G-protein signaling leverages subunit-dependent PNAS PLUS membrane affinity to differentially control βγ translocation to intracellular membranes Patrick R. O’Neilla, W. K. Ajith Karunarathnea, Vani Kalyanaramana, John R. Silviusb,1, and N. Gautama,c,1 Departments of aAnesthesiology and cGenetics, Washington University School of Medicine, St. Louis, MO 63110; and bDepartment of Biochemistry, McGill University, Montreal, QC, Canada H3G 1Y6 Edited by Melvin I. Simon, University of California at San Diego, La Jolla, CA, and approved November 5, 2012 (received for review April 11, 2012) Activation of G-protein heterotrimers by receptors at the plasma by live cell imaging (4, 6). Second, mammals express 5 β-and12 membrane stimulates βγ-complex dissociation from the α-subunit γ-subunit types and the βγ-complex exhibits differential trans- and translocation to internal membranes. This intermembrane move- location kinetics that vary by over an order of magnitude, depending ment of lipid-modified proteins is a fundamental but poorly under- on the specific γ-subunit type (6, 8). The interactions responsible stood feature of cell signaling. The differential translocation of for these differential translocation kinetics are not known. Amino fi γ G-protein βγ-subunit types provides a valuable experimental model acid sequences vary signi cantly between different -subunits, but to examine the movement of signaling proteins between membranes each is highly conserved across species, indicating their functional specificity. We used mutational approaches based on differences in a living cell. We used live cell imaging, mathematical modeling, and γ in vitro measurements of lipidated fluorescent peptide dissociation in evolutionarily conserved residues between -subunit types to from vesicles to determine the mechanistic basis of the intermem- identify the mechanistic basis of the translocation. brane movement and identify the interactions responsible for differ- We used live cell imaging to investigate the reversible nature of βγ translocation as well as intermembrane exchange of free βγ ential translocation kinetics in this family of evolutionarily conserved during a continually stimulated cycle of G-protein activation. Our proteins. We found that the reversible translocation is mediated by findings led to the development of a mathematical model of βγ the limited affinity of the βγ-subunits for membranes. The differential translocation in which G-protein heterotrimers bind stably to the CELL BIOLOGY kinetics of the βγ-subunit types are determined by variations among βγ γ plasma membrane but free -dimers dynamically interact with a set of basic and hydrophobic residues in the -subunit types. G- both the plasma membrane and intracellular membranes. Insight protein signaling thus leverages the wide variation in membrane provided by this model motivated the hypothesis that direct in- dissociation rates among different γ-subunit types to differentially teraction of the γ-subunit with lipid bilayers controls differential βγ control βγ-translocation kinetics in response to receptor activation. translocation. Translocation kinetics of mutant γ-subunits, together The conservation of primary structures of γ-subunits across mamma- with prenylated peptide rates of dissociation from lipid vesicles in lian species suggests that there can be evolutionary selection for vitro, supported this hypothesis and showed that hydrophobic and primary structures that confer specific membrane-binding affinities basic residues in the γ-subunit C-terminal domain determine and consequent rates of intermembrane movement. βγ-membrane dissociation rates and translocation kinetics. protein–membrane interaction | spatio-temporal dynamics | Results G protein-coupled receptors Forward and Reverse βγ Translocations Have Similar γ-Subunit– Dependent Rates. In the classical model of the G-protein activa- α βγ ompared with the vast biochemical information available for tion cycle (3), inactive GDP heterotrimers reside at the plasma signaling proteins from cell-disruptive techniques, much less membrane. Receptor activation with an agonist triggers nucle- C α α is known about their spatiotemporal dynamics in the 3D space otide exchange on the -subunit and the GTP dissociates from of an intact living cell. Many important signaling proteins are βγ. Both α and βγ act on downstream effectors. GTP hydrolysis posttranslationally modified with lipid groups that facilitate on the α-subunit, accelerated by interaction with GTP hydrolysis their interactions with membranes. Live cell imaging has so far accelerating proteins (GAPs), deactivates the α-subunit and α βγ γ revealed important roles for constitutive cycles that maintain GDP reassembles with . This cycle suggests -subunit de- proper localization of lipidated proteins (1) and for dynamic pendence of three potential interactions to explain differential βγ i protein redistribution throughout the cell in response to a signal -translocation rates: ( ) the rate of heterotrimer dissociation, ii βγ (2), but the detailed nature of lipid-modified protein movement ( ) the rate at which dissociates from the activated receptor, iii βγ between membranes is generally not well understood. and ( ) the rate at which dissociates from the plasma Heterotrimeric G proteins consist of lipid-modified α- and membrane. To help distinguish between these possibilities, we βγ βγ-subunits (3). They are central transducers of the most im- investigated the reversible nature of translocation. βγ portant signaling pathways in mammals, yet little is known about Receptor activation with an agonist triggers translocation from the plasma membrane to intracellular membranes (4). their intracellular movement. Inactive heterotrimers consist of βγ a GDP-bound α-subunit in complex with a stable βγ-dimer and The intracellular distribution of -dimersismaintainedinthe localize to the plasma membrane (3). Extracellular signals acti- vate transmembrane G-protein–coupled receptors (GPCRs) that catalyze nucleotide exchange on the G-protein α-subunit (3). βγ α The -complex can then dissociate from the active -subunit Author contributions: P.R.O., W.K.A.K., J.R.S., and N.G. designed research; P.R.O., W.K.A.K., and translocate from the plasma membrane to intracellular and J.R.S. performed research; V.K. contributed new reagents/analytic tools; P.R.O., membranes (4–6). βγ translocation has been observed for many W.K.A.K., and J.R.S. analyzed data; and P.R.O., J.R.S., and N.G. wrote the paper. different cell types and receptor types, suggesting that it is The authors declare no conflict of interest. a common feature of G-protein signaling (4–8). This article is a PNAS Direct Submission. βγ Two factors make translocation an important model system 1To whom correspondence may be addressed. E-mail: [email protected] or john.silvius@ for studying intermembrane movement of signaling proteins. First, mcgill.ca. βγ the spatial redistribution of -subunits within a cell is rapidly and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. reversibly driven by extracellular signals, facilitating kinetic analysis 1073/pnas.1205345109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1205345109 PNAS Early Edition | 1of10 Downloaded by guest on September 29, 2021 continued presence of agonist, and βγ returns to the plasma more slowly (Fig. 1B). Similar results were obtained on activa- membrane on receptor deactivation with an antagonist (4, 6). The tion and deactivation of endogenous Gi/o-coupled CXCR4 reverse rates have not been studied in detail. We therefore receptors with SDF-1α (CXCL12, agonist) and AMD-3100 (an- measured forward and reverse βγ-translocation rates for three tagonist), respectively (Fig. S1 and Movies S1 and S2). These different γ-subunits. HeLa cells were transfected with untagged results suggested that both forward and reverse translocations γ – αo, β1, and a fluorescent protein (FP)-tagged γ-subunit (γ9, γ5, share the same -subunit dependent rate-limiting step and raised γ the possibility that γ-dependent rates of βγ-dissociation from lipid or 3) and imaged by spinning-disk confocal microscopy (Fig. 1). βγ We recently used fluorescence complementation to show that bilayers may explain the differential -translocation kinetics. FP-tagged γ-subunits can be used to accurately measure the Free βγ Dimers Diffuse Throughout the Cell, Exploring both the translocation kinetics of βγ-dimers, consistent with the well- β γ Plasma Membrane and Intracellular Membranes. Next we examined known stable association of - and -subunits under physiological βγ α the steady-state intermembrane dynamics of free -subunits to conditions (8). Endogenous Gi/o-coupled 2 adrenergic recep- determine whether they were consistent with the possibility that tors (α2ARs) were activated with 10 μM norepinephrine (ago- βγ βγ membrane dissociation rates determine -subunit translocation nist) to stimulate forward translocation and deactivated with rates. The ability of intracellular βγ to return rapidly to the plasma 60 μM yohimbine (antagonist) to stimulate reverse translocation. membrane on receptor deactivation suggests that free βγ, which is For each γ-subunit, the reverse translocation rate was similar to not bound to the α-subunit, continually explores the cytosolic the forward rate. Fast-translocating γ9 returned quickly on re- surfaces of both intracellular membranes and the plasma mem- ceptor deactivation, and slower-translocating γ5 and γ3 returned brane. Fluorescence recovery after photobleaching