Interaction of small G proteins with photoexcited rhodopsin

Abstract

Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23-27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinurn C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, P*P]ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed[32P]ADP-ribosy1ation of the a-subunit of transducin.These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.GTP-binding protein; Rhodopsin; Botulinum C3 ADP-ribosyl-transferase; Transducin; (Bovine rod outer segment)

Interaction of recombinant rho A GTP‐binding proteins with photoexcited rhodopsin

“…As was reported for the endogenous C3 substrates [14], the recombinant rho A protein migrated in the SDS-PAGE as a double band, with the less intensively labelled band exhibiting a somewhat larger apparent size. Similar data have been reported for rho C protein expressed in E. coil and was attributed to formation of 1-3) and of small G proteins by C3 (lanes 4-6) was performed as in [14] two conformational isomers of the same protein [4].…”

“…Transducin was eluted from illuminated ROS membranes by repeated (3 x ) hypotonic elution in the presence of 100 #M GTP [14]. Urea-treated ROS membranes were prepared according to [17].…”

“…[32p]ADP-ribosylation of ROS membrane-bound or released transducin by pertussis toxin and of endogenous small G proteins by C3 was performed as in [14].…”

“…Reaction was started by addition of urea-treated ROS membranes 05 #g protein/tube) and rho proteins, pre-incubated with each other for 5 min on ice either in dim red light or in bright white light. SDS-PAGE and autoradiography were as in [14].…”

“…We have recently reported that bovine rod outer segment (ROS) membranes contain two substrates for the ADP-ribosyltransferase activity of C3 [14]. Most interestingly, ADP-ribosylation of these proteins was regulated by light and guanine nucleotides in a manner similar to the ADP-ribosylation of the high molecular mass, heterotrimeric G protein transducin by pertussis toxin.…”