Biomedical subjects

A E Remmers

Publications and source records attributed to A E Remmers.

At least 19 recordsLinked to original sources

PubMed2006

Effects of three cytochrome P450 inhibitors, ketoconazole, fluconazole, and paroxetine, on the pharmacokinetics of lasofoxifene.

AIMS: Two studies were conduced to assess the effects of ketoconazole, a CYP3A4/5 inhibitor; fluconazole, a CYP2C9 inhibitor; and paroxetine, a CYP2D6 inhibitor, on lasofoxifene pharmacokinetics. METHODS: The first parallel group study was conducted in 45 healthy postmenopausal women (15 per group) to compare the pharmacokinetics of a single dose of lasofoxifene (0.25 mg) administered alone and in combination with ketoconazole (400 mg daily x 20 days) or fluconazole (400 mg daily x 20 days). Lasofoxifene was administered on day 2 and blood samples were collected serially for up to 456 h postdose (20 days). The second study enrolled 20 healthy postmenopausal women (10 per group) to compare the pharmacokinetics of a single dose of lasofoxifene (0.25 mg) alone and in combination with paroxetine (30 mg qd x 21 days). Lasofoxifene was given on day 8 of paroxetine treatment and blood samples were collected serially for up to 336 h postdose. RESULTS: All subjects completed the study and the treatments were well tolerated. Lasofoxifene C(max) and AUC ratios [90% confidence interval (CI)] with/without ketoconazole were 111% (98.4, 127) and 120% (105, 136), respectively, and were 91.3% (80.3, 104) and 104% (91.4, 118), respectively, with/without fluconazole. Lasofoxifene C(max) and AUC ratios (90% CI) with/without paroxetine were 118% (95.4, 146) and 135% (120, 152), respectively. CONCLUSIONS: Coadministration of potent inhibitors of CYP3A4/5 and CYP2D6, but not CYP2C9, resulted in a moderate increase in lasofoxifene exposure. No dosage adjustment should be required when lasofoxifene is coadministered with ketoconazole, fluconazole, paroxetine or other agents that inhibit these CYP enzymes.

Adult↗

PubMed2000

Activation of G protein by opioid receptors: role of receptor number and G-protein concentration.

The collision-coupling model for receptor-G-protein interaction predicts that the rate of G-protein activation is dependent on receptor density, but not G-protein levels. C6 cells expressing mu- or delta-opioid receptors, or SH-SY5Y cells, were treated with beta-funaltrexamine (mu) or naltrindole-5'-isothiocyanate (delta) to decrease receptor number. The time course of full or partial agonist-stimulated ¿35SGTPgammaS binding did not vary in C6 cell membranes containing <1-25 pmol/mg mu-opioid receptor, or 1. 4-4.3 pmol/mg delta-opioid receptor, or in SHSY5Y cells containing 0. 16-0.39 pmol/mg receptor. The association of ¿35SGTPgammaS binding was faster in membranes from C6mu cells than from C6delta cells. A 10-fold reduction in functional G-protein, following pertussis toxin treatment, lowered the maximal level of ¿35SGTPgammaS binding but not the association rate. These data indicate a compartmentalization of opioid receptors and G protein at the cell membrane.

Enkephalin, Ala(2)-MePhe(4)-Gly(5)-↗

PubMed1999

Interdomain interactions regulate GDP release from heterotrimeric G proteins.

The role of interdomain contact sites in basal GDP release from heterotrimeric G proteins is unknown. G(alpha)(o) and G(alpha)(i1) display a 5-fold difference in the rate of GDP dissociation with half-times of 2.3 +/- 0.2 and 10.4 +/- 1.3 min, respectively. To identify molecular determinants of the GDP release rate, we evaluated the rate of binding of the fluorescent guanine nucleotide 2'(3')-O-(N-methyl-3'-anthraniloyl)guanosine 5'-O-(3-thiotriphosphate) (mGTPgammaS) to chimers of G(alpha)(o) and G(alpha)(i1). Although no one region of the G protein determined the GDP dissociation rate, when the C-terminal 123 amino acids in G(alpha)(i1) were replaced with those of G(alpha)(o), the GDP release rate increased 3.3-fold compared to that of wild-type G(alpha)(i1). Within the C-terminal portion, modification of four amino acids in a coil between beta4 and the alpha3 helix resulted in GDP release kinetics identical to those of wild-type G(alpha)(o). Based on the G(alpha)(i1)-GDP crystal structure of this region, Leu(232) appeared to form a hydrophobic contact with Arg(144) of the helical domain. The role of this interaction was confirmed by G(alpha)(i1) L232Q and G(alpha)(i1) R144A which displayed 2-5-fold faster GDP release rates compared to wild-type G(alpha)(i1) (t(1/2) 4.7 and 1.5 min, respectively), suggesting that interdomain bridging contacts partially determine the basal rate of GDP release from heterotrimeric G proteins.

Amino Acid Sequence↗

PubMed1999

Membrane microviscosity modulates mu-opioid receptor conformational transitions and agonist efficacy.

The influence of membrane microviscosity on mu-opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH-SY5Y cells and from a C6 glioma cell line stably expressing the rat mu-opioid receptor (C6mu). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) for the mu-opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS. The effect of CHS on agonist binding was reversed by subsequent addition of cis-vaccenic acid, suggesting that the effect of CHS is the result of increased membrane microviscosity and not a specific sterol-receptor interaction. CHS addition increased the potency of DAMGO to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate binding by fourfold, whereas the potency of nalbuphine was unaffected. However, nalbuphine efficacy relative to that of the full agonist DAMGO was strongly increased in CHS-treated membranes compared with that in control membranes. Membrane rigidification also resulted in an increased efficacy for the partial agonists meperidine, profadol, and butorphanol relative to that of DAMGO as measured by agonist-stimulated GTPase activity in control and CHS-modified membranes. These findings support a regulatory role for membrane microviscosity in receptor-mediated G protein activation.

Animals↗

PubMed1999

Opioid efficacy in a C6 glioma cell line stably expressing the human kappa opioid receptor.

A C6 glioma cell line stably transfected with the human kappa opioid receptor (kappaOR) was used to characterize receptor binding and G protein activation via the kappaOR by a comprehensive series of opioid ligands. The ligand-binding affinity for [3H]5alpha,7alpha, 8beta(-)-N-methyl-N-(7-Cl-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl)benzene acetamide (U69593) was similar to that observed in monkey brain membranes and was 10-fold lower in the presence of sodium and GDP. Both peptide and nonpeptide agonists maximally stimulated [35S]GTPgammaS binding. The stimulation of [35S]GTPgammaS binding was blocked by pretreatment of cells with pertussis toxin. Partial stimulation of [35S]GTPgammaS binding via the kappaOR was observed for several ligands that are antagonists at the mu opioid receptor, suggesting an additional mechanism of drug action. The ability of isomers of tifluadom and levallorphan to stimulate [35S]GTPgammaS binding indicates that the chiral carbon of levallorphan, a benzomorphan derivative, imparts a greater degree of stereoselectivity than does the chiral carbon in the benzodiazepine derivative tifluadom. In addition, (-)tifluadom, the less potent isomer of tifluadom, which is also a gamma-aminobutyric acidA receptor agonist, stimulated [35S]GTPgammaS binding. In contrast, d-pentazocine, (+)SKF10047, (+)cyclazocine, and d-ethylketocyclazocine displayed no agonist activity. kappaOR-selective antagonist norbinaltorphimine competitively inhibited the stimulation of [35S]GTPgammaS binding by the active isomers of ethylketocyclazocine, cyclazocine, and nalorphine to the same degree, indicating that all three ligands are eliciting an effect via the kappaOR. The results suggest that these cells express a homogeneous population of kappaOR, and that their [35S]GTPgammaS-binding properties make them an excellent means to assess kappaOR efficacy.

Analgesics, Opioid↗

PubMed1998

Determinants of gi1alpha and beta gamma binding. Measuring high affinity interactions in a lipid environment using flow cytometry.

G protein heterocomplex undergoes dissociation and association during its functional cycle. Quantitative measurements of alpha and betagamma subunit binding have been difficult due to a very high affinity. We used fluorescence flow cytometry to quantitate binding of fluorescein-labeled Gi1alpha (F-alpha) to picomolar concentrations of biotinylated G beta gamma. Association in Lubrol solution was rapid (kon = 0.7 x 10(6) M-1 s-1), and equilibrium binding revealed a Kd of 2.9 +/- 0.8 nM. The binding showed a complex dependence on magnesium concentration, but activation of F-alpha with either GDP/aluminum fluoride or guanosine 5'-O-(3-thiotriphosphate) completely prevented formation of the heterocomplex (Kd > 100 nM). The binding was also influenced by the detergent or lipid environment. Unlabeled betagamma reconstituted in biotinylated phospholipid vesicles (pure phosphatidylcholine or mixed brain lipids) bound F-alpha approximately 2-3-fold less tightly (Kd = 6-9 nM) than in Lubrol. In contrast, beta gamma in ionic detergents such as cholate and 3-[(cholamidopropyl)diethylammonio]-1-propanesulfonate exhibited substantially lower affinities for F-alpha. Dissociation of F-alpha from beta gamma reconstituted in lipid vesicles was observed upon addition of aluminum fluoride or excess unlabeled alpha subunit, indicating that myristoylated alpha subunit has only a weak interaction with lipids without the beta gamma subunit. The kinetics of aluminum fluoride-stimulated dissociation were slower than those of the alpha subunit conformational change detected by intrinsic fluorescence. These results quantitatively demonstrate G protein subunit dissociation upon activation and provide a simple but powerful new approach for studying high affinity protein/protein interactions in solution or in a lipid environment.

Flow Cytometry↗

PubMed1998

Detection and quantitation of heterotrimeric G proteins by fluorescence resonance energy transfer.

N-Methyl-3'-O-anthranoyl (mant) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for detection of heterotrimeric guanine nucleotide binding proteins. The mant derivative of GTP gamma S, mGTP gamma S, is synthesized and purified by modification of a method initially described by Hiratsuka (1983, Biochim. Biophys. Acta 742, 496-508). The binding affinity of mGTP gamma S for G proteins Gi and G(o) is comparable to that of GTP gamma S. The rate of binding is determined by the dissociation rate of the endogenously bound GDP. The large fluorescence increase observed upon mGTP gamma S binding to G protein is due, in part, to resonance energy transfer from tryptophans in the G protein to the mant guanine nucleotide. The magnitude of the fluorescence increase is dependent upon the concentration of G protein. Therefore, mGTP gamma S binding can be used to quantitate and locate G proteins during the protein purification process. This method is rapid compared to the [35S]GTP gamma S binding assay in that (i) the bound ligand does not need to be separated from the free ligand thus avoiding vacuum filtration and (ii) the time required to measure fluorescence in each sample is less than that required for scintillation counting. In addition, the use of radioactivity can be avoided. Thus, the mGTP gamma S binding assay for the detection of Gi and G(o) represents a rapid, reliable alternative to assays based on radiolabeled GTP gamma S binding or ADP-ribosylation with pertussis toxin.

Biopolymers↗

PubMed1998

Roles of G(o)alpha tryptophans in GTP hydrolysis, GDP release, and fluorescence signals.

Single tryptophan mutants of a histidine-tagged G(o)alpha (W132F and W212F) were prepared to examine the functional and spectroscopic role of tryptophan in G(o)alpha. The mutants bound GTP gamma S with high affinity and showed only modest changes in GDP affinity. GTP gamma S-stimulated intrinsic fluorescence changes were completely abolished by removal of W212 but were not affected by elimination of W132. In contrast, both W132 and W212 contributed to the fluorescence signal from binding of methylanthraniloyl-GTP gamma S (mGTP gamma S). W132F and W212F mutants showed 57% and 34% of the mGTP gamma S fluorescence change of wild type (WT), respectively. The decreased fluorescence signals were not due to reduced activation of the W212F protein by nucleotide as protection from tryptic digestion was unchanged. The kinetics of nucleotide binding and hydrolysis were also altered in both mutants. GDP dissociation was slower (0.14 min-1) for W132F and faster (0.54 min-1) for W212F than for WT (0.25 min-1). As expected, the steady-state Vmax for GTPase was lower for W132F, but surprisingly it was also lower for W212F despite faster GDP release. Single turnover kinetics revealed a lower kcat for W212F (0.52 min-1) compared to WT (1.39 min-1) and W132F (1.0 min-1). Thus, W212 in G(o)alpha makes a dominant contribution to both intrinsic and extrinsic fluorescence signals upon alpha subunit activation. In addition, both tryptophans modulate the kinetics of nucleotide binding and hydrolysis.

Amino Acid Substitution↗

PubMed1998

Delta opioid receptor down-regulation is independent of functional G protein yet is dependent on agonist efficacy.

Chronic treatment of C6 glioma cells stably expressing the rat delta opioid receptor (C6delta) with full agonists resulted in receptor down-regulation. Chronic [D-Ser2,L-Leu5]enkephalyl-Thr treatment caused a decrease in cell surface as well as a decrease in agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding. Treatment with full agonists for 12 hr resulted in a 90% decrease in receptor number that was paralleled by a decrease in the ability of agonist to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate binding and inhibit forskolin-stimulated adenylyl cyclase. Of the remaining receptors, a smaller fraction of receptors (41 +/- 4 vs. 56 +/- 4% in control) exhibited high affinity for agonist as compared to receptors in control membranes. Elimination of functional guanosine triphosphate binding protein (G protein) by Pertussis toxin pretreatment did not alter the ability of agonist to down regulate receptor. We hypothesized that agonist affinity (not efficacy) would be a predictor of an agonist's ability to down-regulate receptor. However, we found that only full agonists were able to down-regulate receptor number, G protein activation and adenylyl cyclase inhibition. Chronic exposure to partial agonist 7-spiroindinooxymorphone, which has a very high affinity for the receptor, as well as morphine, did not cause receptor down-regulation. Taken together, these results suggest that full agonists alter receptor conformation such that the altered conformation is recognized by G protein as well as proteins involved in receptor down-regulation. In addition, down-regulation is independent of agonist-mediated G protein activation and subsequent down-stream signaling.

Adenylyl Cyclase Inhibitors↗

PubMed1997

Novel form of crosstalk between G protein and tyrosine kinase pathways.

Neuronal Ca2+ channels are inhibited by a variety of transmitter receptors coupled to Go-type GTP-binding proteins. Go has been postulated to work via a direct interaction between an activated G protein subunit and the Ca2+ channel complex. Here we show that the inhibition of sensory neuron N-type Ca2+ channels produced by gamma-aminobutyric acid involves a novel, rapidly activating tyrosine kinase signaling pathway that is mediated by Galphao and a src-like kinase. In contrast to other recently described G protein-coupled tyrosine kinase pathways, the Galphao-mediated modulation requires neither protein kinase C nor intracellular Ca2+. The results suggest that this pathway mediates rapid receptor-G protein signaling in the nervous system and support the existence of a previously unrecognized form of crosstalk between G protein and tyrosine kinase pathways.

Animals↗

PubMed1997

Opioid efficacy in a C6 glioma cell line stably expressing the delta opioid receptor.

A C6 glioma cell line stably transfected with the rat delta opioid receptor (C6delta) was used to characterize receptor binding and G protein activation by both peptide and nonpeptide delta opioid ligands. The ligand binding affinities for [3H]naltrindole and [3H]pCl-[D-Pen2,D-Pen5]enkephalin (DPDPE) were similar to those observed in monkey brain membranes. The nonpeptide agonists, BW373U86 and SNC80, as well as peptide agonist [D-Ser2, L-Leu5]enkephalyl-Thr maximally stimulated [35S]GTPgammaS binding by 640, 654 and 576%, respectively, over basal. The peptide agonists, DPDPE and deltorphin II, both stimulated [35S]GTPgammaS binding by 375%. Etorphine, diprenorphine, oxymorphindole and 7-spiroindanyloxymorphone were also partial agonists in this assay, although they were less efficacious than deltorphin II. Stimulation of [35S]GTPgammaS binding by agonists was blocked completely by pertussis toxin pretreatment. Both delta-1 and delta-2 selective antagonists 7-benzylidenenaltrexone and a benzofuran analog of naltrindole displayed high affinity for the cloned receptor (0.04 and 0.08 nM) and antagonized the stimulation of [35S]GTPgammaS binding by BW373U86 and DPDPE with similar potencies. Other evidence suggesting the lack of receptor subtypes includes the finding that stimulation of [35S]GTPgammaS binding by receptor subtype selective ligands DPDPE and deltorphin II was not additive. BW373U86, SNC80 and DPDPE maximally inhibited forskolin-stimulated adenylyl cyclase. These cells highly express a homogeneous population of delta opioid receptor that couple to inhibitory Go/Gi proteins. Ligand affinity for the delta opioid receptor correlates with ligand EC50 values for stimulation of [35S]GTPgammaS binding.

Adenylate Cyclase Toxin↗

PubMed1996

Partial G protein activation by fluorescent guanine nucleotide analogs. Evidence for a triphosphate-bound but inactive state.

N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Previously, we showed that MANT fluorescence intensity when bound to G protein was related to the degree of G protein activation where MANT-guanosine-5'-O-(3-thiotriphosphate) (mGTP gammaS) had the highest fluorescence followed by mGTP and mGDP, respectively (Remmers, A. E., Posner, R., and Neubig, R. R. (1994) J. Biol. Chem. 269, 13771-13778). To directly examine G protein conformations with nucleotide triphosphates bound, we synthesized several nonhydrolyzable MANT-labeled guanine nucleotides. The relative maximal fluorescence levels observed upon binding to recombinant myristoylated Goalpha (myrGoalpha) and myrGialpha1 were: mGTPgammaS > MANT-5'-guanylyl-imidodiphosphate > MANT-guanylyl-(beta,gamma-methylene)-diphosphonate > MANT-guanosine 5'-O-2-(thio)diphosphate. Using protection against tryptic digestion as a measure of the activated conformation, the ability of the MANT guanine nucleotides to maximally activate myrGo alpha correlated with maximal fluorescence. Biphasic dissociation kinetics were observed for all of the MANT guanine nucleotides. The data were consistent with the following model, [formula: see text] where G protein activation (G*-GXP) is determined by a conformational equilibrium between two triphosphate bound states as well as by the balance between binding and hydrolysis of the nucleotide triphosphate. Compared with myrGialpha1, maximal mGTP fluorescence was only 2-fold higher for the myrGialpha1 Q204L mutant, a mutant with greatly reduced GTPase activity, and only 24% that of mGTPgammaS, indicating that partial activation by mGTP was not just due to hydrolysis of mGTP. These results extend our previous conclusion that GTP analogs do not fully activate G protein.

Acyltransferases↗

PubMed1994

Rapid kinetics of G protein subunit association: a rate-limiting conformational change?

G protein subunit association and dissociation are thought to play an important role in signal transduction. We measured alpha beta gamma heterocomplex formation using resonance energy transfer. Fluorescein-labelled alpha(F-alpha) emission was quenched approximately 10% on mixing with eosin-labelled beta gamma(E-beta gamma). Unlabelled beta gamma did not quench F-alpha fluorescence. Stopped-flow kinetics showed a t1/2 ranging from 2.5 s to 0.25 s for 50 nM to 1200 nM E-beta gamma. The rate saturated at high E-beta gamma concentrations consistent with a two-step mechanism. We report the first rapid-mix studies of G protein subunit association kinetics which suggest that alpha and beta gamma combine by a two-step process with a maximal rate of 4.1 +/- 0.4 s-1.

Energy Transfer↗

PubMed1994

Binding of an alpha 2 adrenergic receptor third intracellular loop peptide to G beta and the amino terminus of G alpha.

The structural basis of receptor-G protein interactions was examined using a photoaffinity derivative of a G protein-activating receptor-derived peptide (Q peptide) from the carboxyl-terminal region of the third intracellular loop of alpha 2 adrenergic receptor. A diazopyruvoyl photoaffinity derivative of this peptide (DAP-Q) was cross-linked to purified bovine brain Go. Specific, competable cross-linking of 750 nM DAP-Q to sites on both the alpha o and beta subunits was observed. No specific cross-linking was seen with non-target proteins or heat-denatured G protein subunits. 125I-DAP-Q labeled the 2-kDa amino-terminal fragment of alpha o as determined by protease digestion of the cross-linked G protein followed by gel electrophoresis or h igh pressure liquid chromatography purification and mass spectroscopy of the radiolabeled proteolysis fragment. The functional significance of incorporation into beta gamma subunit is supported by the absolute requirement of beta gamma subunit for DAP-Q stimulation of Go/Gi GTPase. Thus, specific interactions of G protein-coupled receptors with the beta subunit of G protein, in addition to those with the alpha subunit, appear to be important for receptor-G protein coupling.

Amino Acid Sequence↗

PubMed1994

Fluorescent guanine nucleotide analogs and G protein activation.

The N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Both MANT-GTP gamma S (mGTP gamma S) and MANT-GTP (mGTP) displayed a magnesium-dependent increase in fluorescence upon binding to bovine brain G(o). A much greater increase in MANT-guanine nucleotide fluorescence was observed with excitation at 280 nm compared with 350 nm, due to energy transfer from tryptophan in G(o). G(o)-bound mGTP gamma S displays a blue-shift in its emission spectrum indicating a nonpolar environment for the G(o)-bound MANT. For the hydrolyzable analog, mGTP, the increase in fluorescence is followed by a decrease as it is hydrolyzed to mGDP. Unexpectedly, dissociation of mGDP was fast (t1/2 1.7 s) by comparison with GDP itself (t1/2 120 s). Binding of mGTP gamma S to G(o) was slow, but mastoparan increased the rate approximately 4-fold. For mGTP, mastoparan increased both the rate of binding and the peak fluorescence, even at saturating mGTP concentrations. Modeling the mGTP fluorescence kinetics in the presence and absence of mastoparan results in two novel conclusions. First, mGTP does not fully activate the G protein, even when bound. Second, mastoparan appears to increase the rate of the G protein conformational activation step, in addition to its known effect on GDP release.

Animals↗

PubMed1993

Synthesis and characterization of fluorescently labeled bovine brain G protein subunits.

G proteins play an important role in transmitting hormonal signals, and fluorescence techniques would be useful to study their cellular distribution and mechanisms. To prepare active fluorescent G protein Go/Gi or beta gamma subunits were reacted with fluorescein isothiocyanate (FITC) to label the alpha (F-alpha) and gamma (F-gamma/beta) subunits or with (iodoacetamido)tetramethylrhodamine (TMR-IAA) to label the beta subunit (TMR-beta gamma). Unreacted dye was removed from the labeled proteins by ultrafiltration, followed by further purification using HPLC gel filtration. The molar ratios of dye to protein were 0.96 +/- 0.15, 0.59 +/- 0.07, and 1.37 +/- 0.09 for labeled alpha,beta, and gamma subunits, respectively. GTP gamma S binding to F-alpha and ADP-ribosylation by pertussis toxin of F-alpha were reduced to 63% and 78% of control, respectively. F-alpha was a heterogeneous population of alpha subunits. Active F-alpha containing less than one (0.7) label/subunit (F-alpha-Mono Q) was separated from unlabeled and multiply labeled F-alpha by Mono Q anion-exchange chromatography. F-alpha-Mono Q displayed reduced GTPase activity (turnover number was 46% of control), while GTP gamma S binding and ADP-ribosylation by pertussis toxin were only decreased to 78% and 82% of control, respectively. TMR-beta gamma and F-gamma/beta retain full function compared to native beta gamma, as measured by three methods: (1) TMR-beta gamma and F-gamma/beta are able to form heterotrimers with alpha o subunits, (2) TMR-beta gamma and F-gamma/beta support the ADP ribosylation of alpha o subunits by pertussis toxin, and (3) TMR-beta gamma and F-gamma/beta inhibit forskolin-stimulated adenylyl cyclase activity. The fluorescent G protein subunits will be valuable tools to study G protein mechanisms in reconstituted membranes and intact cells.

Animals↗

PubMed1993

Resonance energy transfer between guanine nucleotide binding protein subunits and membrane lipids.

Resonance energy transfer was used to estimate the distances of closest approach between fluorescent labels on G protein subunits (alpha, beta, and gamma) and the phospholipid bilayer surface. Fluorescein-labeled alpha, beta, and gamma subunits were the energy transfer donors and hexadecylaminoeosin (HAE) in phospholipid vesicles was the acceptor. Bovine brain G protein (alpha o, beta, and gamma subunits) were individually labeled with fluorescein as described in the accompanying paper [Kwon et al. (1993) Biochemistry (preceding paper in this issue)]. Fluorescein-labeled subunits were combined with the appropriate unlabeled G protein subunits and reconstituted into phospholipid vesicles. HAE quenched the fluorescein emission in a concentration-dependent manner which was dependent upon the G protein subunit labeled (gamma = beta > alpha o). From steady-state quenching data with hexadecylaminofluorescein (HAF) as a standard to determine the density of HAE in the phospholipid bilayer, the calculated distances between fluorescein-alpha o, -beta, and -gamma and HAE are 46 +/- 2, 38 +/- 3, and 37 +/- 2 A, respectively. Energy transfer was identified as the means of fluorescence quenching by two methods: (1) addition of 0.1% Lubrol reversed the quenching of fluorescein-labeled G protein subunits by HAE and (2) sensitized HAE emission was observed in the presence of fluorescein-labeled G protein subunits. These results are the first physical measurements of the distances between sites on G protein subunits and the lipid bilayer. These data demonstrate that the label in the beta and gamma subunits is closer to the lipid bilayer than that in the alpha subunit.

Animals↗

PubMed1991

Reconstitution of high-affinity opioid agonist binding in brain membranes.

In synaptosomal membranes from rat brain cortex, the mu selective agonist [3H]dihydromorphine in the absence of sodium, and the nonselective antagonist [3H]naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with Kd values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 microM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP[gamma-35S] binding sites by 90% and low Km, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP[gamma S] was diminished. High-affinity (Kd, 0.72 nM), guanine nucleotide-sensitive agonist binding was reconstituted by polyethylene glycol-induced fusion of the alkali-treated membranes with (opioid receptor devoid) C6 glioma cell membranes. Also restored was opioid agonist-stimulated, naltrexone-inhibited GTPase activity. In contrast, antagonist binding in the fused membranes was unaltered. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low Km GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor-guanine nucleotide binding protein complex.

Animals↗