Monomeric rhodopsin is sufficient for normal rhodopsin kinase (GRK1) phosphorylation and arrestin-1 binding.
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.
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
Timothy H
TH
Bayburt
Sergey A
SA
Vishnivetskiy
Mark A
MA
McLean
Takefumi
T
Morizumi
Chih-Chin
CC
Huang
John J G
JJ
Tesmer
Oliver P
OP
Ernst
Stephen G
SG
Sligar
Vsevolod V
VV
Gurevich
D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D004563:Electrochemistry; D005453:Fluorescence; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism / Q000494:pharmacology; D008055:Lipids / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014316:Tritium; D014785:Vision, Ocular / Q000502:physiology*; D000071557:beta-Arrestins
D019390:Arrestins; D008055:Lipids; D000071557:beta-Arrestins; D014316:Tritium; D012243:Rhodopsin; D051606:G-Protein-Coupled Receptor Kinase 1; D007930:Leucine