Nucleotide exchange and cGMP phosphodiesterase activation by pertussis toxin inactivated transducin.
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)
Ramdas
L
L
Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Disher
R M
RM
Wensel
T G
TG
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D037342:Pertussis Toxin; D012374:Rod Cell Outer Segment / Q000378:metabolism; D013329:Structure-Activity Relationship; D015293:Transducin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014364:Tryptophan; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D009711:Nucleotides; D010566:Virulence Factors, Bordetella; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D014364:Tryptophan; D037342:Pertussis Toxin; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin