Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process

Abstract

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the "phosphate-sensor" of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins.

Fig. 1.

Structures of arrestin and rhodopsin. (A) Crystal structural models of rhodopsin (gray, PDB entry 1U19) and arrestin (blue: N-domain, green: C-domain, PDB entry 1CF1), generated with MOLSCRIPT (49). Rhodopsin: blue- three possible phosphorylation sites S334, S338, and S343, red- H8, violet- palmitoylated cysteines (palm-C322, palm-C323). Arrestin: dark red—interacting charged residues in the polar core of arrestin, yellow- helix αI and β-strand βI of the three-element interaction, dark yellow—external phosphate-sensors K14 and K15. Loop V-VI of arrestin is presented in its extended (solid line) and “closed” (dashed line) conformation (coordinates are taken from different molecules in the asymmetric unit of the arrestin crystal). The labeling positions C316 (rhodopsin), S106C and S60C (arrestin) are indicated in black. (B) Time traces of Meta-II formation at 380 nm (3 °C, pH 7.5). The increase in Meta-II concentration of P-Rh* membranes corresponds to an average of three phosphates per rhodopsin. (C) Close-up of the C-terminal region of rhodopsin, including part of transmembrane helix 7 (gray), helix 8 (red), and three phosphorylated serines, p-S334, p-S338, and p-S343 (blue). Dark yellow- residues belonging to the NPxxY motif. Green- side chains of the hydrophobic residues F313, M317, and L321. (D) Chemical structure of 5-IAF (SF) and Alexa594 (LF) bound to C316.

Fig. 2.

Kinetics of arrestin binding and conformational changes after activation of rhodopsin by a flash of light. (A) Fluorescence lifetime curves of LY covalently bound to S106C of arrestin in the absence and presence of P-Rh and P-Rh*, respectively. (B) Time trace of Meta-II formation (black). Time trace of arrestin binding to P-Rh* as measured by kinetic light scattering (LS, green). Time trace of arrestin conformational changes as monitored by the integral fluorescence changes of bound LY to position 106 of arrestin (LY, red). The solid lines represent multiexponential fits. Arrows and numbers indicate the transitions explained in the text. Conditions: 1 μM rhodopsin, 10 μM arrestin, 150 mM NaCl, 50 mM potassium phosphate buffer pH 7.5, 20 °C. (C) Time trace of arrestin binding as shown in (B) together with the control (light scattering without arrestin) on a linear time scale. (D) Time trace of arrestin binding to P-Rh* as measured by the increase in final anisotropy r_∞ of the covalently bound dye Atto647N to position 60 of arrestin (top; τ = 4 s). Conformational changes of arrestin at position 60 and 106 as revealed by the changes in integral fluorescence intensity and lifetime of LY, respectively (bottom; ArrCA-S106C-LY: τ₁ = 4 s, τ₂ = 24 s, ArrCA-S60C-LY: τ = 29 s). The fluorescence decay times of ArrCA-S106C-LY are 0.1 ns, 1.6 ns, and 7.8 ns and the changes of the slowest decay time (lifetime) upon binding to P-Rh* are shown. Conditions: 1 μM arrestin, 3–5 μM rhodopsin, 150 mM NaCl, 50 mM potassium phosphate buffer pH 7.5, 20 °C. Fluorescence excitation was at 428 nm (LY) or 617 nm (Atto647N) and the emission was detected after passing through a cut-off filter OG495 and RG665, respectively.

Fig. 3.

H8 dynamics in response to rhodopsin phosphorylation and arrestin interaction. Time-resolved anisotropy decay curves of SF covalently bound to rhodopsin in position Cys316 for dark (A–C) and light-activated rhodopsin membranes (D–F): (A), (D) phosphorylated and unphosphorylated rhodopsin, (B), (E) phosphorylated and unphosphorylated rhodopsin in the presence of arrestin or the arrestin mutant R175E, (C) phosphorylated rhodopsin in the absence and presence of arrestin, (F) rhodopsin in the absence and presence of arrestin. The corresponding fluorescence anisotropy decay parameters are presented in (G–I). (G) rotational correlation time of H8 (ϕ₂), (H) conformational space of H8 expressed as relative mobility in percentage (), (I) steric restriction r_∞ of H8. Black—dark (inactive) rhodopsin, red—light-activated rhodopsin, blue—phosphorylated rhodopsin, green—presence of arrestin. Conditions: 5 μM rhodopsin, 50 μM arrestin, 150 mM NaCl, 50 mM potassium phosphate buffer pH 7.5, 20 °C. The fluorescence excitation was at 470 nm and the emission was detected after passing through a cut-off filter OG515.

Fig. 4.

Arrestin binding based on Meta-II stabilization. Binding affinity of (A) arrestin WT, and (B) Arrestin-R175E. The arrestin binding affinity to unlabeled P-Rh* was set to 100%. Red—unphosphorylated rhodopsin, blue—phosphorylated rhodopsin, black hatched—SF-labeled rhodopsin, white hatched—LF-labeled rhodopsin.

Fig. 5.

Model for the activation of arrestin (Arr). After phosphorylation of Rh* by rhodopsin kinase (not shown), the prebinding and high-affinity binding state are successively formed. See Discussion for details. The conformational changes around α-helix I in the prebinding state of arrestin are indicated by a transparent blue coloring. H8 dynamics is visualized by the size and color of the cone representing H8 conformational space (mobility) and motion (ϕ < 2.5 ns: red, ϕ > 2.5 ns: blue), respectively. The dynamics changes compared to the preceding state are indicated by the corresponding anisotropy parameters φ (correlation time), β′ (conformational space/relative mobility), and r_∞ (steric restriction) presented as arrows. The arrow length represents the magnitude of the change and the direction indicates the increase or decrease of the respective value.