From Atomic Structures to Neuronal Functions of G Protein–Coupled Receptors

“N eu ro g by C as e W es te GPCR: G protein–coupled receptor 7-TM: 7-transmembrane”

“…the GPCR field began in 2007 including studies that produced the first structures of GPCRs bound to various diffusible and covalently bound ligands (Cherezov et al. 2007, Rasmussen et al. 2007, Rosenbaum et al. 2007), the structure of squid rhodopsin (Murakami & Kouyama 2008, Shimamura et al.…”

“…of how an activated GPCR binds to G proteins came with the recent crystal structure of an active state complex composed of (a) agonist-occupied monomeric β2-adrenergic receptor-T4L-lyzosome fusion, (b) nucleotidefree Gs heterotrimer, and (c) a nanobody (Rasmussen et al. 2011) (Figure 4a).”

“Serotonin 2A receptor expression is readily detectible in the retina, and its activation leads mainly to elevations in cytosolic Ca2+ through PLC activation (Hoyer et al. 1994).”

“…FQ (N/OFQ) peptide receptor structure with bound antagonists provide insight into the conserved elements of opioid ligand recognition and also reveal those features associated with ligand-subtype selectivity (Granier et al. 2012, Manglik et al. 2012, Thompson et al. 2012, Wu et al. 2012).”

“…that produced the first structures of GPCRs bound to various diffusible and covalently bound ligands (Cherezov et al. 2007, Rasmussen et al. 2007, Rosenbaum et al. 2007), the structure of squid rhodopsin (Murakami & Kouyama 2008, Shimamura et al. 2008), and a glimpse into the GPCR activation…”

“As predicted from biochemical studies (Oldham & Hamm 2008), the amino- and carboxy-terminal α-helices of the G protein exhibited conformational changes that propagated to its nucleotidebinding pocket.”

“Opsin crystals can bind all-trans-retinal without significant conformational changes (Choe et al. 2011, Scheerer et al. 2008) and can also bind the Gtα C-terminal peptide (Choe et al. 2011) (Figure 3c,d).”

“In addition to bound water molecules, sodium ions can play an important role in GPCR function as recently proposed for the highest resolution X-ray structure (1.8 Å) of a GPCR solved to date (Liu et al. 2012).”

“Several different analyses of the activation mechanism have been published but are beyond the scope of this review (Hofmann et al. 2009, Katritch et al. 2012, Park et al. 2008b, Salon et al. 2011).”

“In neuronal systems, GPCRs fine-tune cellular functions by activating a heterotrimeric G protein–dependent cascade of signals (Gainetdinov et al. 2004).”

“AFM measurements yielded a density of 30,000–55,000 rhodopsin molecules/μm2 and ∼108 rhodopsin molecules per rod, partially organized in para-crystalline arrays composed of rows of rhodopsin (Fotiadis et al. 2003, Liang et al. 2003).”

“Park and colleagues (Park et al. 2008b) proposed that GPCR action can be analogous in some respects to regulatory enzymes where the ligand-binding site is discrete from the active site (Changeux & Edelstein 2005).”

“F or p er so na l u se o nl y. GPCR ligands: agonists, antagonists/ inverse agonists, or neutral ligands that bind to orthosteric GPCR sites, often causing GPCR activation or inhibition (Li et al. 2004, Okada et al. 2002, Salom et al. 2006).”

“…preparations, either monomeric or multimeric forms of GPCRs can activate their cognate G-proteins ( Jastrzebska et al. 2006), but they assemble into homo- and heterodimers or larger oligomers in native tissues (Palczewski 2010, Park et al. 2004, Prezeau et al. 2010, Terrillon & Bouvier 2004).”

“…FQ (N/OFQ) peptide receptor structure with bound antagonists provide insight into the conserved elements of opioid ligand recognition and also reveal those features associated with ligand-subtype selectivity (Granier et al. 2012, Manglik et al. 2012, Thompson et al. 2012, Wu et al. 2012).”

“Opsin crystals can bind all-trans-retinal without significant conformational changes (Choe et al. 2011, Scheerer et al. 2008) and can also bind the Gtα C-terminal peptide (Choe et al. 2011) (Figure 3c,d).”

“Because the amino terminal ligand-binding domains of some GPCRs fold properly and bind ligand independently of the TM α-helical region, researchers have elucidated several structures of these domains (reviewed in Park et al. 2008b, Pin et al. 2003).”

“F or p er so na l u se o nl y. GPCR ligands: agonists, antagonists/ inverse agonists, or neutral ligands that bind to orthosteric GPCR sites, often causing GPCR activation or inhibition (Li et al. 2004, Okada et al. 2002, Salom et al. 2006).”

“Moreover, researchers noted a similar phenomenon for the β2-adrenergic receptor in cell cultures (Liggett 2011, Nobles et al. 2011).”

“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).”

“Investigators have also successfully probed the dynamics of GPCRs during the activation process using hydrogen-deuterium exchange (Lodowski et al. 2010, Orban et al. 2012b, West et al. 2011) and long-time scale computational simulations (Dror et al. 2011).”

“AFM measurements yielded a density of 30,000–55,000 rhodopsin molecules/μm2 and ∼108 rhodopsin molecules per rod, partially organized in para-crystalline arrays composed of rows of rhodopsin (Fotiadis et al. 2003, Liang et al. 2003).”

“For example, Doré et al. (2011) determined the structures of a 146 Palczewski · Orban A nn u.”

“The size of ROS discs is dictated by the level of rhodopsin expression (Liang et al. 2004), and absence of rhodopsin prevents ROS formation (Humphries et al. 1997).”

“Figure adapted from Katritch et al. (2012). range of biological processes.”

“Invertebrate and vertebrate visual systems involving phototransduction are model systems that have provided molecular and functional information about GPCR signaling for decades (Yau & Hardie 2009).”

“…diffusible and covalently bound ligands (Cherezov et al. 2007, Rasmussen et al. 2007, Rosenbaum et al. 2007), the structure of squid rhodopsin (Murakami & Kouyama 2008, Shimamura et al. 2008), and a glimpse into the GPCR activation process provided by the structure of bovine opsin (Park et…”

“…FQ (N/OFQ) peptide receptor structure with bound antagonists provide insight into the conserved elements of opioid ligand recognition and also reveal those features associated with ligand-subtype selectivity (Granier et al. 2012, Manglik et al. 2012, Thompson et al. 2012, Wu et al. 2012).”

“These multipart protein assemblages could influence pharmacological properties of these receptors (Ritter & Hall 2009).”

“Two different constitutively active mutants of rhodopsin (E113Q and M257Y) with a bound C-terminal fragment of a G protein α-subunit were used to trap the activated state of rhodopsin with the agonist all-trans-retinal present in the native-binding pocket (Deupi et al. 2012, Standfuss et al. 2011).”

“Another strategy employed the fusion of GPCRs with fast-folding proteins such as T4 lysozyme (Cherezov et al. 2007, Rosenbaum et al. 2007) or thermostabilized apocytochrome b562 (Chun et al. 2012, Thompson et al. 2012).”

“F or p er so na l u se o nl y. GPCR ligands: agonists, antagonists/ inverse agonists, or neutral ligands that bind to orthosteric GPCR sites, often causing GPCR activation or inhibition (Li et al. 2004, Okada et al. 2002, Salom et al. 2006).”

“…rhodopsin, with its covalently bound ligand 11-cis-retinal, undergoes changes that involve both deprotonation of the Schiff base and protonation/deprotonation at the cytoplasmic surface, which intuitively suggests a role for internal water molecules in proton transfer (Hofmann et al. 2009).”

“…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).”

“…its life cycle (Fichter et al. 2010) or follow a ligand that binds tightly to these receptors (Zhou et al. 2007). allowed investigators to observe a dynamic equilibrium for the N-formyl peptide receptor in live cells, indicating that dimers rapidly fall apart and reassemble (Kasai et al. 2011).”

“F or p er so na l u se o nl y. (Mirzadegan et al. 2003) and perhaps all GPCR classes.”

“Adapted to native tissues, the same strategy also demonstrated the presence of oxytocin receptor dimers and/or oligomers in the mammary gland (Albizu et al. 2010).”

“For disorders such as schizophrenia and dementia, metabotropic glutamate 2 receptor/serotonin 2A receptor– mediated changes in Gi and Gq activity could affect the psychoactive behavioral effects of a variety of pharmacological compounds (Fribourg et al. 2011).”

“For example, a putative octopamine GPCR in sensory neurons actively suppresses innate immune responses by downregulating the expression of noncanonical unfolded-protein response genes to control stress-response pathways (Sun et al. 2011).”

“The cytoplasmic space used for phototransduction represents only ∼30% of the space inside a ROS [see a recent review for a summary of this structure (Palczewski 2012)].”

“For static imaging, atomic force microscopy, used to resolve metal orbitals on hard surfaces, has provided the highest resolution images of several membraneembedded or reconstituted proteins in various activated states (Engel & Gaub 2008, Müller et al. 2008).”

“The main protein of ROS internal discs is rhodopsin, which constitutes ∼50% of the membrane volume (Filipek et al. 2003a).”

“Cholesterol and palmitoyl moieties present on many GPCR C-terminal regions could play a critical role in forming the dimer interface (Maeda et al. 2010, Oates & Watts 2011, Thompson et al. 2011, Zheng et al. 2012).”

“Two different constitutively active mutants of rhodopsin (E113Q and M257Y) with a bound C-terminal fragment of a G protein α-subunit were used to trap the activated state of rhodopsin with the agonist all-trans-retinal present in the native-binding pocket (Deupi et al. 2012, Standfuss et al. 2011).”

“The number of water molecules within the TM α-helical domain is highly conserved among GPCRs, suggesting that functional roles for water-mediated contacts can also be conserved across members of this receptor family (Angel et al. 2009a, Pardo et al. 2007).”

“However, simpler cultured insect (Schneider & Seifert 2010) and mammalian cells (Li et al. 2005) are most often employed for expressing heterologous GPCRs because they can perform the appropriate complex posttranslational modifications and their membranes mimic native membranes.”

“…preparations, either monomeric or multimeric forms of GPCRs can activate their cognate G-proteins ( Jastrzebska et al. 2006), but they assemble into homo- and heterodimers or larger oligomers in native tissues (Palczewski 2010, Park et al. 2004, Prezeau et al. 2010, Terrillon & Bouvier 2004).”

“Interrelated oxidative stress and Ca2+ imbalance both contribute to the pathology of retinal degeneration (Chen et al. 2012).”

“These rows of rhodopsin dimers were used to derive spatial constraints that were used to construct a molecular model of the oligomeric structure of rhodopsin in the rod outer segment (ROS) membrane (Fotiadis et al. 2004).”

“These TM helices are connected by three extracellular (ECL) and three intracellular (ICL) loops denoted as ECL-I to -III and ICL-I to -III, respectively (Salon et al. 2011).”

“N eu ro g by C as e W es te GPCR: G protein–coupled receptor 7-TM: 7-transmembrane”

“The number of water molecules within the TM α-helical domain is highly conserved among GPCRs, suggesting that functional roles for water-mediated contacts can also be conserved across members of this receptor family (Angel et al. 2009a, Pardo et al. 2007).”

“Investigators have observed specific diversity in the GPCR ligand-binding sites, which indicates that these receptors can respond to different groups of chemicals, peptides, or proteins (Granier & Kobilka 2012).”

“Another study also showed that the ghrelin conformation stabilized by a G protein/agonist is distinct from that stabilized by arrestin-based agonists (Rahmeh et al. 2012).”

“Some of these advances were facilitated by using protein engineering techniques to increase GPCR stability—a much sought after property for crystallization trials (Tate & Schertler 2009).”

“The number of water molecules within the TM α-helical domain is highly conserved among GPCRs, suggesting that functional roles for water-mediated contacts can also be conserved across members of this receptor family (Angel et al. 2009a, Pardo et al. 2007).”

“As do rhodopsin and Gt, the M3 muscarinic receptor forms inactive-state complexes with Gq heterotrimers in intact cells (Qin et al. 2011).”

“GPCRs in taste buds and olfactory receptor neurons are also expressed at high concentrations, but receptors in these tissues are scarce (DeMaria & Ngai 2010), making purification difficult.”

“The size of ROS discs is dictated by the level of rhodopsin expression (Liang et al. 2004), and absence of rhodopsin prevents ROS formation (Humphries et al. 1997).”

“Rhodopsin kinase phosphorylates rhodopsin at different subsets of Ser and Thr residues at the C-terminal tail, producing phosphorylated receptor variants with divergent functional properties (Maeda et al. 2003).”

“This finding was expanded in more recent studies ( Jastrzebska et al. 2006, 2011a), showing that communication between the chromophore and the nucleotide-binding site on a G protein can be uncoupled once the complex is formed.”

“Moreover, researchers noted a similar phenomenon for the β2-adrenergic receptor in cell cultures (Liggett 2011, Nobles et al. 2011).”

“Investigators have also successfully probed the dynamics of GPCRs during the activation process using hydrogen-deuterium exchange (Lodowski et al. 2010, Orban et al. 2012b, West et al. 2011) and long-time scale computational simulations (Dror et al. 2011).”

“…from studies of opsin at low pH that was assumed to have achieved an activated conformation (Park et al. 2008a, Scheerer et al. 2008), even though its catalytic properties were known to be extremely low compared with those of photoactivated rhodopsin (Buczylko et al. 1996, Jäger et al. 1996).”

“The main protein of ROS internal discs is rhodopsin, which constitutes ∼50% of the membrane volume (Filipek et al. 2003a).”

“Pharmacological interventions targeting specific reactions in this pathway could provide novel therapeutic strategies for treating blinding retinal disorders such as Stargardt’s disease and age-related macular degeneration (Chen et al. 2012, Maeda et al. 2012).”

“Several different analyses of the activation mechanism have been published but are beyond the scope of this review (Hofmann et al. 2009, Katritch et al. 2012, Park et al. 2008b, Salon et al. 2011).”

“Activated rhodopsin can form a stable complex with Gt when the guanyl nucleotidebinding site in the G protein α-subunit is unoccupied (Bornancin et al. 1989).”

“GPCRs have been successfully expressed in yeast and cell-free systems, whereas expression in bacteria typically requires refolding (Lundstrom 2006, McCusker et al. 2007).”

“…and covalently bound ligands (Cherezov et al. 2007, Rasmussen et al. 2007, Rosenbaum et al. 2007), the structure of squid rhodopsin (Murakami & Kouyama 2008, Shimamura et al. 2008), and a glimpse into the GPCR activation process provided by the structure of bovine opsin (Park et al. 2008a).”

“GPCR crystal formation has also been successfully facilitated by the formation of complexes between GPCRs and antibody Fab fragments or nanobodies (Day et al. 2007).”

“The number of water molecules within the TM α-helical domain is highly conserved among GPCRs, suggesting that functional roles for water-mediated contacts can also be conserved across members of this receptor family (Angel et al. 2009a, Pardo et al. 2007).”

“An elegant application of fluorescence correlation spectroscopy with photon counting from histograms led to the recent conclusion that GPCR dimers represent the basic signaling unit (Herrick-Davis et al. 2006).”

“…retina critical for metabolite transport and photoreceptor cell maintenance Substrates favoring the activated ghrelin state initiated by agonist-induced arrestin recruitment cause ghrelin to adopt a different conformation from that observed in the absence of arrestin (Mary et al. 2012).”

“…preparations, either monomeric or multimeric forms of GPCRs can activate their cognate G-proteins ( Jastrzebska et al. 2006), but they assemble into homo- and heterodimers or larger oligomers in native tissues (Palczewski 2010, Park et al. 2004, Prezeau et al. 2010, Terrillon & Bouvier 2004).”

“For example, Park et al. (2012) demonstrated a functional heterodimer for monomeric GPCRs known as DAF-37 and Daf-38 in live C. elegans (Park et al. 2012).”

“Near-field scanning optical microscopy, fluorescence correlation spectroscopy, and other advanced confocal and 2-photon microscopy techniques also permit static visualization of GPCRs in membranes (HerrickDavis et al. 2012, Ianoul et al. 2005).”

“…probes can monitor movement of a single GPCR during its life cycle (Fichter et al. 2010) or follow a ligand that binds tightly to these receptors (Zhou et al. 2007). allowed investigators to observe a dynamic equilibrium for the N-formyl peptide receptor in live cells, indicating that dimers…”

“F or p er so na l u se o nl y. (Mirzadegan et al. 2003) and perhaps all GPCR classes.”

“In the dimeric form, GPCR monomers are not functionally equivalent because only one monomer will bind to the C-terminal helix of the cognate G protein, and ligand affinities could differ between the monomer and the dimer (Fuxe et al. 2012, Jastrzebska et al. 2013, Maurice et al. 2011).”

“…preparations, either monomeric or multimeric forms of GPCRs can activate their cognate G-proteins ( Jastrzebska et al. 2006), but they assemble into homo- and heterodimers or larger oligomers in native tissues (Palczewski 2010, Park et al. 2004, Prezeau et al. 2010, Terrillon & Bouvier 2004).”

“Cholesterol and palmitoyl moieties present on many GPCR C-terminal regions could play a critical role in forming the dimer interface (Maeda et al. 2010, Oates & Watts 2011, Thompson et al. 2011, Zheng et al. 2012).”

“Near-field scanning optical microscopy, fluorescence correlation spectroscopy, and other advanced confocal and 2-photon microscopy techniques also permit static visualization of GPCRs in membranes (HerrickDavis et al. 2012, Ianoul et al. 2005).”

“Cholesterol and palmitoyl moieties present on many GPCR C-terminal regions could play a critical role in forming the dimer interface (Maeda et al. 2010, Oates & Watts 2011, Thompson et al. 2011, Zheng et al. 2012).”

“…functional significance of heterodimerization, such as variants of the serotonin receptor or the metabotropic glutamate receptor, is questionable on the basis of pharmacological assays using the heterologous expression system (Delille et al. 2012) (see sidebar, Heterologous Expression of GPCRs).”

“Sci. USA 109:6733–38 Rall TW, Sutherland EW.”

“…as guanine-nucleotide-binding protein-coupled membrane receptors, seven-transmembrane (7-TM) receptors, or heptahelical receptors] are evolutionarily highly conserved (Strotmann et al. 2011) and are widely expressed in eukaryotic organisms where they control a vast 140 Palczewski · Orban A nn u.”

“This innovative approach allowed researchers to visualize and measure protein interactions in neuronal cells (Ciruela et al. 2010, Vidi et al. 2010).”

“In the dimeric form, GPCR monomers are not functionally equivalent because only one monomer will bind to the C-terminal helix of the cognate G protein, and ligand affinities could differ between the monomer and the dimer (Fuxe et al. 2012, Jastrzebska et al. 2013, Maurice et al. 2011).”

“This strategy identified M3 muscarinic receptor−Gαq contact sites that help build low- and high-resolution models of the complex (Hu et al. 2010).”

“…average fragments per kilobase of exon per million fragments mapped (FPKM) value of 2 or greater that are categorized as GPCRs by gene ontology (see Mustafi et al. 2011 for details) Gene B6 Eye Average FPKM Gene B6 Eye Average FPKM Gene B6 Eye Average FPKM Rho 6162.04 Adra2c 8.96 Gpr158 3.72 Rgr…”

“Sum-frequency generation vibrational spectroscopy was used to determine the membrane orientation of the GPCR kinase 2-G-βγ complex (Boughton et al. 2011).”

“Once internalized, these GPCRs can produce cellular responses distinct from those elicited at the cell surface ( Jalink & Moolenaar 2010).”

“Another idea, derived from a nuclear magnetic resonance study of rhodopsin in membranes, proposes that the retinal ligand initiates collective helix fluctuations on a microsecond to millisecond timescale between activated GPCR conformers at equilibrium (Struts et al. 2011).”

“( f ) Image of the RPE and a photoreceptor cell in extrafoveal rhesus monkey retina (from Anderson et al. 1980).”

“A mathematical model was recently created to reflect this asymmetric organization and its impact on different signaling pathways (Rovira et al. 2010).”

“Because membranes are integral to GPCR signaling (Inagaki et al. 2012, Jastrzebska et al. 2011a, Kaya et al. 2011), this idea deserves serious consideration.”

“GPCRs expressed by adult neural stem cells have also emerged as potential modulators of adult neurogenesis that are critical for self-repair of neuropathologic conditions (Doze & Perez 2012).”

“Only limited information is available about the dynamics and stability of GPCR dimers (Lambert 2010).”

“However, simpler cultured insect (Schneider & Seifert 2010) and mammalian cells (Li et al. 2005) are most often employed for expressing heterologous GPCRs because they can perform the appropriate complex posttranslational modifications and their membranes mimic native membranes.”

“Gt binds to dark-state rhodopsin prior to photoactivation (Hamm et al. 1987), suggesting that this nonproductive complex does not achieve the necessary conformational changes to allow nucleotide exchange.”

“More recently, radiolytic footprinting analysis of bound waters revealed that internal water was redistributed when rhodopsin was activated and the rod photoreceptor–specific G protein (transducin or Gt) was subsequently bound (Angel et al. 2009b, Orban et al. 2012b).”

“Quantum dot (QD) probes can monitor movement of a single GPCR during its life cycle (Fichter et al. 2010) or follow a ligand that binds tightly to these receptors (Zhou et al. 2007). allowed investigators to observe a dynamic equilibrium for the N-formyl peptide receptor in live cells, indicating…”

“The chromophore can be added in either all-transor 11-cis- configurations, but dissociation of the G protein restores the receptor back to the inactive intermediate ( Jastrzebska et al. 2009).”

“It appears that the M3 muscarinic receptor also exists in multiple dimeric/oligomeric arrangements (McMillin et al. 2011).”

“More than 50% of all publications in the past three years that discuss GPCRs explore the exciting possibility of expanding the GPCR pool by homo- and hetero-oligomerization (Park & Palczewski 2005).”

“Because membranes are integral to GPCR signaling (Inagaki et al. 2012, Jastrzebska et al. 2011a, Kaya et al. 2011), this idea deserves serious consideration.”

“For example, a mouse ROS contains ∼800 membranous discs stacked on top of each other, increasing the total membrane surface area of the cell ∼1,500-fold (Mayhew & Astle 1997).”

“Cholesterol and palmitoyl moieties present on many GPCR C-terminal regions could play a critical role in forming the dimer interface (Maeda et al. 2010, Oates & Watts 2011, Thompson et al. 2011, Zheng et al. 2012).”

“( g) The tip of the outer segment of a foveal cone engulfed by RPE apical processes in rhesus monkey retina (from Anderson & Fisher 1979).”

“On the basis of theoretical considerations, Neri et al. (2010) proposed that one monomer of rhodopsin is responsible for light detection, whereas the other serves as the G protein activator.”

“For example, activation of Arf6 leads to β2-adrenergic receptor degradation and accumulation and also negatively controls Rab4-dependent fast recycling to prevent the resensitization of β2-adrenergic receptors (Macia et al. 2012).”

“In addition to Ca2+ influx and direct interaction with Ca2+binding proteins (Navarro et al. 2012), rapid depolarization-induced charge movement in GPCRs is needed for neurotransmitter control (Kupchik et al. 2011).”

“Sci. USA 109:6733–38 Rall TW, Sutherland EW.”

“The retinal transcriptome has now been analyzed, and most of the ROS proteome components have been identified by mass spectrometry (e.g., Wong et al. 2009).”

“In addition to Ca2+ influx and direct interaction with Ca2+binding proteins (Navarro et al. 2012), rapid depolarization-induced charge movement in GPCRs is needed for neurotransmitter control (Kupchik et al. 2011).”

“More recently, researchers achieved overexpression of heterologous GPCRs in C. elegans (Cao et al. 2012, Salom et al. 2012).”

“Because membranes are integral to GPCR signaling (Inagaki et al. 2012, Jastrzebska et al. 2011a, Kaya et al. 2011), this idea deserves serious consideration.”

“The chromophore can be added in either all-transor 11-cis- configurations, but dissociation of the G protein restores the receptor back to the inactive intermediate ( Jastrzebska et al. 2009).”

“For these reasons, we developed an in vivo system to express heterologous GPCRs in rod photoreceptors of Xenopus (Zhang et al. 2005) and mice (Li et al. 2007, Salom et al. 2008).”

“This innovative approach allowed researchers to visualize and measure protein interactions in neuronal cells (Ciruela et al. 2010, Vidi et al. 2010).”

“These methods are also used for imaging applications (Harrison & van der Graaf 2006).”

“GPCR oligomerization could be responsible for fine-tuning receptor function and signaling (Tadagaki et al. 2012).”

“Opioid receptors form complexes with transient receptor potential (TRP) Ca2+ channels as well (Yekkirala 2013).”

“Investigators have also successfully probed the dynamics of GPCRs during the activation process using hydrogen-deuterium exchange (Lodowski et al. 2010, Orban et al. 2012b, West et al. 2011) and long-time scale computational simulations (Dror et al. 2011).”

“More recently, researchers achieved overexpression of heterologous GPCRs in C. elegans (Cao et al. 2012, Salom et al. 2012).”

“For these reasons, we developed an in vivo system to express heterologous GPCRs in rod photoreceptors of Xenopus (Zhang et al. 2005) and mice (Li et al. 2007, Salom et al. 2008).”

“More recently, radiolytic footprinting analysis of bound waters revealed that internal water was redistributed when rhodopsin was activated and the rod photoreceptor–specific G protein (transducin or Gt) was subsequently bound (Angel et al. 2009b, Orban et al. 2012b).”

“These receptors likely share a common mode of activation (Smith 2012).”

“For these reasons, we developed an in vivo system to express heterologous GPCRs in rod photoreceptors of Xenopus (Zhang et al. 2005) and mice (Li et al. 2007, Salom et al. 2008).”

“Another important physiological example is the interaction of GPCRs with voltage-gated calcium channels that can synergize neurotransmitter release (Altier & Zamponi 2011).”

“Moreover, GPCR homodimer activity can be controlled by an allosteric ligand-binding site when different ligands bind to each receptor monomer (Zylbergold & Hébert 2009).”

“The first class encodes more than 100 orphan G-protein coupled receptors (Palczewski and Orban, 2013).”

“GPCRs have multiple effects on neuronal development, including neuronal differentiation, axon guidance, and targeting (25).”

“The hsa04080 (neuroactive ligand-receptor interaction) pathway contains many GPCRs, including growth hormone secretagogue receptor (GHSR), gonadotropin-releasing hormone receptor (GNRHR), leucine-rich repeat-containing G protein-coupled receptor 7/8 (LGR7/8), corticotrophin-releasing hormone receptor 1/2 (CRHR1/2), ga…”

“Preliminary results have been published (47, 83, 88).”

“atRAL along with its derivative products likely are the initiators of photoreceptor cell pathology for several reasons: (i) phototransduction is the only light-sensitive pathway in ROS and this process involves the conversion of retinoids and generation of atRAL (62, 63); (ii) such light-induced degeneration can be pr…”

“This receptor family is structurally defined by a seven alpha-helix transmembrane span topology [4, 7, 12].”

“The vertebrate visual excitation and recovery are mediated by intricately connected protein complexes localized to the disk membranes in the outer segments of photoreceptor cells (1, 2).”

“12; Supplementary Movies 1, 2, 3).”

“The primary sequences of GPCRs are highly diverse [46] but structurally very similar [43], with frequently conserved specific features.”

“2021; Lämmermann and Kastenmüller 2019; Palczewski and Orban 2013).”

“…such as hormones, neurotransmitters and light and for the transmission of that information inside the cell via interaction with the membraneassociated heterotrimeric G-proteins to regulate various intracellular second messenger pathways (Palczewski and Orban, 2013; Manglik and Kobilka, 2014).”

“While these integral membrane proteins have relatively similar structural features in their intracellular part, large structural diversity appears in the extracellular part including the GPCR ligand-binding sites, which explains why these receptors recognize so many extracellular physical and chemical signals [5, 6].”

“1, no. 60) is expressed in the brain and contributes to G Protein-Coupled Receptor (GPCR)- mediated signaling, 60 and Brs3 (Fig.”