Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding

Timothy H. Bayburt; Sergey A. Vishnivetskiy; Mark A. McLean; Takefumi Morizumi; Chih-chin Huang; John J. G. Tesmer; Oliver P. Ernst; Stephen G. Sligar; Vsevolod V. Gurevich

doi:10.1074/jbc.M110.151043

Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding*

From the ^‡Department of Biochemistry, University of Illinois, Urbana, Illinois 61801,
the ^§Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232,
^¶Charité-Universitätsmedizin Berlin, D-10117 Berlin, Germany, and
the ^‖Life Sciences Institute and
^**Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-2216

1 To whom correspondence may be addressed: Dept. of Biochemistry, University of Illinois, 116 Morrill Hall, 505 S. Goodwin, Urbana, IL 61801. Tel.: 217-244-7395; Fax: 217-265-4073; E-mail: s-sligar{at}uiuc.edu.
2 To whom correspondence may be addressed: Dept. of Pharmacology, Vanderbilt University, 2200 Pierce Ave., Rm. 417D, Nashville, TN 37232. Tel.: 615-322-7070; Fax: 615-343-6532; E-mail: vsevolod.gurevich{at}vanderbilt.edu.

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.

Footnotes

↵* This work was supported, in whole or in part, by National Institutes of Health Grants EY011500, GM081756, and GM077561 (to V. V. G.), GM033775 (to S. G. S.), and HL071818 (to J. J. T.). This work was also supported by Deutsche Forschungsgemeinschaft Grants Sfb740 and ER 294/1-1 (to O. P. E.).
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.