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Phosphorylation modulates the affinity of light-activated rhodopsin for G protein and arrestin

S K Gibson1, J H Parkes, P A Liebman

  • 1Department of Biochemistry and Biophysics, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6059, USA.

Biochemistry
|May 10, 2000
PubMed

Insights

GPCR phosphorylation stoichiometry quantitatively impacts G protein and arrestin binding. Increased phosphorylation favors arrestin binding over G protein binding to activated rhodopsin.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Signaling

Background:

  • G protein-coupled receptor (GPCR) phosphorylation is known to reduce effector activity and arrestin binding.
  • The quantitative impact of GPCR multiphosphorylation on G protein activation and arrestin binding has not been thoroughly investigated.

Purpose of the Study:

  • To quantitatively examine the effect of receptor multiphosphorylation on G protein and arrestin binding parameters for light-activated rhodopsin.
  • To determine how phosphorylation stoichiometry influences the binding constants of G protein and arrestin to rhodopsin.

Main Methods:

  • Utilized rod disk membranes with varying average receptor phosphorylation stoichiometries.
  • Titrated membranes with G protein (Gt) or arrestin using brief light flashes.
  • Analyzed titration data using nonlinear least-squares (Simplex) reaction modeling to determine binding constants.

Main Results:

  • Receptor phosphorylation altered both G protein and arrestin binding constants for activated rhodopsin in proportion to phosphorylation stoichiometry.
  • G protein binding affinity decreased, while arrestin binding affinity increased with added phosphates.
  • At phosphorylation levels ≤2 phosphates/rhodopsin, Gt bound more tightly; at higher levels, arrestin binding was favored.
  • Arrestin binding was significantly slower than G protein binding across all phosphorylation levels.

Conclusions:

  • GPCR phosphorylation stoichiometry directly modulates G protein and arrestin binding affinities.
  • The balance shifts towards arrestin binding at higher phosphorylation levels, potentially regulating signaling duration.
  • Membrane electrostatics play a crucial role in modulating these binding interactions during receptor phosphorylation.

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