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. 2016 Oct 18;113(42):11961-11966.
doi: 10.1073/pnas.1606347113. Epub 2016 Oct 4.

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

Christopher T Schafer  1 , Jonathan F Fay  1 , Jay M Janz  1 , David L Farrens  2
Affiliations

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

Christopher T Schafer et al. Proc Natl Acad Sci U S A. .

Abstract

Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on time-resolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.

Keywords: GPCR; conformational dynamics; fluorescence; retinal; rhodopsin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Conformational selection model for retinal binding to opsin (8). The focus of the current work is on the process of ATR dissociation from opsin (enclosed in the dashed rounded rectangle) testing two hypotheses: first, that ATR will bind in equilibrium depending on the conformation of the opsin (i), and second, that the reversion of Ops* to Ops is distinct from the ligands presence, resulting in the possibility of an active Ops* state can transiently persist in the absence of ligand (ii).
Fig. 2.
Fig. 2.
Evidence that ATR released during decay of photoactivated MII rhodopsin can rebind in an Ops*-dependent manner. (A) Cartoon depiction of how retinal release following light activation can be monitored by an increase in intrinsic protein fluorescence. (B) Release trace from WT opsin shows a monoexpontential rise to a plateau. HA treatment yields no additional release, indicating full ATR dissociation. (C) Release from the CAM instead results in some ATR remaining bound to the receptor in an equilibrium that is only relieved when HA is added. (D) Photoactivation of GtF, a rhodopsin sample with the Gtα C terminus fused on its end, shows no apparent ATR release until HA is added. (E) Addition of tbHA, an HA derivative that cannot enter the binding pocket (16), had no effect on retinal release for the WT sample (compare with B). (F) In contrast, tbHA induces full retinal release from the CAM, resulting in data nearly identical to the WT opsin. (G) Similarly, the presence of tbHA induced a (slower) retinal release from GtF. Subsequent assays measured in the presence of increasing amounts of added ATR show a shift in the equilibrium to more bound ATR. (H) Results for increasing [ATR] for WT rhodopsin release. (I) Extra ATR further shifts the CAM toward the bound state. (J) Experiments with GtF and increasing amounts of ATR show no change, because the receptor is already fully bound at the lowest ATR concentration. Release experiments were conducted at 20 °C in 0.05% N-dodecyl-β-d-maltopyranoside (DDM) at pH 6.0.
Fig. 3.
Fig. 3.
ATR produced inside rhodopsin by light bleaching can exchange with an equimolar amount of exogenously added, radioactive [3H]ATR. (A) Cartoon depiction of the ATR exchange experiments. (B) The amount of radioligand exchange for WT, CAM, and GtF rhodopsins at 2 h after photoactivation correlates with the amount of active Ops* and retinal trapping seen in Fig. 2. (C) Time-course measurement for the GtF construct shows complete and rapid ATR exchange (t½ ∼4.7 min). (D) Surprisingly, although GtF clearly shows exchange with exogenous [3H]ATR, acid protonation experiments of identical samples show that the light-activated ATR–Schiff base linkage in GtF remains “stable” for the entire (2-h) length of the exchange experiment [note the ∼440-nm absorbing species indicating a protonated retinal Schiff base (PSB)]. Experiments were performed at room temperature in 0.05% DDM at pH 6.0.
Fig. S1.
Fig. S1.
Radioactive binding to GtF comparing different conditions. The left two bars observe the exchange and binding of externally added [3H]ATR with the nonlabeled, cold retinal inside the receptor before (−hν) and after (+hν) photobleaching. The right two bars display the ability of GtF opsin to maximally bind the radiolabeled ligand without (MAX) and in the presence of a massive dilution with cold ATR (nonspecific binding, NSB). Accounting for the 1/2 isotopic dilution of the ligand that occurs in the exchange experiments (equimolar amounts of cold and [3H]-labeled ATR), the total counts are about equal to the maximal value observed that was obtained in the undiluted MAX sample. Applying this analysis to the results in Fig. 3 indicates the light-activated samples underwent near-maximal ATR exchange.
Fig. 4.
Fig. 4.
Time-resolved fluorescence assay based on TrIQ for simultaneously monitoring receptor conformational changes and retinal release after rhodopsin photoactivation. (A) Model showing activation moves the bimane fluorophore (green) on TM6 into near contact with the quenching Trp (purple) on TM3. Reversion back to the inactive conformation relieves the TrIQ, causing a rise in the bimane emission. (B and C) Absorbance spectra indicate both V250B (Trp-less control, B) and V139W/V250B (C) are WT-like in their photoactivation properties. Spectra taken before and then every 5 min after photoactivation. (D and E) Bimane emission spectra following photoactivation of V250B and V139W/V250B. Spectra were taken first in the dark then every 5 min after bleach. The immediate increase for V250B is due to relief of bimane FRET to the 11CR. Note that for V139W/V250B the initial increase is absent, and instead the emission only slowly increases over time. (F and G) Time course for tryptophan ATR release (Trp fluorescence monitored at 330 nm, black trace) with simultaneous measurement of bimane fluorescence (at 460 nm, red trace) for V250B and V139W/V250B. A log time scale is used to enable comparison over a wide time range. For V139W/V250B, note the ATR release rate and TM6 movement are very similar, with a very small time lag. Models produced using Chimera and Protein Data Bank ID codes 1GZM and 3PXO (–46). Experiments were conducted in the same conditions as Fig. 2.
Fig. 5.
Fig. 5.
The time lag between ATR release and TM6 movement back to the inactive state is increased when retinal release is accelerated. As with Fig. 4 F and G, the plots show simultaneous measurement of tryptophan (black traces) and bimane fluorescence (red traces) as a function of time (note log time scale). (A) Data for “WT” (V139W/V250B) receptor. (B) Arrhenius analyses show similar activation energies for both events. (C) Addition of HA accelerates retinal release faster than the TM6 movement. (D) HA does not have a drastic effect on the activation energies of either process. (E) Same measurements carried out on rhodopsin mutant, A295S, reveal a similar separation of ATR release and TM6 movement. (F) Arrhenius analysis of this construct again shows little change in the activation energy of the two events. Errors reported as SDs. Experiments were conducted in the same conditions as Fig. 2.
Fig. S2.
Fig. S2.
Measurement of the retinal release and TM6 movement of rhodopsin mutations Y223A and Y223F. These constructs also show a distinct time lag between the agonist dissociation and the collapse back to an Ops conformation. (A) Fluorescence traces for the two events for Y223A. (B) Arrhenius analysis shows that the two events retain similar activation energies, suggesting the mechanism for release and conformational change is the same. (C) The traces for Y223F show a more rapid release of ATR, which exaggerates the time lag between the dissociation and the Ops*-to-Ops transition. The more rapid ATR release is consistent with previous reports (24). (D) Arrhenius analysis of Y223F confirms that the activation energies have remained the same. See Table S1 for rates and activation energies.
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