Functional homology between signal-coupling proteins. Cholera toxin inactivates the GTPase activity of transducin.

M E Abood,J B Hurley,M C Pappone,H R Bourne,L Stryer

doi:10.1016/s0021-9258(18)33851-1

Abstract

Both the light-stimulated cGMP phosphodiesterase of retinal rod outer segments (ROS) and hormone-stimulated adenylate cyclase are regulated by guanine nucleotide-binding regulatory proteins (N). Transducin serves as the signal-carrying regulatory protein in ROS, and the N protein (also called G or G/F) performs this role in the adenylate cyclase system. The GTP form of these regulatory proteins activates the corresponding enzyme, whereas the GDP form does not. Both transducin and the N protein possess a GTPase activity that restores the regulatory protein to the unstimulated state. Cholera enterotoxin catalyzes the transfer of ADP-ribose from NAD+ to the N protein, which inhibits its GTPase activity and activates adenylate cyclase. We report here that the toxin also catalyzes ADP-ribosylation of the alpha-subunit of transducin in ROS membranes. This modification of the guanine nucleotide-binding subunit of transducin is markedly enhanced by the bleaching of rhodopsin and by the addition of guanosine-5'-(beta, gamma-imino)triphosphate. In contrast, GDP, GTP, and guanosine-5'-(3-O)thiotriphosphate inhibit the reaction, while GMP and ATP have no effect. Under optimal conditions, toxin catalyzes labeling of 0.7 mol of the alpha-subunit of transducin/mol of bound [3H]guanosine-5'-(beta, gamma-imido)triphosphate and causes 70% inhibition of the light-dependent GTPase activity of transducin in ROS. These results indicate close functional homology between transducin of ROS and the N protein of adenylate cyclase.

Highlights

Both the light-stimulated cGMP phosphodiesterase of retinal rod outer segments (ROS) and hormone-stimulated adenylate cyclase are regulated by guanine nucleotide-binding regulatory proteins (N)
Materials-[3 2P]NAD+ and [y- 32 P]GTP were obtained from New England Nuclear or Amersham Corp.; [3H]Gpp(NH)p from Amersham Corp.; cholera toxin from Schwarz-Mann; ATP, GTP, GDP, Tris, NAD+, thymidine, arginine HCI, w-amino octyl agarose, NeurosporacrassaNADase, and calf intestine alkaline phosphatase from Sigma; and guanosine-5'-(3-O)thiotriphosphate, Gpp(NH)p was omitted from the ADP-ribosylation reaction mixture, only 0.05 mol of 32p was incorporated into the 39kDa band after 8 h of incubation
Our experiments reveal a series of striking parallels between transducin and the N component of adenylate cyclase as substrates for cholera toxin

Summary

CHOLERA TOXIN INACTIVATES THE GTPase ACTIVITY OF TRANSDUCIN*

Toxin catalyzes labeling of 0.7 mol of the a-subunit of tranducin/mol of bound [3 l]guanosine-5'-(f,y-imido)triphosphate and causes 70% inhibition of the light-dependent GTPase activity of transducin in ROS These results indicate close functional homology between transducin of ROS and the N protein of adenylate cyclase. The toxin stimulates cAMP synthesis by catalyzing ADP-ribosylation of a guanine nucleotide-binding polypeptide subunit of the N protein [11,12,13, 17, 20] This reaction results in inhibition of the GTPase activity associated with hormonal stimulation of cAMP synthesis and augmentation of GTP-dependent adenylate cyclase activity [12, 13, 20]. We find that the GTPase activity of transducin is blocked by ADP-ribosylation

EXPERIMENTAL PROCEDURES

DISCUSSION