Regulation of Gαi Palmitoylation by Activation of the 5-Hydroxytryptamine-1A Receptor

Transfection of either the ␣ 1b -adrenoreceptor or G␣ 11 into a fibroblast cell line derived from a G␣ q /G␣ 11 double knockout mouse failed to produce elevation of intracellular [Ca 2؉ ] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the ␣ 1b -adrenoreceptor with the palmitoylation-resistant C9S,C10S G␣ 11 also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S G␣ 11 or C10S G␣ 11 . Expression of a fusion protein between the ␣ 1b -adrenoreceptor and G␣ 11 allowed [Ca 2؉ ] i elevation, and this was also true for a fusion protein between the ␣ 1b -adrenoreceptor and C9S,C10S G␣ 11 , since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin ␣, as a ␤⅐␥-sequestering agent, fully attenuated the Ca 2؉ signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type G␣ 11 were also targets for agonist-regulated

Section: Discussionsupporting

confidence: 70%

Coordinated Agonist Regulation of Receptor and G Protein Palmitoylation and Functional Rescue of Palmitoylation-deficient Mutants of the G Protein G11α following Fusion to the α1b-Adrenoreceptor

Stevens¹,

Pediani

Carrillo

et al. 2001

“…The co-distribution of PAT and its substrates in lipid rafts would facilitate repalmitoylation. Agonists stimulate palmitate turnover on G ␣s and G ␣i , and this may have a regulatory role in desensitization of G-protein responses (43)(44)(45)(46). In vitro, palmitoylated G ␣i is resistant to deactivation by RGS proteins (47).…”

Section: Discussionmentioning

confidence: 99%

Enrichment of G-protein Palmitoyltransferase Activity in Low Density Membranes

Dunphy

Greentree

Linder

2001

Many signaling proteins are targeted to low density, sphingomyelin-and cholesterol-enriched membranes, also called lipid rafts. These domains organize receptormediated signaling events at the plasma membrane. Fatty acylation is one mechanism for targeting proteins to rafts. It was therefore of interest to determine if protein palmitoyltransferase activity is also present in these domains. In this study, protein palmitoyltransferase activity, assayed using G-protein alpha subunits as a substrate, was found to be highly enriched in low density membranes derived from cells that express caveolin as well as those that do not. Depletion of cellular cholesterol with the drug methyl-␤-cyclodextrin resulted in inhibition of palmitoyltransferase activity and a redistribution of the remaining activity to membranes of higher density. This effect was reversed by adding cholesterol to cyclodextrin-treated cells. When reconstituted into cell membranes, the population of purified recombinant G ␣i that was palmitoylated was highly enriched in the low density membrane fractions, whereas the bulk unmodified G ␣i -protein was largely excluded. This effect required palmitoyltransferase activity and was abolished if the palmitoylated cysteine was mutated. Thus, palmitoyltransferase facilitates the enrichment of fatty acylated signaling molecules in plasma membrane subdomains.

“…Shuttling and Translocation Are Inhibited by 2BP-The G protein ␣ subunits belonging to the G i/o , G s , and G q families are palmitoylated (5), and the palmitoyl moiety can be turned over in the basal deactivated state (t1 ⁄ 2 Ͼ 60 min for ␣ i ; 90 min for ␣ s ) (23)(24)(25). We tested whether acylation played a role in the shuttling of G protein subunits and the translocation of the ␤␥ complex between PM and endomembranes by examining the effect of 2BP, an inhibitor that has been used for identifying palmitoylation (13) of proteins.…”

Section: G Protein Shuttling Is Likely Diffusion-mediated-mentioning

confidence: 99%

Shuttling of G Protein Subunits between the Plasma Membrane and Intracellular Membranes

Chisari

Saini

Kalyanaraman

et al. 2007

Heterotrimeric G proteins (␣␤␥) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1 ⁄ 2 < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.Heterotrimeric G proteins are known to function on the cytosolic surface of the plasma membrane (PM) 2 in response to the stimulation of transmembrane receptors by most of the extracellular signals a mammalian cell senses (1-4). The PM localization of a G protein is facilitated by individual lipid modifications of the ␣ and ␤␥ subunits (5). Consistent with a model of G protein action on the PM, activated G protein subunits have been shown to modulate the functions of effectors such as adenylyl cyclase, phospholipase C, and ion channels which are also PM-localized (1-4). However, the spatiotemporal dynamics of G protein localization in a living cell has not been examined to determine whether G protein subunits are stably constrained to the PM. Although there is longstanding evidence for the existence of G protein subunits in intracellular membranes such as the Golgi complex and suggestions that they affect protein trafficking (6 -11), it has been unclear as to how they reach endomembranes and if they are native to these membranes or in transit to the PM.Here we used a variety of imaging methods on live cells with fluorescent protein-tagged G protein subunits including photoswitchable Dronpa (12) to observe G protein movement. A fluorescence resonance energy transfer (FRET)-based G protein sensor was used to examine whether G proteins reside constitutively in the endomembranes. The results show that G protein subunits shuttle rapidly between the PM and endomembranes in cells in the basal state maintaining a pool of G proteins in the endomembranes. We show that the shuttling is likely diffusive and not vesicle-mediated. Furthermore, 2-bromopalmitate (2BP), an inhibitor of palmitoylation (13), inhibited shuttling, suggesting that it may be regulated by acylation. MATERIALS AND METHODS Details of chemicals, expression constructs, cell lines, transfection, and treatment of cells are as in Saini et al. (41). Dronpa fluorescent proteins were introduced downstream of Gly-92 in␣ o as in the ␣ o -CFP construct that we have shown previously to possess normal...