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R R Neubig

Publications and source records attributed to R R Neubig.

At least 37 records · Page 2Linked to original sources
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

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

Cotransfection of second and third intracellular loop fragments inhibit angiotensin AT1a receptor activation of phospholipase C in HEK-293 cells.

Peptides from the intracellular regions of G protein-coupled receptors are useful probes of receptor-G protein coupling mechanisms. As a first step toward the genetic delivery of such "G protein inhibitors," we describe inhibition of angiotensin II (AII) receptor responses by expressed fragments of the second and third intracellular loops of the AT1a receptor (AT1a/i2 and AT1a/i3). Transient transfection of human embryonic kidney 293 cells with DNA encoding the rat AT1a receptor resulted in AII-dependent increases of inositol phosphates (maximum 4.5-fold). Cotransfection of AT1a/i2 and AT1a/i3 fragments raised the EC50 for AII stimulation of phospholipase C activity 5-fold (from 0.18 nM to 0.99 nM, n = 12, P < .001) and 3-fold (from 0.38 nM to 1.2 nM, n = 8, P < .002), respectively. The combined effect of AT1a/i2 and AT1a/3 was additive, and transfection of an alpha-1b adrenergic receptor third intracellular loop (alpha1b/i3) fragments also increased the EC50 for AII. Neither AT1a/i1 nor C-terminus (AT1a/Ct) constructs had significant effects on angiotensin responses. These data confirm a role for the second and third intracellular loops in angiotensin receptor responses and show the potential of this approach to blocking multiple phospholipase C-linked receptors.

Animals↗
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

Modulation of K+ and Ca2+ currents in cultured neurons by an angiotensin II type 1a receptor peptide.

Angiotensin II (ANG II) inhibits delayed rectifier K+ current (IK) and stimulates total Ca2+ current (ICa) in neurons cocultured from newborn rat hypothalamus and brain stem, effects mediated via ANG II type 1 (AT1) receptors. Here, we identify potential G protein activator regions of the AT1 receptor responsible for initiating the intracellular changes that lead to alterations in these currents. Intracellular application into cultured neurons of a peptide corresponding to the third cytoplasmic loop of the AT1 receptor (AT1a/i3) mimicked the actions of ANG II on IK and ICa, whereas application of a peptide corresponding to the second cytoplasmic loop (AT1a/i2) did not alter these currents. This modulation of IK and ICa by AT1a/i3 involves intracellular messengers (G alpha q, protein kinase C, and intracellular Ca2+) that are identical to those involved in the modulation of IK and ICa following ANG II activation of AT1 receptors. These data provide functional evidence for a role of the third cytoplasmic loop of the AT1 receptor in G protein coupling and subsequent modulation of ion channel effectors.

Amino Acid Sequence↗
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↗
PubMed1996

Receptor and membrane interaction sites on Gbeta. A receptor-derived peptide binds to the carboxyl terminus.

The functional organization of Gbetagamma is poorly understood. Regions of bovine brain Gbetagamma that interact with a photoaffinity derivative of an alpha2-adrenergic receptor-derived peptide from the third intracellular loop (diazopyruvoyl-modified peptide Q (DAP-Q)) and a hydrophobic membrane probe (3-trifluoromethyl-3-(m-iodophenyl)diazirine (TID)) were examined. We previously showed that DAP-Q cross-links to specific, competable sites on both the alpha and beta subunits of Go/Gi but not on the gamma subunit and that betagamma subunit was required for stimulation of Go/Gi GTPase activity (Taylor, J. M., Jacob Mosier, G. G., Lawton, R. G., Remmers, A. E., and Neubig, R. R. (1994) J. Biol. Chem. 269, 27618-27624). Similarly, we show here that the membrane-associated photoprobe [125I]TID labels alpha and beta but not gamma. We have now mapped the sites of incorporation of DAP-Q and TID into the beta subunit. TID labels both the 14-kDa amino-terminal and the 23-kDa carboxyl-terminal fragments from a partial tryptic digest of beta while DAP-Q labels only the carboxyl-terminal fragment. Further mapping with endopeptidase Lys C reveals substantial labeling of multiple fragments by TID while DAP-Q labels predominantly a approximately 6-kDa fragment within the carboxyl-terminal 60 amino acids of beta1. Thus, regions within the 7th (or possibly 6th) WD-40 repeat of the beta subunit of G protein interact with the receptor-derived peptide while membrane interaction involves multiple sites throughout the beta subunit.

Animals↗
PubMed1996

Structural requirements for G(o) activation by receptor-derived peptides: activation and modulation domains of the alpha 2-adrenergic receptor i3c region.

Synthetic peptides are important tools for understanding the sites and mechanisms of receptor/G protein interactions. We examined the structural determinants of receptor-fragment peptides for G protein binding and activation. A dimer of peptides from the carboxyl-terminal (i3c) and amino-terminal (i3n) regions of the alpha 2A-adrenergic receptor is most potent in stimulating guanine-nucleotide exchange of any peptides studied. Stimulation of GTPase by i3n is partially blocked by pertussis toxin treatment, whereas stimulation by i3c is not, which is consistent with action of i3c at the amino terminus of Gi. Both peptides inhibit adenylyl cyclase in Chinese hamster ovary cell membranes, but only the i3c effect is consistent with a pure Gi stimulation. We also examined the mechanism and defined a minimal structural subset of i3c required for G protein activation. Residues 361-365 from the receptor were essential for GTPase stimulation, whereas determinants in the region 368-373 modulated that activity. A specific role for arginines is defined beyond just their positive charge. Complex effects of modifications of Thr373 suggest a regulatory or conformational role of that residue in the previously defined constitutive activation of the alpha 2-adrenergic receptor [J. Biol. Chem. 268:16483-16487 (1993)]. Thus, our data plus recent mutagenesis results support a role for hydrophobicity in the i3n region and a positively charged/arginine-rich region approximately 15-20 residues from the sixth transmembrane span in G protein activation. In contrast, the immediate perimembrane region of i3c seems to have largely conformational effects in producing constitutive activation of the receptors.

Adenylate Cyclase Toxin↗
PubMed1995

Lack of association of G-protein beta 2- and gamma 2-subunit N-terminal fragments provides evidence against the coiled-coil model of subunit-beta gamma assembly.

The association between peptides from bovine G-protein beta 2- and gamma 2-subunits was studied by CD spectroscopy and cross-linking. Both peptides had approximately 25% stable alpha-helical structure at 25 degrees C, but there was no increase on mixing subunits as expected for coiled-coil formation. Also, disulphide cross-linking gave more beta 2 beta 2 homodimer than beta 2 gamma 2 heterodimer. These data do not support the proposed N-terminal coiled-coil model of beta gamma-subunit association.

Amino Acid Sequence↗
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↗
PubMed1994

Coupling an alpha 2-adrenergic receptor peptide to G-protein: a new photolabeling agent.

A photoreactive derivative of a tetradecapeptide G-protein activator (peptide Q) derived from the alpha 2-adrenergic receptor was designed and used to label purified G-protein (Go/Gi). N-bromoacetyl-N'-(3-diazopyruvoyl)-m-phenylene-diamine (Br-DAP) was conjugated to the C-terminal cysteine of peptide Q. The DAP-modified peptide Q (DAP-Q) specifically incorporated into the a subunit of Go. The incorporation of DAP-Q into alpha o was blocked by unmodified Q peptide (IC50 = 15 +/- 6 microM; n = 4). Photolysis of sixfold higher concentrations of DAP-Q with ovalbumin or bovine albumin failed to produce cross-linked products. Br-DAP should prove useful in detecting mutual contact sites between peptides and their binding proteins.

Affinity Labels↗
PubMed1994

Peptides as probes for G protein signal transduction.

Triggered by agonist binding to cell surface receptors, the heterotrimeric G proteins dissociate into alpha and beta gamma subunits, each activating distinct second messenger pathways. Peptides from the primary sequences of receptors, G proteins, and effectors have been used to study the molecular interactions between these proteins. Receptor-derived peptides from the second, third and fourth intracellular loops and certain naturally occurring peptides antagonize G protein interactions and can directly activate G protein. These peptides bind to G protein sites that include the N and C terminal regions of the alpha subunit and a yet to be identified region of the beta subunit. Peptides have also been useful in characterizing G protein-effector interactions. The identification of the contact sites between proteins involved in G protein signal transduction should aid in the development of non-peptide mimetic therapeutics which could specifically modify G protein-mediated cellular responses.

Animals↗
PubMed1994

Lateral mobility of tetramethylrhodamine (TMR) labelled G protein alpha and beta gamma subunits in NG 108-15 cells.

Multi-step signal transducing events, such as those mediated by G proteins, have been difficult to study in intact cells. We prepared fluorescently labelled G protein subunits, tetramethylrhodamine-alpha o (TMR-alpha o) and TMR-beta gamma, in order to study their subcellular distribution and lateral mobility. Heterotrimeric G proteins labelled in the alpha (TMR-alpha o/beta gamma) or beta (TMR-beta gamma/alpha o) subunit were reconstituted into lipid vesicles and fused to NG-108-15 cells using polyethylene glycol (PEG). Vesicles fused completely to the cells as determined by dequenching of a fluorescent lipid probe, octadecyl rhodamine B. The orientation of G protein beta gamma subunits after fusion followed the expected random distribution; the quenching of surface fluorescence with anti-fluorescein antibodies showed that about 50% of the label was accessible extracellularly. G proteins incorporated by the fusion method were able to couple to endogenous alpha 2 adrenergic receptors based on the restoration of high affinity agonist binding to pertussis toxin-treated cells. The subcellular localization of TMR-alpha o and TMR-beta gamma determined by differential centrifugation and confocal microscopy indicated that TMR-alpha o was present in the plasma membrane and in intracellular membranes, whereas TMR-beta gamma was mainly localized in the plasma membrane. The lateral mobility of TMR-alpha o and TMR-beta gamma measured using fluorescence recovery after photobleaching (FRAP) demonstrated low mobile fractions of 0.34 +/- 0.03 and 0.16 +/- 0.03, respectively. The translational diffusion coefficients of the mobile components were similar, 4.0 x 10(-9) and 2.0 x 10(-9) cm2/s, for alpha and beta gamma respectively. Neither activation of Gi-linked receptors nor cytoskeletal disruption with nocodozole or cytochalasin D changed the mobile fraction or diffusion coefficient of the alpha or beta gamma subunits. The FRAP data combined with the localization of fluorescent subunits by confocal microscopy suggest that the beta gamma subunits are highly constrained to localized regions of the plasma membrane while the alpha subunit may diffuse in intracellular regions to transmit signals from receptors to effector proteins.

Cell Membrane↗
PubMed1994

Membrane organization in G-protein mechanisms.

A prevailing view of receptor and G-protein function in cells includes random collisions between the proteins with a great specificity at the sites of protein-protein interaction. Recent evidence suggests that receptors, G-proteins, and effectors may be less mobile and that these systems are more highly organized than previously appreciated. Several types of evidence suggest that receptors do not have free access to all G-protein with which they are capable of coupling. Also, the specificity of signaling in intact cells appears to be significantly greater than in reconstituted systems. The distribution and mobility of G-proteins in cells are restricted to a surprising degree. Thus, complex interactions of the receptors and G-proteins with their effectors and cell membrane machinery appear to play an important role in their function. A full understanding of G-protein-coupled receptors must include a better description of the organization of these systems in cell membranes. Possible roles for noncoated pits (caveolae) and a novel pleckstrin homology domain need to be examined.

Animals↗
PubMed1994

The novel alpha-2 adrenergic radioligand [3H]-MK912 is alpha-2C selective among human alpha-2A, alpha-2B and alpha-2C adrenoceptors.

We have determined the binding affinities of the novel alpha-2 adrenoceptor antagonist radioligand [3H]-MK912 for the cloned human alpha-2A, alpha-2B and alpha-2C adrenoceptors. The KD-values were 1.25 nM, 1.36 nM and 0.086 nM for the alpha-2A, alpha-2B and alpha-2C subtypes, respectively. Thus, the selectivity of [3H]-MK912 for the human alpha-2C adrenoceptor vs. the human alpha-2A and alpha-2B adrenoceptors is 14-fold and 16-fold, respectively. The alpha-2C selectivity, and the very high affinity of [3H]-MK912 for the alpha-2C adrenoceptor subtype (KD = 86 pM) makes this radioligand a promising tool for studying the role of alpha-2C adrenoceptors in the human. A selection of antagonists useful for differentiating between the human alpha-2A, alpha-2B and alpha-2C adrenoceptor subtypes is discussed.

Adrenergic alpha-Antagonists↗