G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes

Abstract

Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. Gprotein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.protein-membrane interaction | spatio-temporal dynamics | G protein-coupled receptors C ompared with the vast biochemical information available for signaling proteins from cell-disruptive techniques, much less is known about their spatiotemporal dynamics in the 3D space of an intact living cell. Many important signaling proteins are posttranslationally modified with lipid groups that facilitate their interactions with membranes. Live cell imaging has so far revealed important roles for constitutive cycles that maintain proper localization of lipidated proteins (1) and for dynamic protein redistribution throughout the cell in response to a signal (2), but the detailed nature of lipid-modified protein movement between membranes is generally not well understood.Heterotrimeric G proteins consist of lipid-modified α-and βγ-subunits (3). They are central transducers of the most important signaling pathways in mammals, yet little is known about their intracellular movement. Inactive heterotrimers consist of a GDP-bound α-subunit in complex with a stable βγ-dimer and localize to the plasma membrane (3). Extracellular signals activate transmembrane G-protein-coupled receptors (GPCRs) that catalyze nucleotide exchange on the G-protein α-subunit (3). The βγ-complex can then dissociate from the active α-subunit and translocate from the plasma membrane to intracellular membranes (4-6). βγ translocation has been observed for many different cell types and receptor types, suggesting that it is a common feature of G-protein signaling (4-...

Gγ identity dictates efficacy of Gβγ signaling and macrophage migration

“…Although Gβγ is classically considered PM bound, recent work has shown that, upon GPCR activation, Gβγ translocates from the PM to internal membranes (IMs) until an equilibrium is reached (25). Interestingly, translocation half time (Tt 1/2) and the extent |T| are governed by the type of accompanying Gγ subunit (28,29). These results further suggest that the PMaffinity of a Gβγ is Gγ subtype dependent.…”

“…Extensive mutagenesis to the pre-CaaX region of Gγ suggested that this 5 or 6 -residue region modulates Gβγ-PM interactions, in which positively charged and hydrophobic amino acids strengthen the PM-affinity. Previously reported translocation data of Gγ mutants with altered preCaaX residues further validate the role of this motif in controlling the PM-affinity (29). The complete loss of PM localization observed in Gγ9 upon cysteine removal from CaaX motif indicates that, pre-CaaX region only serves as a strong modulator of PM-affinity, while prenylation is essential for primary PM anchoring of Gβγ.…”

Activated G Protein Gαs Samples Multiple Endomembrane Compartments

“…These results rule out a role for the classical secretory pathway in the return of activated G␣ s to the plasma membrane, and argue against a role for vesicular trafficking in general. It is possible that free G␣ s subunits sample all endomembrane compartments and passively redistribute back to the plasma membrane upon deactivation, as is thought to be the case for translocating G␤␥ dimers (34). However, the rate at which G␣ s recycles to the plasma membrane is 3-fold slower than the rate at which it leaves this compartment, suggesting that different mechanisms are involved in recycling and the initial translocation.…”

Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

“…Consistent with previous observations (Karunarathne et al. , 2012; O'Neill et al. , 2012), activation of endogenous CXCR4 receptors with 50 ng/ml SDF-1α triggered βγ9 translocation from the plasma membrane to intracellular membranes, which was detected as a loss of YFP fluorescence from the plasma membrane (Figure 2, B and C).…”