Specific isoprenyl group linked to transducin gamma-subunit is a determinant of its unique signaling properties among G-proteins.
Among 11 subtypes of heterotrimeric G-protein gamma-subunit, gamma1 (rod), gamma8 (cone) and gamma11 are modified with farnesyl while the others are modified with geranylgeranyl at the C-terminus. To understand the role of specific isoprenylation (farnesylation) of retinal transducin, we examined how and to what extent the type of isoprenyl group affects transducin-beta gamma (beta1 gamma1) functions such as interactions with membranes, Galpha/receptor, and effectors. To this end, the C-terminal farnesylation signal sequence (CVIS) of gamma1 was replaced by a geranylgeranylation signal (CVIL), and the resultant mutant (S74L) or wild-type (WT) gamma1 was coexpressed with beta1 in the baculovirus-Tn5 insect cell system. Both gamma1WT and gamma1S74L expressed as a beta gamma complex were mixtures modified with farnesyl and geranylgeranyl groups. The ratio of farnesyl to geranylgeranyl in preparations of beta1 gamma1WT and beta1 gamma1S74L purified from the Tn5 cell membrane fraction was about 1:2 and 1:6, respectively. These two forms of recombinant beta1 gamma1 and retinal beta1 gamma1 were different in their abilities to associate with rod outer segment membranes with the following rank order: beta1 gamma1S74L > beta1 gamma1WT > retinal beta1 gamma1. Functionally, beta1 gamma1S74L was the most potent to promote pertussis toxin-catalyzed ADP ribosylation of transducin-alpha (Talpha), to stimulate metarhodopsin II-catalyzed GTPgammaS-binding reaction to Talpha and to modulate adenylyl cyclase and phospholipase C activities. All of the beta1 gamma1 functions absolutely required the isoprenylation of the gamma-subunit. As for the interaction with Goalpha and adenylyl cyclase, predominantly geranylgeranylated beta1 gamma1S74L was less effective than geranylgeranylated beta1 gamma2 purified from bovine brain. These results demonstrate that the properties of Gbeta gamma are strongly affected by the type of functionally indispensable isoprenylation in addition to the amino acid sequence of Ggamma. The relative contribution of the two factors depends on proteins with which Gbeta gamma interacts.
Matsuda
T
T
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Japan.
Hashimoto
Y
Y
Ueda
H
H
Asano
T
T
Matsuura
Y
Y
Doi
T
T
Takao
T
T
Shimonishi
Y
Y
Fukada
Y
Y
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism; D046911:Macromolecular Substances; D009036:Moths / Q000235:genetics; D017368:Protein Prenylation / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D018411:Spodoptera / Q000235:genetics; D015293:Transducin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012243:Rhodopsin; D015293:Transducin