slow or accelerate both uptake and release, often with opposite effects, and mutations closer to the Lys296 active site show larger effects. The mutations do not probe local channel permeability but affect global protein dynamics, with the focal point in the ligand pocket. Piechnick et al. (2012) proposed a model for retinal/receptor interaction in which the active receptor conformation sets the open state of the channel for 11-cis-retinal and all-trans-retinal, with positioning of the ligand at the active site as the kinetic bottleneck. Although other G protein-coupled receptors lack the covalent link to the protein, the access of ligands to their binding pocket may follow similar schemes.

Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor and a fused ion channel, engineered to couple channel gating to ligand binding. These biological entities have potential use in drug screening and functional characterization, in addition to providing new tools in the synthetic biology repertoire as synthetic K⁺-selective ligand-gated channels. The ICCR concept has been validated with fusion proteins between the K⁺ channel Kir6.2 and muscarinic M₂ or dopaminergic D₂ receptors. Caro et al. (2011) extended the concept to the longer β₂-adrenergic receptor which, unlike M₂ and D₂ receptors, displayed barely detectable surface expression and did not couple to Kir6.2 when unmodified. However, a Kir6.2-binding protein, the N-terminal transmembrane domain of the sulfonylurea receptor, greatly increased plasma membrane expression of β₂ constructs.

The generalized reaction catalyzed by opsin is:

lipid (inner leaflet) ⇌ lipid (outer leaflet)

This family belongs to the Rhodopsin Superfamily.