17 research outputs found
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Gi- and Gs-coupled GPCRs show different modes of G-protein binding.
More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand-receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR-G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin-G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin-Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR-Gs structures, providing insight into specificity determinants
Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology
Engineering of an artificial light-modulated potassium channel
Ion Channel-Coupled Receptors (ICCRs) are artificial receptor-channel fusion proteins designed to couple ligand binding to channel gating. We previously validated the ICCR concept with various G protein-coupled receptors (GPCRs) fused with the inward rectifying potassium channel Kir6.2. Here we characterize a novel ICCR, consisting of the light activated GPCR, opsin/rhodopsin, fused with Kir6.2. To validate our two-electrode voltage clamp (TEVC) assay for activation of the GPCR, we first co-expressed the apoprotein opsin and the G protein-activated potassium channel Kir3.1F137S (Kir3.1*) in Xenopus oocytes. Opsin can be converted to rhodopsin by incubation with 11-cis retinal and activated by light-induced retinal cis→trans isomerization. Alternatively opsin can be activated by incubation of oocytes with all-trans-retinal. We found that illumination of 11-cis-retinal-incubated oocytes co-expressing opsin and Kir3.1* caused an immediate and long-lasting channel opening. In the absence of 11-cis retinal, all-trans-retinal also opened the channel persistently, although with slower kinetics. We then used the oocyte/TEVC system to test fusion proteins between opsin/rhodopsin and Kir6.2. We demonstrate that a construct with a C-terminally truncated rhodopsin responds to light stimulus independent of G protein. By extending the concept of ICCRs to the light-activatable GPCR rhodopsin we broaden the potential applications of this set of tools
Use of a G protein-activated channel as a reporter of opsin activation.
<p>(<b>A</b>) Activation by all-<i>trans</i>-retinal. Direct application of all-<i>trans</i>-retinal to <i>Xenopus</i> oocytes expressing opsin and Kir3.1* (Kir3.1<sub>F137S</sub>) leads to Kir3.1* activation via G proteins (Gαßγ). (<b>B</b>) Activation by light. Oocytes were pre-incubated with 20 µM 11-<i>cis</i> retinal for >30 min in the dark to form rhodopsin. Visible light exposure isomerizes 11-<i>cis</i> retinal to all-<i>trans</i>-retinal (red), thus activating rhodopsin and, in turn, G proteins which release Gßγ to open Kir3.1*. The traces are representative TEVC recordings from <i>Xenopus</i> oocytes expressing opsin and Kir3.1*. Current amplitude was recorded at −50 mV. Dashed line indicates the Ba<sup>2+</sup>-sensitive current baseline.</p
ICCRs report conformational changes of receptors deficient in G protein coupling.
<p>(<b>A</b>) Representative TEVC traces. Yellow and blue bars correspond to oocyte illumination and Ba<sup>2+</sup> (3 mM) application, respectively. PTX (Pertussis Toxin) ADP-ribosylates G<sub>αi/o</sub> proteins, thus preventing G<sub>α</sub>-G<sub>βγ</sub> dissociation. OpsΔicl3 is a rhodopsin mutant with a deletion in the third intracellular loop (Δ244–249) known for its inability to activate G proteins. Dashed line indicates the Ba<sup>2+</sup>-sensitive current baseline. Measurements were done at −50 mV. (<b>B</b>) Percent change in current induced by light after 11-<i>cis</i> retinal incubation in the dark. Numbers below bars indicate the number of oocytes tested. The responses of OpsΔicl3-K<sub>-16–25</sub> and Ops-K<sub>-16–25</sub>+ PTX were not statistically different (Student t-test; P>0.5) while those of OpsΔicl3-K<sub>-16–25</sub> and Ops-K<sub>-16–25</sub> were different (P<0.002). (<b>C</b>) Oocytes were co-injected with Ops-K<sub>-16–25</sub>+ Kir3.1* or OpsΔicl3-K<sub>−16–25</sub>+ Kir3.1*. Ability of opsin to activate G proteins was determined by the addition of all-<i>trans</i>-retinal at 10 µM and measurement of Kir3.1* activation as a percent change in current. Numbers above bars indicate the number of oocytes tested.</p
PTX-sensitive activation of Kir3.1* by opsin and all-<i>trans</i>-retinal, or light-activated rhodopsin.
<p> (<b>A</b>) <i>Xenopus</i> oocytes were injected with opsin and Kir3.1* mRNAs. Current amplitude was recorded at −50 mV. Black bars represent the average current measured prior to all-<i>trans</i>-retinal application or in the dark (after 11-<i>cis</i> retinal incubation) in the case of light activation. White bars represent the average current induced by 5 µM all-<i>trans</i>-retinal or light stimulation, respectively. Numbers above bars denote the number of oocytes tested. (<b>B</b>) Percent change in current induced by application of either 5 µM all-<i>trans</i>-retinal or light (after 11-<i>cis</i> retinal incubation) in control (black bars) and in the presence of co-expressed catalytic subunit S1 of pertussis toxin (PTX-S1) (white bars). Changes in current were computed for each oocyte and then averaged (The resulting average changes are different from the changes in average current represented in panel A). (<b>C</b>) Concentration-dependent response to all-<i>trans</i>-retinal. Average data computed as in panel B. Line corresponds to Hill equation fit with h = 4 and EC<sub>50</sub> = 2.5 µM. Each point represents the average of 7 to 40 measurements.</p
Design strategy of rhodopsin-based ICCRs and functional coupling.
<p>(<b>A</b>) Sequence alignment of the fusion area between GPCRs and Kir6.2ΔN25. Alignment of GPCR C-terminal sequences were based on the presence of the H8 Helix. (<b>B</b>) Percent change in current for each construct in response to 10 µM all-<i>trans</i>-retinal or light (after 11-<i>cis</i> retinal incubation). Oocytes were co-injected with the specified Ops-Kir6.2 and TMD0, a SUR transmembrane domain. Numbers below bars indicate the number of oocytes tested. The changes induced by all-<i>trans</i>-retinal and light were statistically significant for Ops-K<sub>−16–25</sub> (Student t-test; P<0.04 & P<0.0001, respectively), but not for Ops-K<sub>0–25</sub> (P>0.4 & P>0.05, respectively). (<b>C</b>) Representative TEVC recordings for each construct in the case of photoactivation. Yellow bar represents oocyte illumination. Blue bar corresponds to Ba<sup>2+</sup> application at 3 mM. Dashed line indicates the Ba<sup>2+</sup>-sensitive current baseline. (<b>D</b>) Opsin ability to activate G proteins within the fusion Ops-Kir6.2. Both constructs were co-expressed with Kir3.1* and change in current was measured in response to 10 µM all-<i>trans</i>-retinal. All measurements were done at −50 mV. Numbers above bars indicate the number of oocytes tested.</p
Recommended from our members
Gi- and Gs-coupled GPCRs show different modes of G-protein binding.
More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand-receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR-G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin-G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin-Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR-Gs structures, providing insight into specificity determinants