Interaction of the C-terminal region of the Ggamma protein with model membranes

Abstract

Heterotrimeric G-proteins interact with membranes. They accumulate around membrane receptors and propagate messages to effectors localized in different cellular compartments. G-protein-lipid interactions regulate G-protein cellular localization and activity. Although we recently found that the Gbetagamma dimer drives the interaction of G-proteins with nonlamellar-prone membranes, little is known about the molecular basis of this interaction. Here, we investigated the interaction of the C-terminus of the Ggamma(2) protein (P(gamma)-FN) with model membranes and those of its peptide (P(gamma)) and farnesyl (FN) moieties alone. X-ray diffraction and differential scanning calorimetry demonstrated that P(gamma)-FN, segregated into P(gamma)-FN-poor and -rich domains in phosphatidylethanolamine (PE) and phosphatidylserine (PS) membranes. In PE membranes, FN increased the nonlamellar phase propensity. Fourier transform infrared spectroscopy experiments showed that P(gamma) and P(gamma)-FN interact with the polar and interfacial regions of PE and PS bilayers. The binding of P(gamma)-FN to model membranes is due to the FN group and positively charged amino acids near this lipid. On the other hand, membrane lipids partially altered P(gamma)-FN structure, in turn increasing the fluidity of PS membranes. These data highlight the relevance of the interaction of the C-terminal region of the Ggamma protein with the cell membrane and its effect on membrane structure.

FIGURE 1

FTIR amide I′ band region spectra of P_γ-FN and P_γ peptides. (A and D) Pure peptides and (B, C, E, and F) pellets of DEPE- or DMPA-peptide mixtures centrifugated and resuspended in Hepes buffer prepared with D₂O. The lipid/peptide molar ratio was 10:1 in all cases. The buffer spectrum was subtracted from those of the samples containing the peptides. The traces shown in each plot correspond to the spectra acquired at 20°C (——) and 55°C (- - - -).

FIGURE 2

Linear plots of the x-ray scattering patterns of DEPE/P_γ-FN and DMPS/P_γ-FN mixtures. (A and B) DEPE/P_γ-FN (20:1, mol/mol) and (C) DMPS/P_γ-FN (10:1, mol/mol) samples. The sequence of the patterns were acquired under (A) kinetic conditions with a scan rate of 1°C/min and (B and C) quasiequilibrium conditions, after equilibrating the sample during 15 min at each temperature. Successive diffraction patterns were collected for 15 s each minute. The L_β-to-L_α phase transition was identified by the disappearance of the peak in the WAXS region. (D) The dependence of lattice spacing on the temperature for DEPE alone and in the presence of P_γ-FN or P_γ at a 10:1 (DEPE/peptide) molar ratio. The phases represented are L_β, L_α, and H_II. The coexistence of L_α and H_II phases corresponds to the temperature range defined by the vertical lines. Only the heating sequence from 27°C to 75°C is shown here. (E) Diffraction pattern of DEPE/P_γ-FN sample at 63°C. The arrows indicate the diffractions' peaks corresponding to the two hexagonal phases with an epitaxial relationship.

FIGURE 3

DSC thermograms of DEPE- or DMPS-peptide mixtures. (A), (B) DEPE, and (C) DMPS alone and in the presence of P_γ-FN, P_γ, or FN. The molar ratio of the mixtures is indicated by each thermogram. DSC scans were performed at a scan rate of 1°C/min. (D) Deconvolution analysis of the calorimetric peak: experimental data (––––) and individual components (- - -) of DMPS/P_γ-FN mixtures at the molar ratio specified.

FIGURE 4

FTIR spectra of the phosphate stretching mode region of DMPA-peptide mixtures. DMPA alone (–––) and in the presence of (A) P_γ-FN or (B) P_γ peptides. The lipid/peptide molar ratios were 10:1 (– – –) and 5:1 (- - -). The samples were prepared in aqueous Hepes buffer and measured at 25°C and 60°C.

FIGURE 5

FTIR spectra of the C=O stretching mode region of DEPE and DMPS mixtures. (A) DEPE or (B) DMPS alone and in the presence of P_γ-FN, P_γ, or FN. The lipid/peptide or lipid/FN molar ratio was 10:1 in all cases. (C and D) Temperature profiles derived from the intensity band ratio of the deconvoluted CO stretching modes of DEPE or DMPS alone (▪) and in the presence of P_γ-FN (▴), P_γ (▵), or FN (◊).

FIGURE 6

Temperature profiles of the CH₂ stretching band position. (A) DEPE or (B) DMPS alone (▪) and in the presence of P_γ-FN (▴), P_γ (▵), or FN (◊). The lipid/peptide or lipid/FN molar ratio was 10:1 in all cases. Similar results could also be obtained by plotting the data as the bandwidth at half-height instead of at the band position. (C) Illustration of temperature dependence for the C-H stretching region of the spectra of pure DEPE, 1:10 DEPE + FN, 1:10 DEPE + Pγ, 1:10 DEPE + Pγ-FN at 20°C and 55°C.

FIGURE 7

Fluorescence polarization of TMA-DPH-labeled DEPE and DMPS liposomes. Fluorescence polarization was measured in DEPE or DMPS membranes in the absence (▪) or presence of P_γ-FN (▴), P_γ (▵), or FN (◊). The lipid/peptide or lipid/FN molar ratio was 10:1 in all cases.

FIGURE 8

Schematic model for G-protein-membrane interactions. (A) Based on model membranes containing only one type of phospholipids molecule, this cartoon proposes that the P_γ-FN peptide binds to membranes through electrostatic interactions between its peptide moiety (light gray) and PS (light gray) and by hydrophobic interactions between the FN group (triangle in dark color) and PE (black). Phosphatidylcoline (PC) is shown in dark gray. (B) This panel shows the possible interaction of whole G-proteins with membranes, highlighting the role of the Gγ₂ subunit. A membrane region with membrane receptors (R) and heterotrimeric G-proteins (Gαβγ) is shown. Both the transmembrane region of GPCRs and the FN moieties of G-proteins contribute to the nonlamellar phase propensity (31), which in turn favors the binding of G-proteins (9) and explains the cooperative interactions between these transducers and the regions with high PE content.