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

Artemyev, N.O.

Biochemistry 36(14): 4188-4193

1997


ISSN/ISBN: 0006-2960
PMID: 9100013
DOI: 10.1021/bi963002y
Accession: 008224155

Download citation:  
Text
  |  
BibTeX
  |  
RIS

Article/Abstract emailed within 0-6 h
Payments are secure & encrypted
Powered by Stripe
Powered by PayPal

Abstract
In photoreceptor cells of vertebrates, the GTP-bound alpha-subunit of rod G-protein, transducin (G-talpha), interacts with the cGMP phosphodiesterase inhibitory gamma-subunit (P-gamma) to activate the effector enzyme. The GDP-bound G-talpha can also bind the P-gamma subunit, albeit with a lower affinity than G-talpha-GTP. In this work, interactions between G-talpha-GDP and P-gamma or P-gamma-24-45Cys labeled with the fluorescent probe 3-(bromoacetyl)7-(diethylamino)coumarin (P-gamma-BC, P-gamma-24-45BC) have been investigated. Addition of G-talpha-GDP to P-gamma-BC produced approximately a 6-fold maximal increase in the probe fluorescence, while the fluorescence of P-gamma-24-45BC was enhanced by 2.3-fold. The K-d's for the G-talpha-GDP binding to P-gamma-BC and P-gamma-24-45BC were 75 +- 8 nM and 400 +- 110 nM, respectively. The G-tbeta-gamma subunits had no notable effect on the binding of G-talpha-GDP to P-gamma-BC or P-gamma-24-45BC, suggesting that P-gamma and G-tbeta-gamma bind to G-talpha-GDP noncompetitively. The G-talpha-beta-gamma interaction with the fluorescently labeled P-gamma was effectively blocked in the light-activated rhodopsin (R*)-G-talpha-beta-gamma complex. Furthermore, addition of excess P-gamma or P-gamma-24-45 prevented binding of G-talpha-beta-gamma to R*, indicating that the R* and P-gamma binding surfaces on G-talpha-beta-gamma may overlap. It is likely that R* has a binding site within the alpha-3-beta-5 region of G-talpha, which is a proposed site of G-talpha-GDP binding to P-gamma-24-45. Alternatively, R* may induce conformational changes of the G-talpha alpha-3-beta-5 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.