From atomic structures to neuronal functions of g protein-coupled receptors

Abstract

G protein-coupled receptors (GPCRs) are essential mediators of signal transduction, neurotransmission, ion channel regulation, and other cellular events. GPCRs are activated by diverse stimuli, including light, enzymatic processing of their N-termini, and binding of proteins, peptides, or small molecules such as neurotransmitters. GPCR dysfunction caused by receptor mutations and environmental challenges contributes to many neurological diseases. Moreover, modern genetic technology has helped identify a rich array of mono- and multigenic defects in humans and animal models that connect such receptor dysfunction with disease affecting neuronal function. The visual system is especially suited to investigate GPCR structure and function because advanced imaging techniques permit structural studies of photoreceptor neurons at both macro and molecular levels that, together with biochemical and physiological assessment in animal models, provide a more complete understanding of GPCR signaling.

Figure 1

Current phylogenetic tree for GPCRs. GPCR gene names are located on branches on the basis of their primary sequence similarity. Solved structures are represented using gene names on blue backgrounds. Colored backgrounds indicate groups of GPCRs that share sequence homology with determined X-ray structures that are 35% or higher. Major branches such as the adhesion, secretin, glutamate, Frizzled/TAS2, and rhodopsin are labeled, as well. Figure adapted from Katritch et al. (2012).

Figure 2

Gallery of selected GPCR structures determined by X-ray crystallography. Available X-ray structures are shown in the same orientation for all GPCRs together with an alignment graph of each GPCR pair obtained using flexible structure alignment by chaining aligned fragments (Ye & Godzik 2003). (a) bovine rhodopsin (PDB code: 1U19); (b) β₂-adrenergic receptor/T4 lysozyme chimera (PDB code: 2RH1); (c) thermostabilized β₁-adrenergic receptor (PDB code: 2Y01); (d ) squid rhodopsin (PDB code: 3AYN); (e) adenosine receptor/T4 lysozyme chimera (PDB code: 3EML); ( f ) CXCR4 chemokine/T4 lysozyme chimera (PDB code: 3ODU); ( g) dopamine D3 receptor/T4 lysozyme chimera (PDB code: 3PBL); (h) human histamine H1 receptor/T4 lysozyme chimera (PDB code: 3RZE); (i ) human M2 muscarinic acetylcholine receptor/T4 lysozyme chimera (PDB code: 3UON); ( j) κ-opioid receptor/T4 lysozyme chimera (PDB code: 4DJH); (k) lipid GPCR/T4 chimera (PDB code: 3V2W); (l ) M3 muscarinic receptor/T4 lysozyme chimera (PDB code: 4DAJ); (m) μ-opioid receptor/T4 lysozyme chimera (PDB code: 4DKL); (n) δ-opioid receptor/T4 lysozyme chimera (PDB code: 4EJ4); (o) thermostabilized apocytochrome b₅₆₂ (Bril)-nociceptin/orphanin FQ receptor fusion (PDB code: 4EA3); ( p) chemokine receptor CXCR1 (PDB code: 2LNL); (q) neurotensin receptor NTS1 (PDB code: 4GRV); (r) human protease-activated receptor 1 (PAR1) (PDB code: 3VW7); (s) chimeric protein of 5-HT2B-BRIL (HTR2B) (PDB code: 4IB4); (t) chimeric protein of 5-HT1B-BRIL (HTR1B) (PDB code: 4IAQ). GPCRs are shown in a rainbow-colored scheme: N-terminal (dark blue), C-terminal (red ), and nonnative fusion protein components ( gray). (u) Alignment of fragment pairs derived for each combination of two GPCR structures presented in panels a–t. Blue circles indicate the largest differences resulting from insertion/deletions when comparing the aligned structures of the GPCRs under study.

Figure 3

Signal transduction in GPCRs. (a) Diagrams of the seven transmembrane (TM) helices and hydrogen-bonding network in rhodopsin (PDB ID:1U19). (Left) Helices are colored according to their primary sequence: helix-I (blue); helix-II (blue-green), helix-III ( green); helix-IV (light green); helix-V ( yellow); helix-VI (orange); helix-VII (red ); helix-8 ( purple). (Right) The chromophore is shown with balls and sticks ( pink). Water molecules are displayed as spheres (light blue). The receptor is oriented such that the extracellular space is above and the G protein–interacting cytoplasmic face is below. (b) The superposed surface representations of ground-state rhodopsin (PDB code: 1u19 in red ) and photoactivated-like rhodopsin (PDB code: 4a4m in yellow) viewed from the intracellular side of a plasma membrane. Phospholipids representing the plasma membrane are shown as a gray layer. On the right, 45° rotation reveals major secondary structure elements of rhodopsin and photoactivated rhodopsin visible outside the phospholipid membrane. Locations of intracellular loops (ICLs) and the C-terminal segment are indicated by arrows. (c) An intracellular view of metarhodopsin II ( yellow) with a bound peptide ( green) derived from the primary sequence of Gt. (d ) Superposition of the structure of ground-state rhodopsin (red ) on the structure of photoactivated rhodopsin ( panel c). The overlap of the peptide derived from Gt with ground-state rhodopsin suggests that interaction of the two would be unlikely.

Figure 4

GPCR: G protein complex. (a) Superposed structures of photoactivated rhodopsin in complex with Gt (red ) (Jastrzebska et al. 2011b) and the β₂-adrenergic receptor in complex with Gs (blue) (Chung et al. 2011) (dark gray, T4 lysozyme present in the β₂-adrenergic receptor-Gs structure; gray transparent layer, the phospholipid membrane). The section highlighted in detail (large black circle) shows superposed Gt (PDB code: 1GOT, red ) and Gs (PDB code: 4A4M, blue). The averaged angle between the Gsα-AH and Gtα-AH domains evaluated from several measurements was ~95°, whereas distances between corresponding atoms of the two domains had an average value of ~40 Å (Jastrzebska et al. 2011b). Superposition of the Gsα-AH subdomain with the Gtα-AH subdomain is achieved only after a full 180° rigid body rotation around the axis shown on the left. (b) Differences in normalized hydrogen-deuterium exchange evaluated for photoactivated rhodopsin, photoactivated rhodopsin-Gt, and Gt. Heat maps (dark blue to rose) were used to evaluate differences in normalized hydrogen-deuterium exchange for free Gt and Gt in complex with photoactivated rhodopsin and for free photoactivated rhodopsin and photoactivated rhodopsin in complex with Gt (Orban et al. 2012b). Heat maps were then placed on the three-dimensional structure of the photoactivated rhodopsin-Gt complex model (Jastrzebska et al. 2011b). Negative differences in hydrogen-deuterium exchange are shown as 0–9% ( green), 10–19% (cyan), 20–29% (light blue), 30–39% (blue), and 40–50% (darker blues). Positive differences are displayed as 0–9% ( yellow), 10–19% (light orange), 20–29% (orange), 30–39% (red ), and 40–50% (magenta to purple). The lipid bilayer where photoactivated rhodopsin is embedded is presented as a transparent gray layer. Abbreviation: AH, α-helical.

Figure 5

Hypothetical model for assembly on a cell surface of major proteins required for effective visual signal transduction. Visual signal transduction is carried out by a multitude of proteins and messengers. (Top left) Structures of proteins involved in phototransduction. (Bottom left) A phospholipid cell membrane represented as a gray transparent layer containing a dimer and monomer of rhodopsin (red ). Various complexes of these proteins assembled on the membranes are modeled on the right. Following activation, heterotrimeric Gt ( pink) is recruited to the cytoplasmic cell surface where it binds to photoactivated rhodopsin, forming the photoactivated rhodopsin-Gt complex. Gt-bound guanosine diphosphate (GDP) is then exchanged for guanosine-5′-triphosphate (GTP). Next, phosphodiesterase 6 holoenzyme (PDE6, green) is recruited to the membrane and carries out hydrolysis of cyclic guanosine monophosphate (cGMP). The activity of Gt is, in turn, suppressed by RGS9 (orange), which promotes its inactivation. Finally, arrestin ( yellow) and GRK1 (blue) both help deactivate photoactivated rhodopsin, a step required for resetting the dark state of rhodopsin prior to another light stimulus.

Figure 6

High-resolution images and pictorial representation of mammalian photoreceptors and the retinal pigment epithelium (RPE). (a–d ) Fluorescence images obtained by two-photon excitation of endogenous fluorophores. Scale bars represent 20 μm. (a) Double-nucleated RPE cells obtained from an ex vivo unfixed C57BL/6J-Tyr^C-2J mouse eye. Highly fluorescent retinosomes are visible as bright spots located along the plasma membranes. (b) A transverse (xz) image of cones and rods in cynomolgus monkey peripheral retina assembled from a series of en face images. Ellipsoids of two cones directly on the xz axis are indicated by yellow arrows. (c) An en face (xy) image of the rod and cone outer segment mosaic. Outer segments of two cones are indicated with yellow arrows. (d ) En face (xy) image of the rod and cone inner segment mosaic. Two-photon images courtesy of G. Palczewska. (e) Pictorial representation of the RPE-photoreceptor interface. Melanosomes ( purple) are located in the RPE cell and processes. Other elements: lipofuscin (smaller dark red circles), phagosomes (larger blue circles), cone photoreceptor cell ( green), and rod cell (blue). The dashed line represents the external limiting membrane separating the nuclei and the ellipsoid with its abundant mitochondria. ( f–i ) Electron microscopic images. ( f ) Image of the RPE and a photoreceptor cell in extrafoveal rhesus monkey retina (from Anderson et al. 1980). ( g) The tip of the outer segment of a foveal cone engulfed by RPE apical processes in rhesus monkey retina (from Anderson & Fisher 1979). Some distal discs appear separate from the cell membrane. (h) The cone outer segment base from a rhesus monkey retina (from Anderson et al. 1978). Black arrows indicate a new evagination of the membrane. (i ) The nucleus of a Müller cell and the external limiting membrane. Müller cell villous processes extend beyond the external limiting membrane between rods and cones. ( j ) The cytotoxic effect of all-trans-retinal in light-induced photoreceptor degeneration. The illustrated signaling cascade implicates GPCRs, PLC/IP₃/Ca²⁺ signaling, and NADPH oxidase in this process. Elevated signaling of G_q-coupled GPCRs is involved in mediating all-trans-retinal toxicity during light-induced photoreceptor degeneration, but the precise mechanism has yet to be clarified (black arrow with dotted line). Activation of G_q-coupled GPCRs stimulates PLC/IP₃/Ca²⁺ pathways, which then lead to NADPH oxidase-mediated ROS production and photoreceptor degeneration (black arrows). Pharmacological interventions targeting G_q-coupled GPCRs, PLC/IP₃/Ca²⁺, and NADPH oxidase protect photoreceptors from light-induced, all-trans-retinal-mediated degeneration (red bars). Modified illustration based on Chen et al. (2012, figure 8). APO/DPI, Apocynin/diphenylleneiodonium; ER, endoplasmic reticulum; NADPH, nicotinamide adenine dinucleotide phosphate; PLC, Phospholipase-C; ROS, reactive oxygen species