Targeted Mutagenesis of the Farnesylation Site of Drosophila Gγe Disrupts Membrane Association of the G Protein βγ Complex and Affects the Light Sensitivity of the Visual System

Abstract

Activation of phototransduction in the compound eye of Drosophila is mediated by a heterotrimeric G protein that couples to the effector enzyme phospholipase C␤. The ␥ subunit of this G protein (G␥e) as well as ␥ subunits of vertebrate transducins contain a carboxyl-terminal CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) with a consensus sequence for protein farnesylation. To examine the function of G␥e farnesylation, we mutated the farnesylation site and overexpressed the mutated G␥e in Drosophila. Mass spectrometry of overexpressed G␥e subunits revealed that nonmutated G␥e is modified by farnesylation, whereas the mutated G␥e is not farnesylated. In the transgenic flies, mutated G␥e forms a dimeric complex with G␤e, with the consequence that the fraction of non-membrane-bound G␤␥ is increased. Thus, farnesylation of G␥e facilitates the membrane attachment of the G␤␥ complex. We also expressed human G␥rod in Drosophila photoreceptors. Despite similarities in the primary structure between the transducin ␥ subunit and Drosophila G␥e, we observed no interaction of human G␥rod with Drosophila G␤e. This finding indicates that human G␥rod and Drosophila G␥e provide different interfaces for the interaction with G␤ subunits. Electroretinogram recordings revealed a significant loss of light sensitivity in eyes of transgenic flies that express mutated G␥e. This loss in light sensitivity reveals that post-translational farnesylation is a critical step for the formation of membrane-associated G␣␤␥ required for transmitting light activation from rhodopsin to phospholipase C␤.

Phototransduction and retinal degeneration in Drosophila

“…The effector for the light-activated rhodopsin is the heterotrimeric G-protein, G q and it is encoded by Gα q [108][109][110], Gβ e [111,112], and Gγ e [113,114], all of which are eyeenriched. After exchange of the guanosine diphosphate (GDP) bound to the Gα q for GTP, the Gα q and Gβγ subunits dissociate and the activated Gα q -GTP binds to the PLC encoded by norpA (no receptor potential A) [5,115].…”

“…A strong allele of Gβ e mutant, Gβ 1 e , displays a ∼100-fold reduction in light sensitivity [111], and a mutation in the farnesylation site of Gγ e also results in a reduction in light sensitivity [114]. Moreover, light-simulated PLC activity is deficient in the Gβ 1 e mutant [116].…”

Over-expression of a truncated Arabidopsis thaliana heterotrimeric G protein γ subunit results in a phenotype similar to α and β subunit knockouts

“…Functional characterization of four independent 35S-γ ⁎ lines has consistently shown that they behave in the same way as α-and β-deficient mutants, albeit with less severity. The reduction of severity was predicted as a possible outcome of our approach, which is not as dominant in nature as the complete α or β knockouts and was also observed in a similar study using Drosophila (Schillo et al, 2004). Expression levels of the endogenous AGG1 and AGG2 genes are unchanged in 35S-γ ⁎ plants and it is therefore possible that a small proportion of the β/γ dimers in the transgenic plants contain a WT γ subunit, making the dimer functional.…”

“…In mammals and insects it is known that a prenyl group is posttranslationally added (isoprenylation) to a CAAX motif on the C-terminus of the γ subunit and functions as a membrane anchor for the dimer (Matsuda et al, 1998). Studies have shown that isoprenylation of the γ subunit is essential for interactions with the plasma membrane (Simonds et al, 1991;GarciaHiguera et al, 1996;Lindorfer et al, 1996;Matsuda et al, 1998;Schillo et al, 2004), receptors (Simonds et al, 1991;Myung et al, 1999), and effectors (Simonds et al, 1991;Li et al, 1998;Matsuda et al, 1998;Murga et al, 1998), but not with the β subunit as dimerisation occurs before isoprenylation (Matsuda et al, 1998;Peskan and Oelmuller, 2000;Schillo et al, 2004). It is also known that β or γ subunits are unable to activate effectors individually (Murga et al, 1998;Yoshikawa et al, 2000), and free β or γ monomers are unstable or rapidly degraded by proteases (Simonds et al, 1991;Garcia-Higuera et al, 1998;Jin et al, 1998).…”

Mouse G-protein gamma3 expression in the developing CNS and neural crest cell derivatives

“…This motif is of great importance as it is the site where a prenyl group is post-translationally added to the poylpeptide, which occurs following the binding of a γ to a β subunit, and allows the heterodimer to be targeted to the endoplasmic reticulum prior to its subsequent anchorage at the plasma membrane (reviewed in Marrari et al, 2007). Dimerization between β and γ subunits is not dependent on isoprenylation (Schillo et al, 2004), but this lipidation is required for high affinity α-βγ and α-βγ-receptor interactions and effector activation (Myung et al, 2006, Myung et al, 1999.…”