The Gβγ Dimer Drives the Interaction of Heterotrimeric Gi Proteins with Nonlamellar Membrane Structures

Abstract

Heterotrimeric G proteins are peripheral membrane proteins that propagate signals from membrane receptors to regulatory proteins localized in distinct cellular compartments. To facilitate signal amplification, G proteins are in molar excess with respect to G protein-coupled receptors. Because G proteins are capable of translocating from membrane to cytosol, protein-lipid interactions play a crucial role in signal transduction. Here, we studied the binding of heterotrimeric G proteins (Gαβγ) to model membranes (liposomes) and that of the entities formed upon receptor-mediated activation (Gα and Gβγ). The model membranes used were composed of defined membrane lipids capable of organizing into either lamellar or nonlamellar (hexagonal HII) membrane structures. We demonstrated that although heterotrimeric Gi proteins and Gβγ dimers can bind to lipid bilayers of phosphatidylcholine, their binding to membranes was markedly and significantly enhanced by the presence of nonlamellar phases of phosphatidylethanolamine. Conversely, activated G protein α subunits showed an opposite membrane binding behavior with a marked preference for lamellar membranes. These results have important consequences in cell signaling. First, the binding characteristics of the Gβγ dimer account for the lipid binding behavior and the cellular localization of heterotrimeric G proteins. Second, the distinct protein-lipid interactions of heterotrimeric G proteins, Gβγ dimers, and Gα subunits with membrane lipids explain, in part, their different cellular mobilizations during signaling upon receptor activation. Finally, their differential interactions with lipids suggest an active role of the membrane lipid secondary structure in the propagation of signals through G protein-coupled receptors. Heterotrimeric G proteins are peripheral membrane proteins that propagate signals from membrane receptors to regulatory proteins localized in distinct cellular compartments. To facilitate signal amplification, G proteins are in molar excess with respect to G protein-coupled receptors. Because G proteins are capable of translocating from membrane to cytosol, protein-lipid interactions play a crucial role in signal transduction. Here, we studied the binding of heterotrimeric G proteins (Gαβγ) to model membranes (liposomes) and that of the entities formed upon receptor-mediated activation (Gα and Gβγ). The model membranes used were composed of defined membrane lipids capable of organizing into either lamellar or nonlamellar (hexagonal HII) membrane structures. We demonstrated that although heterotrimeric Gi proteins and Gβγ dimers can bind to lipid bilayers of phosphatidylcholine, their binding to membranes was markedly and significantly enhanced by the presence of nonlamellar phases of phosphatidylethanolamine. Conversely, activated G protein α subunits showed an opposite membrane binding behavior with a marked preference for lamellar membranes. These results have important consequences in cell signaling. First, the binding characteristics of the Gβγ dimer account for the lipid binding behavior and the cellular localization of heterotrimeric G proteins. Second, the distinct protein-lipid interactions of heterotrimeric G proteins, Gβγ dimers, and Gα subunits with membrane lipids explain, in part, their different cellular mobilizations during signaling upon receptor activation. Finally, their differential interactions with lipids suggest an active role of the membrane lipid secondary structure in the propagation of signals through G protein-coupled receptors. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; HII, hexagonal II; PE, phosphatidylethanolamine; PC, phosphatidylcholine.1The abbreviations used are: GPCR, G protein-coupled receptor; HII, hexagonal II; PE, phosphatidylethanolamine; PC, phosphatidylcholine. constitute the main class of membrane receptors and form the widest gene family known in the mammalian genome. Therefore, signal transduction via G proteins represents one of the most important ways of cell signaling (for a review, see Ref. 1Morris A.J. Malbon C.C. Physiol. Rev. 1999; 79: 1373-1430Crossref PubMed Scopus (395) Google Scholar). Upon activation by agonists, GPCRs undergo conformational changes that induce the activation of heterotrimeric G proteins, promoting the exchange of the GDP bound to the Gα subunit for GTP. This exchange provokes the dissociation of the Gα subunit from the Gβγ dimer and enables both molecular entities to modulate the activity of specific effectors or other signaling proteins (e.g. G protein receptor kinases). The association of both GPCRs and G proteins to the plasma membrane makes them susceptible to their lipid environment so that lipid-protein interactions are crucial to their function. In recent years, evidence has accumulated showing that the plasma membrane organization is more complex than a simple “chaotic sea” of bipolar lipid molecules in a liquid-crystalline state that only serves to support membrane proteins (2Vereb G. Szollosi J. Matko J. Nagy P. Farkas T. Vigh L. Matyus L. Waldmann T.A. Damjanovich S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8053-8058Crossref PubMed Scopus (417) Google Scholar). In fact, differentiated membrane domains with specific protein and lipid compositions can exist in a cell such as the basal and apical membranes of epithelial/endothelial cells, the pre- and postsynaptic membranes of neuronal synapses, or lipid rafts and caveolae. Moreover, the extracellular and cytosolic leaflets of the plasma membrane differ in their lipid composition (3Rothman J.E. Lenard J. Science. 1977; 195: 743-753Crossref PubMed Scopus (729) Google Scholar), further demonstrating the relevance of lipids in the organization and function of the membrane. Natural membranes are composed of different amphitropic molecules that differ in their propensity to form secondary lipid structures that influence the structural properties of the membrane (for a review, see Ref. 4Epand R.M. Biochim. Biophys. Acta. 1998; 1376: 353-368Crossref PubMed Scopus (324) Google Scholar). In this context, hexagonal (HII) structures regulate the localization and activity of some key membrane signaling proteins (5Giorgione J. Epand R.M. Buda C. Farkas T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9767-9770Crossref PubMed Scopus (72) Google Scholar, 6Escribá P.V. Ozaita A. Ribas C. Miralles A. Fodor E. Farkas T. Garcia-Sevilla J.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11375-11380Crossref PubMed Scopus (99) Google Scholar). Indeed, we have recently been able to show that changes in the lipid composition of erythrocyte cell membranes can influence the membrane association of G proteins and protein kinase C in vivo (7Escribá P.V. Sanchez-Dominguez J.M. Alemany R. Perona J.S. Ruiz-Gutierrez V. Hypertension. 2003; 41: 176-182Crossref PubMed Scopus (64) Google Scholar). Moreover, the neural membranes of cold-adapted fishes contain higher levels of phosphatidylethanolamine (PE) species in winter than in summer (8Buda C. Dey I. Balogh N. Horvath L.I. Maderspach K. Juhasz M. Yeo Y.K. Farkas T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8234-8238Crossref PubMed Scopus (94) Google Scholar). These lipid changes probably lower the solid-to-liquid and lamellar-to-hexagonal phase transition temperatures of membranes to maintain protein function and cell signaling in neurons and other cells. These data suggest that hexagonal phase propensity plays a major role in regulating physiological processes and might also participate in the control of other pivotal cellular events influenced by membrane proteins, such as cell growth or energy metabolism. Our main goal was to elucidate the role of the membrane lipid structure in the association to membranes of heterotrimeric G proteins and the molecular entities formed after their receptor-mediated activation. For this purpose we used synthetic membranes (liposomes) and purified G proteins as model systems because their lipid composition can be precisely controlled. We show that heterotrimeric Gi proteins (αβγ) and Gβγ dimers have protein-membrane lipid interactions distinct from those of Gαi monomers. These differences may explain their distinct cellular localizations after dissociation. We also show that the binding of the Gβγ dimer accounts for the binding characteristics and cellular localization of the complete heterotrimeric G protein. To our knowledge, this is the first work describing this new role of the G protein βγ dimer. 31P NMR—Multilamellar lipid vesicles (15%; w/w) were prepared mixing 56 mg of phospholipids with deuterated water (D2O). Either egg phosphatidylcholine (PC), bovine brain PE, or mixtures of both were prepared, keeping constant the lipid phosphorus content. The lipid suspensions were hydrated and homogenized with a pestle-type mini-homogenizer (Sigma) and submitted to vortex shaking until homogeneity. The suspensions were then submitted to 10 heating (60 °C) and freezing (–80 °C) cycles and equilibrated prior to data acquisition, as reported previously (9Funari S.S. Barceló F. Escribá P.V. J. Lipid Res. 2003; 44: 567-575Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). 31P NMR measurements were made in 5-mm tubes on an Advance-300 multinuclear NMR spectrometer (Bruker Instruments). Samples were equilibrated at the assay temperatures (4 or 25 °C) for 15 min before data was acquired. 31P NMR free induction decays were accumulated for 64 transients by employing a 4.4-μs, 90° radio-frequency pulse, a 24.3-kHz sweep width, and 65,000 data points. The delay between transients was 2 s, and the spectra were obtained by scanning from lower to higher temperatures. Liposome Preparation and G Protein Binding—Liposomes containing PC were prepared, with or without PE, by dissolving the lipids in chloroform and mixing the appropriate volumes in glass vials. The solvent was removed under argon, and the residue was dried for at least 3 h under vacuum. Subsequently, the lipid films were hydrated in 20 mm Tris-HCl at pH 7.4 containing 100 mm KCl and 0.1 mm EDTA at 42 °C for 1 h and subjected to vigorous vortex shaking every 15 min. Following five freeze/thaw cycles, this lipid emulsion was passed 11 times through a 400-nm polycarbonate membrane using a mini-extruder (Avanti Polar Lipids). The liposome emulsions were then diluted to a final concentration of 600 μm in lipid phosphorus, as determined by the method of Chen et al. (10Chen Jr., P.S. Toribara T.Y. Warner H. Anal. Chem. 1956; 28: 1756-1758Crossref Scopus (5834) Google Scholar). Subsequently, the liposomes were incubated with either 500 ng of purified heterotrimeric G proteins, 100 ng of the Gβγ dimer, or 150 ng of the Gαi1 subunit in a total volume of 300 μl for 30 min at 25 °C. Incubation of heterotrimeric G proteins or the Gαi1 subunit was carried out in the presence of 50 μm GDPβS or GTPγS, respectively. The unbound G proteins were then separated from the membranes by centrifugation at 62,000 × g for 45 min at 25 °C. Finally, the membrane pellets were resuspended in electrophoresis loading buffer (84 mm Tris-HCl (pH 6.8), 4% SDS, 1% 2-mercaptoethanol, 5% glycerol, and 0.01% bromphenol blue) and boiled for 5 min. Immunoblot Analysis and Quantification of Specific Immunoreactivity—Immunoblotting was performed as described elsewhere (11Escribá P.V. Sastre M. Garcia-Sevilla J.A. Arch. Gen. Psychiatry. 1994; 51: 494-501Crossref PubMed Scopus (82) Google Scholar). Briefly, proteins were submitted to electrophoresis on SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. These membranes were blocked with phosphate-buffered saline containing 5% nonfat dry milk, 0,5% bovine serum albumin, and 0.02% Tween 20. The membranes were then incubated with the specific primary antibodies polyclonal anti-Gαi1/2 (dilution 1:5000) and anti-Gβ1 (1:1000) in fresh blocking solution. The primary antibodies were detected with horseradish peroxidase-linked secondary antibodies and visualized with the ECL Western blot detection system and ECL Hyperfilm (Amersham Biosciences). Quantification was performed by image analysis of the immunoreactive bands on the film. Values from liposomes composed of PC:PE were normalized to the values of pure PC liposomes, which were considered as 100%. Materials—Egg PC and liver PE were both purchased from Avanti Polar Lipids. Recombinant, purified, myristoylated Gαi1 protein subunit, purified heterotrimeric Gi protein, and purified Gβγ protein dimers were from Calbiochem. The polyclonal anti-Gαi1/2 antiserum was from PerkinElmer Life Sciences and the monoclonal antibody anti-Gβ1 protein was obtained from BD Biosciences. All other reagents were supplied by Sigma. Data Analysis—The data shown are the mean values ± S.E. from the number of experiments indicated (n). Values were compared by one-way analysis of variance followed by a Bonferroni comparison test against the control values. Differences between experimental groups were considered statistically significant at p < 0.05. Temperature-dependent Formation of Nonlamellar Phases in Model Membranes—For G protein-membrane binding experiments, model membranes of PC or PE or a combination of both (see “Experimental Procedures”) at different proportions were used. At 25 °C, egg PC membranes were organized forming lipid bilayers (lamellar structures), whereas bovine brain PE was mainly organized into hexagonal (HII phase) structures (Fig 1A). Membranes formed with different proportions of both lipids behaved as non-ideal mixtures, as has been described for many binary systems with two membrane lipids or more (for example, see Ref. 12Escribá P.V. Morales P. Smith A. DNA Cell Biol. 2002; 21: 355-364Crossref PubMed Scopus (18) Google Scholar) where membrane domains enriched in one lipid species are formed. In this context, mixtures containing a higher content of PC (PC:PE at 10:0, 8:2, and 6:4; mol/mol) exhibited the peaks corresponding to the lamellar organization of each phospholipid (L1 and L2) (Fig. 1A). In addition, the shoulder at positive chemical shift values indicated the presence of a small proportion of nonlamellar phases in PE-containing samples. At greater proportions of PE (PC:PE at 4:6, 2:8 and 0:10; mol/mol), the occurrence of nonlamellar structures (NL1 and defined by the peaks at and at 25 °C with a of the peaks corresponding to the lamellar phases (Fig. 1A). In at °C were only two peaks corresponding to the lamellar phases of PC and PE, and (Fig Therefore, at °C PC, PE, and their mixtures are organized into lamellar phases into nonlamellar of the Gi to Model have studied the binding of heterotrimeric Gαi proteins to model membranes (liposomes) the state was by the of GDPβS We that the lipid composition of the liposome influenced the binding of Gi proteins. the PE content of the membrane protein binding to membranes. The PE used in model membranes in different and membranes (PC:PE at 10:0, 8:2, 4:6, and 0:10; In mammalian the of PE is although the of the plasma membrane in some the PE content can to of the total membrane The presence of the hexagonal phospholipid PE, which into phases under the experimental an in the binding of Gi to liposomes in a (Fig. containing a of μm or PC:PE μm mammalian plasma membranes or their bound ± and ± of Gi protein respectively. This was between and greater binding than that of vesicles composed of only the lamellar phospholipid PC of PE may membrane domains or greater binding of heterotrimeric Gi proteins, ± at a PC:PE molar of μm and ± for pure PE liposomes μm the other at °C only lamellar and hexagonal phases were formed in our model the binding of Gi to liposomes was the for of PC:PE used 4:6, and at ± ± ± and ± with respect to PC:PE as p in This that the in binding of the Gi is by binding preference to hexagonal membrane structures and by preference for PE of Gαi1 to Model to the with heterotrimeric G proteins, we also studied the binding of G protein subunits to liposomes to the interactions that might upon G protein activation and dissociation. The purified Gαi1 protein used was myristoylated and able to bind to membrane bilayers in a Jr., Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the presence of 50 μm that the protein in the that to activated to the heterotrimeric G proteins, the presence of the nonlamellar lipid PE (HII the binding of Gαi1 subunits to the liposomes (Fig. In fact, an in the membrane content of PE the binding of Gαi1 to membranes. vesicles containing or PC:PE bound only ± or ± the of Gi bound by pure PC membranes (Fig. proportions of PE further Gαi1 protein binding to ± for a of and ± of control values for pure PE liposomes (Fig. At °C, where only lamellar and hexagonal phases are formed in our model binding of the to liposomes with the of PC:PE 4:6, and 0:10; ± ± ± and ± with respect to PC:PE at as showing that the in binding of the Gαi1 subunit was by binding preference for lamellar membrane structures and by preference for PC These results that the Gαi1 with lamellar phases membranes and lipid than with hexagonal (HII phase) structures membrane structures with of Gβγ to Model we the protein-lipid interactions of the Gβγ dimer, which lipid binding characteristics more to those of heterotrimeric Gi proteins than to those of Gαi1 the binding of Gβγ subunits was significantly and markedly as the PE content in the model membrane (Fig. Gβγ subunit binding to model membranes with molar of PC:PE at and 2:8 to ± and ± that of pure PC lipid respectively. These results show that the Gβγ dimer has a marked preference for hexagonal (HII phase) structures. signaling as signal In signals from the first at the membrane where each receptor can G proteins upon than on the cell GPCRs and many other membrane receptors are or in defined membrane R. V. F. J. C. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). Therefore, a number of heterotrimeric G protein molecules be in domains with membrane the protein-lipid interactions between Gα subunits and lamellar membranes have been studied S. L. PubMed Scopus Google Scholar), been on the interactions with nonlamellar lipid Here, we have the different interactions of and proteins with lamellar and nonlamellar (HII) lipid a we have demonstrated that the presence of nonlamellar phases formed by PE the binding of heterotrimeric Gi proteins and Gβγ dimers to whereas Gαi subunits lamellar membrane structures formed by PC Cell membranes are in PE, which is mainly on the of the plasma membrane where of G proteins are bound J. of and Scholar). Indeed, our results show that the greater the hexagonal phase the higher the of heterotrimeric Gi proteins for membranes. domains or with in either a or a as membrane for peripheral proteins. This is in with the of G proteins from brain plasma membranes after the of nonlamellar structures by the P.V. Sastre M. Garcia-Sevilla J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). Nonlamellar lipids modulate the properties of lipid membrane such as changes in the the and the of membranes G. A. PubMed Scopus Google Scholar). The of in membranes under physiological the presence of structures or with in which might be or by membrane proteins Science. PubMed Scopus Google Scholar). In this context, is that with such as the of the of hexagonal phases under T. S. J.A. PubMed Scopus Google Scholar). Moreover, has been shown that facilitate the conformational of the to activated state in model membranes 2002; 41: PubMed Scopus Google Scholar). enriched in hexagonal membrane lipids PE a for the of GPCRs and heterotrimeric G proteins, a role for hexagonal lipids in signal membrane of the signaling and in activation (see In to G protein the activated Gαi1 subunit a marked preference for lamellar structures. This the of activated Gαi1 from the receptor environment where the hexagonal phase propensity is by the domains of GPCRs and the of the subunit This of the Gα protein subunit from the receptor environment may facilitate with proteins (e.g. that might be in other membrane Indeed, Gαi and have been in lipid rafts P. J.E. Biol. PubMed Scopus Google Scholar), domains enriched in and with a lamellar state and Our that Gαi1 subunits have a greater for with lamellar such as those formed in lipid the lipid of G proteins be to specific interactions with phospholipid in a lipid Therefore, we also studied the binding of proteins to the membrane systems at °C. at 25 °C PE nonlamellar at °C is only organized into lipid bilayers both or in combination with At this Gα a preference for PE or PC, respectively. This binding behavior is markedly distinct from that at 25 °C and that G protein binding to membranes is by the membrane structure and by interactions with specific phospholipids (e.g. PC or In keeping with this the G protein that to the also to lipid rafts activated N. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). that the opposite binding of activated Gα subunits them from the receptor environment to other membrane where may with their The that a of proteins of the signal transduction are in lipid rafts this C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Cell Biol. 1995; PubMed Scopus Google Scholar). lipid rafts as to the of the of the signaling by promoting the association between Gα subunits and their The of Gβγ subunits for hexagonal membranes also their in membranes. The results are in with the of Gβγ subunits from synthetic lipid rafts reported by et al. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Our data suggest that the Gβγ dimer is for the lipid-protein properties of heterotrimeric G proteins. In this the Gβγ dimer the hexagonal phase preference of complete the lamellar membrane of the Gα Therefore, one of the of the Gβγ dimer be to the Gα subunit to the of the GPCR, to the to of proteins a localization by proteins containing such a J. J. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, J. F. N. A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). The hexagonal (HII phase) preference of the Gβγ dimer also be important for which after activation. Gβγ dimers can bind G protein-coupled receptor which and The of the Gβγ dimer for nonlamellar membrane membrane receptors are to their the of the signaling by the binding of the to the The of membrane lipids other than PC in the of and this has been demonstrated with the of PC, membrane lipids can regulate G protein-coupled receptor kinase activity J. E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). In the results new signal GPCRs and G protein subunits facilitate the of hexagonal phase membrane domains (Fig. nonlamellar membrane domains be enriched in proteins because of the higher of for membranes with by the Gβγ dimer. Therefore, a of G proteins be membrane receptors. Upon each receptor can G proteins, and the activated Gα subunit from the Gβγ Because of for nonlamellar membrane activated Gα protein from the receptor to membrane lamellar such as lipid In the of proteins facilitate their with activated Gα subunits the propagation of the Because the Gβγ dimer a higher for nonlamellar lipid may regulate signaling effectors or G protein-coupled receptor to with the membrane receptors. The is with receptor which In our results the relevance of structural for signal only in of proteins and also with respect to lipid organization and lipid-protein