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Monomeric rhodopsin in nanodiscs is phosphorylated by rhodopsin kinase and binds arrestin with high affinity in the presence of negatively charged lipids
Author(s) -
Vishnivetskiy Sergey A,
Bayburt Timothy H,
Morizumi Takefumi,
Huang Chihchin,
Tesmer John J,
Ernst Oliver P,
Sligar Stephen G,
Gurevich Vsevolod V
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.707.3
Subject(s) - rhodopsin , arrestin , chemistry , phosphorylation , biophysics , g protein coupled receptor , mutant , monomer , phosphatidylserine , membrane , receptor , biochemistry , stereochemistry , biology , phospholipid , retinal , organic chemistry , gene , polymer
The role of dimerization of class A GPCRs is hotly debated. We show that monomeric rhodopsin in nanodiscs is phosphorylated by rhodopsin kinase (GRK1) and binds arrestin1 as well as in native disc membranes. The efficiency of rhodopsin phosphorylation is the same in phosphatidylcholine (PC) nanodiscs containing 0–50% phosphatidylserine (PS). In contrast, arrestin1 binding to P‐Rh* in pure PC is low. Binding increases with the proportion of PS and plateaus at ~10‐fold higher level at ~30% PS. To investigate the role of PS in the arrestin1‐P‐Rh* interaction, we used structurally different “enhanced” arrestin1 mutants that bind P‐Rh* and Rh* with comparable affinity. Truncated arrestin1(1–378) that has no C‐tail and 3A mutant where the interaction anchoring the C‐tail to the body of the molecule is destabilized were significantly less dependent on PS, demonstrating >50% of maximum binding in pure PC. In contrast, polar core mutants with the same phenotype, R175E and D296R, depend on PS almost as much as WT arrestin1. Thus, monomeric receptor is normally phosphorylated and binds arrestin. Negatively charged lipids promote interaction by “pushing” the arrestin C‐tail away from the receptor‐binding surface. This is the first indication of the function of multiple negative charges present in the C‐tail of all arrestin subtypes. NIH grants EY011500 (VVG), GM033775 (SGS), HL071818 (JJT), and DFG Sfb740 and ER 294/1‐1 (OPE)