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.
Highlights
Phosphorylated rhodopsin (P-Rh*) blocks further transducin activation [1] by steric exclusion [2]
The data show that GRK1, like transducin, does not require negatively charged lipids, phosphorylating monomeric Rh* in nanodiscs containing 0 –50% POPS to the same level of ϳ3 mol of phosphate/mol, which is required for high affinity arrestin-1 binding in vitro [33] and in vivo [41]
Recent reports from several groups show that monomeric rhodopsin is fully capable of activating its cognate G protein, transducin (18 –21), and that forced dimerization of rhodopsin reduces its activity ϳ2-fold [18, 20]
Summary
Phosphorylated rhodopsin (P-Rh*) blocks further transducin activation [1] by steric exclusion [2]. We used two different interaction assays utilizing radiolabeled and purified fluorescently labeled arrestin-1 to show that arrestin-1 binds monomeric P-Rh* with physiologically relevant affinity and stoichiometry.
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