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Nucleotide exchange and cGMP phosphodiesterase activation by pertussis toxin inactivated transducin
L Ramdas1, R M Disher, T G Wensel
1Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Abstract:
Transducin, the signal coupling protein of retinal rod photoreceptor cells, is one of a family of G proteins that can be inactivated by pertussis toxin. We have investigated the nature of this inactivation in order to determine (1) whether it requires the toxin-catalyzed transfer of ADP-ribose from NAD+ to cysteine-347 of the alpha subunit and (2) whether it involves locking the alpha subunit in the inactive conformation characteristic of its GDP-bound state, or is limited to disruption of binding to photoexcited rhodopsin (R*). Our results indicate that all observed effects of pertussis toxin treatment, including a shift in the electrophoretic mobility of transducin's alpha subunit and functional inactivation, require NAD+ and that the appearance of the shift parallels incorporation of ADP-ribose. We have also found that, apart from interactions with photoexcited rhodopsin, the functional properties of ADP-ribosylated transducin are essentially the same as those of unmodified transducin. Normal spontaneous nucleotide exchange kinetics and the ability to activate cGMP phosphodiesterase are preserved following quantitative ADP-ribosylation, as are the abilities to hydrolyze GTP, to bind to a dye affinity column, and to display enhanced fluorescence upon addition of Al3+ and F-. Thus, ADP-ribosylation merely blocks catalysis of transducin nucleotide exchange by R* and does not lock transducin in an inactive state.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Pertussis toxin inactivates transducin, a G protein in retinal rod cells, by ADP-ribosylating its alpha subunit. This modification blocks interaction with photoexcited rhodopsin but does not lock the protein in an inactive state.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Signaling
Background:
- Transducin is a key G protein in retinal rod photoreceptors, mediating phototransduction.
- Pertussis toxin is known to inactivate certain G proteins, including transducin.
Purpose of the Study:
- To elucidate the mechanism of pertussis toxin-induced inactivation of transducin.
- To determine if inactivation requires ADP-ribosylation at cysteine-347.
- To ascertain if inactivation locks transducin in a GDP-bound state or disrupts rhodopsin binding.
Main Methods:
- Investigated the role of NAD+ in pertussis toxin-induced effects.
- Monitored ADP-ribose incorporation into transducin's alpha subunit.
- Assessed functional properties of ADP-ribosylated transducin, including nucleotide exchange, GTP hydrolysis, and cGMP phosphodiesterase activation.
Main Results:
- Pertussis toxin-induced inactivation and electrophoretic mobility shift of transducin's alpha subunit require NAD+.
- ADP-ribosylation parallels the observed mobility shift.
- ADP-ribosylated transducin retains normal GTP hydrolysis, nucleotide exchange kinetics, and cGMP phosphodiesterase activation capabilities.
- The primary effect of ADP-ribosylation is blocking transducin activation by photoexcited rhodopsin.
Conclusions:
- Pertussis toxin inactivates transducin via ADP-ribosylation of the alpha subunit.
- This modification specifically inhibits the interaction with photoexcited rhodopsin.
- ADP-ribosylation does not induce a constitutively inactive state of transducin.