Technical Reference #1642

1642.

A Self-Scaffolding Model for G Protein Signaling Jingting Wang; Urszula Golebiewska; and Suzanne Scarlata, Stony Brook University, Journal of Molecular Biology, 387(1642), (2009)
Link To Paper

Abstract:
Activation of heterotrimericG proteins is generally believed to induce dissociation of Gα and Gβγ subunits which are then free to bind to and change thecatalytic activity of a variety of intracellular enzymes. We have previouslyfound that in cells G

Keywords:
G protein; cell signaling; fluorescence spectroscopy; protein complexes; G protein effectors

Materials & Methods:
Expression and purification of Gαi mutants DNAs of mutant Gαi proteins were provided by Prof. Heidi Hamm (Department of Pharmacology Vanderbilt University). In these proteins all six of the Cys were mutated and single-Cys residues were introduced as indicated. These proteins behaved identically to their wild-type counterparts (see Refs. 3346 for full description of their properties as well as for their expression and purification). Escherichia coli cells were grown to an OD600 (optical density at 600 nm) of 0.5 U and then induced with 30 μM IPTG at room temperature for 16–20 h with gentle shaking at 200 rpm. The pellet was resuspended in buffer containing 50 mM NaH2PO4 pH8.0 300 mM NaCl 5 mM imidazole and fresh protease inhibitors and then disrupted by sonication. The cytosolic fractions were collected after centrifugation and incubated with 5 ml Ni-NTA agarose resin for 60 min at 4 °C and loaded to a column. The column was washed twice by wash buffer containing 5 and 10mMimidazole respectively and then eluted with 10 ml elution buffer containing 40 mM imidazole. An additional pass through an anion-exchange column was sometimes used. Eluted protein was dialyzed with exchange buffer [50 mM Tris pH8.0 50 mM NaCl 1 mM MgCl2 20% glycerol 20 μMGDP 10mMβ-mercaptoethanol (βME) and 100 μM PMSF] and then loaded to a MonoA column (Pharmacia Amersham Piscataway NJ). The protein was eluted by a linear gradient buffer containing 0–200mMNaCl. The identity and purity of pooled proteins were tested by SDS-PAGE. Glycerol was added to purified protein to 10% (v/v) and the protein was aliquoted flash-frozen with liquid nitrogen and stored at −80 °C. Expression of Gβ1γ2 Expression of His6-Gβ1γ2 through baculovirus infection of SF9 cells has been described previously.37 This method allows for post-synthetic modifications. The geranylgeranyl chain on the Gγ2 subunit was assessed by thin-layer chromatography on LK5D linear-k silica gel thin-layer chromatography plates. Expression of PLCβ2 His6-PLCβ2 was expressed in Sf9 cells using a baculovirus vector provided by Alan Smrcka (University of Rochester Medical School). Sf9 cells were grown to 1×106 cells/ml at 27 °C. Cells were infected with His6-PLCβ2 recombinant baculovirus at a multiplicity of 10 for 48 h at 27 °C and shaken at 125 rpm. Cells were harvested at 2500 rpm for 25 min washed in phosphate-buffered saline resuspended and spun at 4 °C. The pellet was suspended in ice-cold lysis buffer (20 mM Tris–HCl pH8.0 300 mM NaCl 10 mM βME and fresh protease inhibitor cocktail) and subjected to prechilled nitrogen cavitation (Parr cell disruption bomb N500 psi for 30 min with intermittent agitation). Cells were exploded into prechilled flask containing 40 ml lysis buffer and the soluble fraction was recovered after ultracentrifugation at 35000 rpm for 1 h at 4 °C. The supernatant was loaded to an equilibrated Ni-NTA column. Bound proteins were eluted with 50 ml elution buffer (20 mM Tris–HCl pH8.0 500 mM NaCl and 10 mM βME) with a gradient of 10–150 mM imidazole. PLC activity assays Activity assays were conducted as described previously. 37 Briefly samples containing substrate {POPC: POPE:POPS (1:1:1) with 2 mol.% PtdIns(45)P2 and 8000 cpm/sample [3H]-PtdIns(45)P2} PLCβ2 and other proteins were incubated at 30 °C for 5 min and the reaction was then initiated by the addition of calcium ions. The reaction was terminated by addition of 200 μl ice-cold 10% trichloroacetic acid and 100 μl 1% bovine serum albumin. After centrifugation for 5 min 300 μl of the supernatant was subjected to scintillation counting. Activity measurements of the PLCβ2–Gβγ–Gα complexes were carried out at 20 nM enzyme and 200 nM G protein concentrations. These high concentrations were required to insure that the ternary complexes would form. However even at 30-s measurements we were close to the edge of the linear range. This resulted in lower values of Gβγ activation and higher intrinsic error in the measurements. Gα activation Gαi was activated by incubation at 30 °C for 30 min with the activation buffer [50 mM Hepes 100 mM (NH4)2SO4 150 mM MgSO4 100 mM ethylenediaminetetraacetic acid (EDTA) and 100 μM GTPγS].3347 Protein labeling The labeling procedure for these proteins has been previously described48 and yields fully functional proteins labeled between 0.3 and 1.2 probe:protein as determined by absorption measurements. Briefly PLCβ2 or Gαi were dialyzed against 150 mM NaCl and 20 mM Hepes buffer for 30 min three times to remove DTT and then labeled with Alexa546 (PLCβ2) or with CPM (Gαi) which labels cysteine side chains. Gβγ was labeled at pH8.0 with Dabcyl succinimidyl ester which reacts with primary amines. Labeling was also verified by the diffusion coefficient using fluorescence correlation spectroscopy. Unreacted probe was removed by dialysis three times for 30 min against a 100-fold excess of buffer containing DTT or by gel chromatography. Fluorescence measurements Fluorescence experiments were carried out on an ISS PC1 spectrofluorometer (ISS Urbana IL); 10 nM CPMlabeled Gαi was reconstituted on 200 μMLUVs composed of POPC:POPS:POPE (1:1:1) and the solution was placed in a 3-mm microcuvette. Spectra were recorded using a 384-nm excitation wavelength and by scanning the emission from 420 to 520 nm. The area under the curves was calculated to give the total emission intensity. In vitro FRET studies FRET measurements were carried out by reconstituting 10 nM CPM-Gαi onto 200 μM POPC:POPS:POPE LUVs 10 nM Dabcyl-Gβγ subunits or 80 nM for activated CPMGαi is added as compared to the changes seen using identical amounts of unlabeled Gβγ subunits. Since we are viewing associations on proteins confined to membrane surfaces there is the possibility that nonspecific association may occur. To determine the importance of nonspecific encounters we note that at 200 μM lipid the proteins diffuse on a surface area of ∼1017 Å2. We assume that the proteins encounter each other diffusing at a length of 50 Å from the membrane surface into the solvent. We also consider that the G protein subunits are at a concentration of 20 nM each and are ∼2.5×105 Å3. If FRET occurs when the proteins are within a radius of 2500 Å2 of each other then the amount of membrane far exceeds the probability of diffusional encounters (almost 60-fold). This point has been previously discussed along with control studies using noninteracting proteins.374950 Accessibility studies Cys accessibility was determined by the increase in fluorescence intensity of CPM which is only fluorescent upon reacting with free thiol groups. To isolate Gαi Cys reactivity we first blocked Gβγ Cys side chains by removing DTT and incubating with iodoactamide at a 1:5 ratio at room temperature for 1 h. Excess iodoactamide was removed by dialysis for 30 min three times. To test the accessibility of CPM to Cys on Gαi mutants we incubated 10 nM unlabeled Gαi together with 200 μM LUV blocked Gβγ and 50 nM CPM in cuvettes. Fluorescence intensity was tested at 0 30 and 60 min respectively. The 384-nm excitation wavelength was used and the emission from 420 to 520 nm was recorded. NBS modification of Trp was performed by measuring the Trp intensity at λexc=280 nm and λem=345 nm after the addition of 20 μM freshly prepared NBS to a cuvette containing 10 nM Gαi and 200 μM lipid when a constant value was reached after 8 min.51 In each case the fluorescence intensity of the same amount of proteins without NBS and the same amount of Gαi with NBS was used as controls. Cell culture and transfection HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum 50 U/ml of penicillin and 50 μg/ml streptomycin sulfate at 37 °C in a 5% CO2 incubator. The cells were transfected using calcium phosphate coprecipitation in which cells were grown on 60-mm dishes for 24–48 h to achieve 80–90% confluence the media was then replaced and 5 μg of eYFP-Gαi and eCFP-Gβ1 and 10 μg of HAGγ7 plasmids were mixed with 120 mM CaCl2 and Hepesbuffered saline buffer (21mMHepes 123mMNaCl 5mM KCl and 0.9 mM Na2HPO4 pH7.1) incubated on ice for 10 min and added to cells dropwise. The cells were then incubated at 37 °C and the media were replaced after 8– 14 h. The cells were allowed to recover for 8–14 h and split into 35-mm glass-bottom MatTek dishes and imaged 48– 72 h later. For PLCβ2–G protein FRET measurements cells were transfected with labeled G proteins 48 h prior to microinjections with Alexa546-labeled PLCβ2. The integrity of the expressed G protein subunits was assessed by preparing membrane fractions from the transfected cells and measuring their ability to support activation of PLCβ2. We find that cells transfected with eYFP-Gαi and eCFP-Gβ1 activate PLCβ2 at an ∼50% higher level than membrane prepared from cells transfected with empty vector. Microinjections Transfected cells were grown in MatTek dishes for 48 h to achieve 70–80% confluence. Prior to microinjecting we changed the media to phenol-free Leibovitz's-15. We used an InjectMan NI2 with FemtoJet pump from Eppendorf to microinject the solutions into the cytoplasm. We typically set the injection pressure (Pi) at 27–25 hPa and kept the compensation pressure (Pc) at 14 hPa. The injection time (t) was 0.4 s. Typically we injected about 10–25 cells within a 10- to 20-min period. We examined the microinjected cells under the phase microscope (Axiovert 200M from Zeiss with 40× phase 2 objective) to select viable cells. We then transferred the cells to the Zeiss LSM 510 META/ConfoCor 2 apparatus (Jena Germany) and collected images. In vivo single-cell FRET measurements In vivo FRET experiments were performed on Zeiss LSM 510 META/ConfoCor 2 apparatus (Jena) by monitoring the sensitized emission. This system uses laser excitation allowing little bleed-through from the CFP channel. The optical settings were as follows: λexc=458 nm and λem=475–525 nm for eCFP λexc=514 nm and λem=560–615 nm for eYFP and λexc=458 nm and λem=560–615 nm for FRET. Details of the experimental setup procedure and analysis have been described previously. 29 For GFP-Alexa546 FRET we used λexc=488 nm and λexc=585–615 nm. FRET measurements entailed taking an image through a donor acceptor and FRET filter at identical instrument settings (detector gain and laser intensity). We expressed fluorescent constructs CFP and YFP (for the CFP/YFP FRET) and expressed GFP only and injected Alexa546 to nontransfected cells (for the GFP-Alexa546 FRET) to determine the amount of bleedthrough between channels. Data were analyzed using Zeiss software that takes into account bleed-through and detector sensitivity.

Microscopic Technique
Phase Microscopy

Cell Type(s)
HEK-293