In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 51 ( 2012-12-18)
Abstract:
Overall, our results suggest that G-protein signaling leverages subunit-dependent membrane affinities to differentially control receptor-induced movement of G-protein subunits in cells. They show that variation in hydrophobic and positively charged residues among these subunits can control their differential movement inside cells. The conservation of the primary structures of γ-subunits across mammalian species shows that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intracellular movement. More therapeutic drugs target GPCRs than any other protein family, and G-protein βγ translocation has so far been observed for many different GPCRs and cell types. The identification of mechanisms at the basis of signaling protein movement triggered by extracellular signals provides potential targets for therapeutic intervention as well as experimental perturbation to understand biological signaling. We tested this hypothesis by mutating the basic and hydrophobic residues located immediately upstream of the lipid-modified cysteine in a slow translocating γ-subunit type, but not in other more rapidly translocating γ-subunits. Basic residues can interact with negatively charged lipid head groups. Hydrophobic residues can interact with the lipid tails. Replacing the basic or the hydrophobic residues with the neutral residue, alanine, increased the rate of βγ translocation almost fourfold. Replacing all four residues increased it sixfold. To test whether these residues exert their effects on translocation rates through direct interaction with lipid membranes, we synthesized lipid-modified peptides corresponding to these γ-subunit domains and measured their dissociation from pure lipid vesicles in vitro. The membrane dissociation rates of these peptides showed a similar dependence on hydrophobic and basic residues to that observed for differential βγ translocation kinetics in intact cells. Both the α- and γ-subunits are modified by lipid moieties. We developed a mathematical model to describe βγ translocation in which it was surmised that the strong hydrophobicity of lipid modifications on the α- and γ-subunits together helps maintain stable association of G-protein heterotrimers with the plasma membrane. On receptor activation, when the βγ-complex dissociates from the α-subunit, the lipid moiety on the γ-subunit alone was surmised to be insufficient to retain βγ on the membrane ( Fig. P1 ). This can lead to its diffusion through the cytosol and continual exchange between the plasma membrane and intracellular membranes. This βγ movement is consistent with the requirement of the palmitoyl lipid moiety of α-subunits for stable membrane binding of prenyl lipid-modified proteins like the γ-subunit ( 2 ). This mechanism can enable an intracellular pool of activated βγ to be maintained by continued cycles of heterotrimer activation at the plasma membrane. It also allows inactive α-subunits to trap βγ-subunits in heterotrimers at the plasma membrane upon receptor deactivation. Comparison of simulations based on the model with results from analyzing live cells suggested that the same rate-limiting step controls both the forward and the reverse translocation kinetics. This suggested that differential membrane affinity of βγ-subunit types controls the differences seen in their translocation kinetics. G proteins are made up of three subunits: α, β, and γ. Each of these subunits comprises families of proteins. The βγ complex undergoes receptor-stimulated translocation from the plasma membrane to internal membranes. By imaging βγ translocation in living cells, we found that the rates of forward translocation to intracellular membranes following receptor activation and reverse translocation to the plasma membrane upon receptor deactivation have the same dependence on the associated γ-subunit family member. We also found that when a pool of free βγ-subunits is maintained by sustained receptor activation, βγ dimers constantly move throughout the cell, exploring the cytosolic surfaces of the plasma membrane and intracellular membranes. Extracellular signals such as light, hormones, and neurotransmitters activate G-protein–coupled receptors (GPCRs) and G proteins on the plasma membrane of cells to regulate critical functions such as vision, heart rate, and hormone secretion. There is limited information on the spatiotemporal dynamics of proteins in this signaling pathway in a living cell. Recent evidence suggests that G-protein subunits undergo constitutive and receptor-stimulated movement between the plasma membrane and intracellular membranes, allowing direct communication between extracellular signals and the cell interior ( 1 ). Here we identify differential affinities of G-protein subunits for membranes as a mechanistic basis for this movement.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1205345109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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