Interacting Targets of the Farnesyl of Transducin γ-Subunit

Abstract

G protein γ-subunits are isoprenylated and carboxyl methylated at the C-terminal cysteine residue, and the set of the posttranslational modifications is indispensable for the function of the photoreceptor G protein transducin (Tα/Tβγ). To explore farnesyl-mediated molecular interactions, we investigated molecular targets of a Tβγ analogue that was engineered to have a photoreactive farnesyl analogue, (3-azidophenoxy)geranyl (POG), covalently bound to the C-terminal cysteine of Tγ. POG-modified Tβγ was further subjected to modification by methylation at the C-terminal carboxyl group, which copies a complete set of the known posttranscriptional modifications of Tβγ. Photoaffinity labeling experiment with the photoreactive Tβγ analogue in its free form indicated that the POG moiety of Tγ interacted with Tβ. In the trimeric Tα/Tβγ complex, the POG moiety was cross-linked with Tα in addition to concurrent affinity labeling of Tβ. When photoreactive Tβγ was reconstituted with Tα and light-activated rhodopsin (Rh*) in rod outer segment (ROS) membranes, the POG moiety interacted with not only Tα and Tβ but also Rh* and membrane phospholipids. The cross-linked phospholipid species was analyzed by ELISA employing a variety of lipid-binding probes, which revealed phosphatidylethanolamine (PE) and phosphatidylserine (PS) as selective phospholipids for POG interaction in the ROS membranes. These results demonstrate that the modifying group of Tγ plays an active role in protein−protein and protein−membrane interactions and suggest that the farnesyl−PE/PS interaction may support the efficient formation of the signaling ternary complex between transducin and Rh*.

G-Protein βγ-Complex Is Crucial for Efficient Signal Amplification in Vision

“…Although the soluble Gtα pool is fully capable of productive interactions with R*, the rate of its activation is limited by binding to the membrane and R* (Heck and Hofmann, 2001). This result is also consistent with a direct involvement of Gtβγ in R* interactions and R*-catalyzed nucleotide exchange on Gtα (Kisselev and Downs, 2006; Katadae et al, 2008). Overall, this leads to the severely compromised rate of Gtα activation without Gtβγ.…”

Phosphatidylserine colocalizes with epichromatin in interphase nuclei and mitotic chromosomes

“… 3 During apoptosis, PS is externalized at the plasma membrane, where it can be detected with Annexin V or a PS-specific monoclonal antibody (1H6). 4 - 6 PS has also been detected at the nucleoplasmic side of the NE with GFP-Annexin V, where it is likely derived from continuity of the NE with the ER. 7 In the present study, we demonstrate by immunostaining that PS is localized within the epichromatin region of interphase nuclei and mitotic chromosomes, exhibiting a location which is conserved in evolution.…”

“…Phosphatidylserine (PS) is the major anionic glycerophospholipid within eukaryotic membranes, being distributed asymmetrically within lipid bilayers and involved in binding to membrane associated polycationic proteins 3 . During apoptosis, PS is externalized at the plasma membrane, where it can be detected with Annexin V or a PS-specific monoclonal antibody (1H6) 4 - 6 . PS has also been detected at the nucleoplasmic side of the NE with GFP-Annexin V, where it is likely derived from continuity of the NE with the ER 7 .…”

GPCRs and G Protein Activation

“…All G subunits are C-terminally prenylated (some with geranyl-geranyl, others with farnesyl groups) and carboxymethylated: this helps to anchor the G subunits to the plasma membrane. The C-terminal sequence determines the nature of the prenyl group (farnesyl or geranyl-geranyl) modifying the G subunit; both the C-terminal sequence and the prenyl group play an active role in the recognition of both rhodopsin and phospholipids (Katadae et al, 2008). Although the literature on this subject is sparse, there is some evidence that other GPCRs also recognize preferentially specific G subunits (Jian et al, 2001;Kisselev and Downs, 2003;Birnbaumer, 2007).…”