Monomeric rhodopsin is sufficient for normal rhodopsin kinase (GRK1) phosphorylation and arrestin-1 binding - PubMed (original) (raw)

Monomeric rhodopsin is sufficient for normal rhodopsin kinase (GRK1) phosphorylation and arrestin-1 binding

Timothy H Bayburt et al. J Biol Chem. 2011.

Abstract

G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.

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Figures

FIGURE 1.

FIGURE 1.

GRK1 efficiently phosphorylates monomeric rhodopsin. Purified rhodopsin from the same batch (50 μg/ml) in original native disc membranes or solubilized and reconstituted into POPC nanodiscs with the indicated fraction of POPS was phosphorylated by purified GRK1 (30 μg/ml) under room light for 10 min at 30 °C. The stoichiometry of phosphorylation was determined, as described under “Experimental Procedures.” Means ± S.D. (error bars) from two experiments performed in duplicate are shown.

FIGURE 2.

FIGURE 2.

Arrestin-1 binding to monomeric P-Rh*. A, phosphorhodopsin from the same batch (0.3 μg) in native disc membranes or solubilized and reconstituted in POPC nanodiscs with 30 or 50% POPS was incubated with radiolabeled arrestin-1 (100 fmol) in 50 μl at 30 °C. The samples were cooled on ice, and bound and free arrestin-1 was separated, as described under “Experimental Procedures.” B, the same assay was performed with the indicated amounts of P-Rh*. Means ± S.D. from three experiments performed in duplicate are shown.

FIGURE 3.

FIGURE 3.

The dependence of arrestin-1 binding to P-Rh* on negatively charged lipids is reduced by increased ionic strength. A, the binding of radiolabeled arrestin-1 to P-Rh* (0.3 μg) in POPC nanodiscs with the indicated fraction of POPS was performed, as in Fig. 2, at varying total salt concentration (50 m

m

Tris-HCl, pH 7.4, supplemented with 100, 200, 300, or 400 m

m

sodium acetate, pH 7.4). Means ± S.D. of two experiments performed in duplicate are shown. B, to facilitate comparison, means from A are plotted as a percentage of maximum binding at each salt concentration (which is reduced by increasing ionic strength).

FIGURE 4.

FIGURE 4.

Fluorescence-based assay of arrestin-1 binding to monomeric P-Rh* in nanodiscs. A, purified recombinant arrestin-1 with unique cysteine in the C-terminal domain (A348C) was covalently labeled with Texas Red maleimide, and the nanodisc MSP is covalently modified with a quenching group. Free arrestin-1 demonstrates bright fluorescence, whereas the signal from arrestin-1 bound to light-activated P-Rh* in nanodisc is quenched by fluorescence energy transfer. B, change of A348C-Texas Red fluorescence upon photoactivation of P-Rh monomer in nanodiscs. Texas Red fluorescence was monitored at 620 nm. Two-second flashes of light were used to photoactivate rhodopsin (asterisks). The addition of hydroxylamine (indicated by the arrow) released bound arrestin-1 and restored fluorescence.

FIGURE 5.

FIGURE 5.

Affinity and stoichiometry of arrestin-1 interaction with monomeric P-Rh* in nanodiscs. A, arrestin-1 binding isotherms. Measurements were taken at 25 °C with 40 n

m

total arrestin-1 (A348C mutant labeled with Texas Red as in Fig. 4) and 196 n

m

rhodopsin monomer in POPC nanodiscs. Calibrated light exposures were used to titrate in photoactivated rhodopsin. Three separate experiments are shown with fitted KD of 2.5 ± 0.3 n

m

(triangles), 4.2 ± 0.4 n

m

(open circles), or 4.0 ± 0.2 n

m

(circles). The inset shows the fluorescence transient from the first light exposure, fit to a second order reaction. B, titration of arrestin with photoactivated rhodopsin. Measurements were made at a total arrestin concentration of 230 n

m

and P-Rh* monomer nanodiscs at a concentration of 510 n

m

. Photoactivated P-Rh* was titrated in, using calibrated light exposures. KD from fitting of the curve is 16 n

m

. The inset shows transformed data with an x intercept of 1.0, representing the number of arrestin binding sites per P-Rh* monomer.

FIGURE 6.

FIGURE 6.

The effect of negatively charged lipids on the affinity of arrestin-1 for P-Rh*. A, dissociation constants obtained from the fluorescence binding assay as a function of POPS content of the nanodiscs. Two preparations of rhodopsin with different phosphorylation levels (as determined by [γ-32P]ATP incorporation) were used in the nanodisc assembly. Binding reactions contained 50 n

m

arrestin and 400 n

m

rhodopsin. B, pseudo-first order kinetic constants were determined from exponential fit to the binding time course. Reactions contained 25 n

m

arrestin. 184 n

m

light-activated rhodopsin in nanodiscs was generated by a 2.5-s light exposure.

FIGURE 7.

FIGURE 7.

Activating mutations differentially change the dependence of arrestin-1 binding to P-Rh* on negatively charged lipids. A, the binding of radiolabeled WT arrestin-1, two polar core mutants (R175E and D296R), and two mutants where the C-tail is either deleted (Tr(1–378)) or detached (arrestin-1 (F375A,V376A,F377A) (3A)) to P-Rh* (0.3 μg) in POPC nanodiscs with the indicated fraction of POPS was performed, as in Fig. 2. Means ± S.D. of two experiments performed in duplicate are shown. B, to facilitate comparison, means are plotted as a percentage of maximum binding of each form of arrestin-1 (which is significantly increased by activating mutations). C, sequence alignment of the C termini of four arrestins with negatively charged residues shown in red. Other highlights are as follows: three bulky hydrophobic residues anchoring the C-tail to the N-domain via β-strand I and α-helix I (olive); arginine that is part of the main phosphate sensor, the polar core (light blue); and the main clathrin-binding site in arrestin-2 (present in all vertebrate non-visual arrestins) (underlined).

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