Binding of transducin to light-activated rhodopsin prevents transducin interaction with the rod cGMP phosphodiesterase gamma-subunit

Biochemistry. 1997 Apr 8;36(14):4188-93. doi: 10.1021/bi963002y.


In photoreceptor cells of vertebrates, the GTP-bound alpha-subunit of rod G-protein, transducin (G(t alpha)), interacts with the cGMP phosphodiesterase inhibitory gamma-subunit (Pgamma) to activate the effector enzyme. The GDP-bound G(t alpha) can also bind the Pgamma subunit, albeit with a lower affinity than G(t alpha)GTP. In this work, interactions between G(t alpha)GDP and Pgamma or Pgamma-24-45Cys labeled with the fluorescent probe 3-(bromoacetyl)-7-(diethylamino)coumarin (PgammaBC, Pgamma-24-45BC) have been investigated. Addition of G(t alpha)GDP to PgammaBC produced approximately a 6-fold maximal increase in the probe fluorescence, while the fluorescence of Pgamma-24-45BC was enhanced by 2.3-fold. The Kd's for the G(t alpha)GDP binding to PgammaBC and Pgamma-24-45BC were 75 +/- 8 nM and 400 +/- 110 nM, respectively. The G(t betagamma) subunits had no notable effect on the binding of G(t alpha)GDP to PgammaBC or Pgamma-24-45BC, suggesting that Pgamma and G(t betagamma) bind to G(t alpha)GDP noncompetitively. The G(t alpha betagamma) interaction with the fluorescently labeled Pgamma was effectively blocked in the light-activated rhodopsin (R*)-G(t alpha betagamma) complex. Furthermore, addition of excess Pgamma or Pgamma-24-45 prevented binding of G(t alpha betagamma) to R*, indicating that the R* and Pgamma binding surfaces on G(t alpha betagamma) may overlap. It is likely that R* has a binding site within the alpha3-beta5 region of G(t alpha), which is a proposed site of G(t alpha)GDP binding to Pgamma-24-45. Alternatively, R* may induce conformational changes of the G(t alpha) alpha3-beta5 region such that the resulting structural changes alter the adjacent consensus sequence for the guanine ring binding of GDP/GTP(NKXD), and lead to a reduction in the affinity of G-protein for guanine nucleotides.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3',5'-Cyclic-GMP Phosphodiesterases / metabolism*
  • Animals
  • Binding Sites
  • Cattle
  • Cell Membrane
  • Coumarins
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation
  • Escherichia coli / genetics
  • Fluorescence
  • Fluorescent Dyes
  • Gene Expression
  • Guanosine Diphosphate / metabolism
  • Guanosine Triphosphate / metabolism
  • Protein Binding
  • Recombinant Proteins / metabolism
  • Rhodopsin / metabolism*
  • Rod Cell Outer Segment / enzymology*
  • Transducin / metabolism*


  • 3-bromoacetyl-7-(diethylamino)coumarin
  • Coumarins
  • Fluorescent Dyes
  • Recombinant Proteins
  • Guanosine Diphosphate
  • Guanosine Triphosphate
  • Rhodopsin
  • 3',5'-Cyclic-GMP Phosphodiesterases
  • Transducin