Biomedical subjects

Richard R Neubig

Publications and source records attributed to Richard R Neubig.

At least 37 records · Page 2Linked to original sources

PubMed2004

RGS-insensitive G-protein mutations to study the role of endogenous RGS proteins.

Regulator of G-protein signaling (RGS) proteins are very active GTPase-accelerating proteins (GAPs) in vitro and are expected to reduce signaling by G-protein coupled receptors in vivo. A novel method is presented to assess the in vivo role of RGS proteins in the function of a G protein in which Galpha subunits do not bind to RGS proteins or respond with enhanced GTPase activity. A point mutation in the switch I region of Galpha subunits (G184S Galpha(o) and G183S Galpha(i1)) blocks the interaction with RGS proteins but leaves intact the ability of Galpha to couple to betagamma subunits, receptors, and downstream effectors. Expression of the RGS-insensitive mutant G184S Galpha(o) in C6 glioma cells with the micro-opioid receptor dramatically enhances adenylylcyclase inhibition and activation of extracellular regulated kinase. Introducing the same G184S Galpha(o) protein into embryonic stem (ES) cells by gene targeting allows us to assess the functional importance of the endogenous RGS proteins using in vitro differentiation models and in intact mice. Using ES cell-derived cardiocytes, spontaneous and isoproterenol-stimulated beating rates were not different between wild-type and G184S Galpha(o) mutant cells; however, the bradycardiac response to adenosine A1 receptor agonists was enhanced significantly (seven-fold decrease EC50) in Galpha(o)RGSi mutant cells compared to wild-type Galpha(o), indicating a significant role of endogenous RGS proteins in cardiac automaticity regulation. The approach of using RGS-insensitive Galpha subunit knockins will reveal the role of RGS protein-mediated GAP activity in signaling by a given G(i/o) protein. This will reveal the full extent of RGS regulation and will not be confounded by redundancy in the function of multiple RGS proteins.

Animals↗

PubMed2004

Ribozyme- and siRNA-mediated suppression of RGS-containing RhoGEF proteins.

Given recent efforts to determine the sequence information on thousands of genes in the human genome, the current challenge is to identify the functions of these genes, including those encoding the regulator of G-protein signaling protein gene superfamily, and to establish their roles in particular signaling pathways in a native system. Increasingly, reverse genetic approaches are being used to address these questions. This article compares two powerful approaches [ribozyme and "short interfering" RNA (siRNA) techniques] under identical conditions for the first report on the suppression of endogenous RGS domain-containing RhoGEFs. The siRNA technique was found to be much more potent than ribozyme targeting at the same mRNA site of RGS-RhoGEFs. Also, the three siRNAs targeting LARG, PDZ-RhoGEF, and p115-RhoGEF are able to discriminate the closely related sequences within this RGS-RhoGEF gene family.

Amino Acid Sequence↗

PubMed2004

Structure-based design, synthesis, and activity of peptide inhibitors of RGS4 GAP activity.

One of the principal roles of the multifunctional regulator of G-protein signaling (RGS) proteins is to terminate G-protein-coupled receptor (GPCR) signaling by binding to the G-protein Galpha subunit, thus acting as GTPase-activating proteins (GAPs). In principle, then, selective inhibitors of this GAP function would have potential as therapeutic agents, as they could be used to augment the effects of endogenous or exogenous GPCR agonists. Using the published RGS4-G(ialpha1) X-ray structure, we have designed and synthesized a series of cyclic peptides, modeled on the G(ialpha) switch I region, that inhibit RGS4 GAP activity, presumably by blocking the interaction between RGS4 and G(ialpha1). These compounds should prove useful for elucidating RGS-mediated activity and serve as a starting point for the development of a novel class of therapeutic agent.

Amino Acid Sequence↗

PubMed2003

Dimerization in aminergic G-protein-coupled receptors: application of a hidden-site class model of evolution.

G-Protein-coupled receptors (GPCRs) are an important superfamily of transmembrane proteins involved in cellular communication. Recently, it has been shown that dimerization is a widely occurring phenomenon in the GPCR superfamily, with likely important physiological roles. Here we use a novel hidden-site class model of evolution as a sequence analysis tool to predict possible dimerization interfaces in GPCRs. This model aims to simulate the evolution of proteins at the amino acid level, allowing the analysis of their sequences in an explicitly evolutionary context. Applying this model to aminergic GPCR sequences, we first validate the general reasoning behind the model. We then use the model to perform a family specific analysis of GPCRs. Accounting for the family structure of these proteins, this approach detects different evolutionarily conserved and accessible patches on transmembrane (TM) helices 4-6 in different families. On the basis of these findings, we propose an experimentally testable dimerization mechanism, involving interactions among different combinations of these helices in different families of aminergic GPCRs.

Amino Acid Substitution↗

PubMed2003

Depicting a protein's two faces: GPCR classification by phylogenetic tree-based HMMs.

Related proteins with similar biological functions generally share common features, allowing us to extract the common sequence features. These common features enable us to build statistical models that can be used to classify proteins, to predict new members, and to study the sequence-function relationship of this protein function group. Although evolution underlies the basis of multiple sequence analysis methods, most methods ignore phylogenetic relationships and the evolutionary process in building these statistical models. Previously we have shown that a phylogenetic tree-based profile hidden Markov model (T-HMM) is superior in generating a profile for a group of similar proteins. In this study we used the method to generate common features of G protein-coupled receptors (GPCRs). The profile generated by T-HMM gives high accuracy in GPCR function classification, both by ligand and by coupled G protein.

Animals↗

PubMed2003

Detection of G proteins by affinity probe capillary electrophoresis using a fluorescently labeled GTP analogue.

An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTPgammaS-Galpha(o) peak heights increased linearly with Galpha(o) up to approximately 200 nM using a 50 nM BGTPgammaS probe. The detection limit for Galpha(o) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (Kd) to be determined. The on-rate and off-rate of BGTPgammaS to Galpha(o) were 0.0068 +/- 0.0004 and 0.000 23 +/- 0.000 01 s(-1), respectively. The half-life of the BGTPgammaS-Galpha(o) complex was 3060 +/- 240 s and Kd was 8.6 +/- 0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTPgammaS was demonstrated with assays for Galpha and Galpha(i1) and for Ras and Rab3A.

Animals↗

PubMed2003

Stimulation of cellular signaling and G protein subunit dissociation by G protein betagamma subunit-binding peptides.

We previously developed peptides that bind to G protein betagamma subunits and selectively block interactions between betagamma subunits and a subset of effectors in vitro (Scott, J. K., Huang, S. F., Gangadhar, B. P., Samoriski, G. M., Clapp, P., Gross, R. A., Taussig, R., and Smrcka, A. V. (2001) EMBO J. 20, 767-776). Here, we created cell-permeating versions of some of these peptides by N-terminal modification with either myristate or the cell permeation sequence from human immunodeficiency virus TAT protein. The myristoylated betagamma-binding peptide (mSIRK) applied to primary rat arterial smooth muscle cells caused rapid activation of extracellular signal-regulated kinase 1/2 in the absence of an agonist. This activation did not occur if the peptide lacked a myristate at the N terminus, if the peptide had a single point mutation to eliminate betagamma subunit binding, or if the cells stably expressed the C terminus of betaARK1. A human immunodeficiency virus TAT-modified peptide (TAT-SIRK) and a myristoylated version of a second peptide (mSCAR) that binds to the same site on betagamma subunits as mSIRK, also caused extracellular signal-regulated kinase activation. mSIRK also stimulated Jun N-terminal kinase phosphorylation, p38 mitogen-activated protein kinase phosphorylation, and phospholipase C activity and caused Ca2+ release from internal stores. When tested with purified G protein subunits in vitro, SIRK promoted alpha subunit dissociation from betagamma subunits without stimulating nucleotide exchange. These data suggest a novel mechanism by which selective betagamma-binding peptides can release G protein betagamma subunits from heterotrimers to stimulate G protein pathways in cells.

Amino Acid Sequence↗

PubMed2003

Endogenous RGS protein action modulates mu-opioid signaling through Galphao. Effects on adenylyl cyclase, extracellular signal-regulated kinases, and intracellular calcium pathways.

RGS (regulators of G protein signaling) proteins are GTPase-activating proteins for the Galpha subunits of heterotrimeric G proteins and act to regulate signaling by rapidly cycling G protein. RGS proteins may integrate receptors and signaling pathways by physical or kinetic scaffolding mechanisms. To determine whether this results in enhancement and/or selectivity of agonist signaling, we have prepared C6 cells stably expressing the mu-opioid receptor and either pertussis toxin-insensitive or RGS- and pertussis toxin-insensitive Galpha(o). We have compared the activation of G protein, inhibition of adenylyl cyclase, stimulation of intracellular calcium release, and activation of the ERK1/2 MAPK pathway between cells expressing mutant Galpha(o) that is either RGS-insensitive or RGS-sensitive. The mu-receptor agonist [d-Ala(2),MePhe(4),Gly(5)-ol]enkephalin and partial agonist morphine were much more potent and/or had an increased maximal effect in inhibiting adenylyl cyclase and in activating MAPK in cells expressing RGS-insensitive Galpha(o). In contrast, mu-opioid agonist increases in intracellular calcium were less affected. The results are consistent with the hypothesis that the GTPase-activating protein activity of RGS proteins provides a control that limits agonist action through effector pathways and may contribute to selectivity of activation of intracellular signaling pathways.

Adenylyl Cyclases↗

PubMed2003

Inverse agonist activity of agouti and agouti-related protein.

Agouti and agouti-related protein (AgRP) are endogenous antagonists of the melanocortin receptors (MCxR). Previous data showed that recombinant full-length agouti and a synthetic fragment of AgRP, AgRP (83-132), are inverse agonists at the MC1R and MC4R, respectively. This study demonstrates the smaller analogs AgRP (87-120) and ASIP [90-132 (L89Y)], and short peptides Yc[CRFFNAFC]Y and Qc[CRFFRSAC]S are also MC4R inverse agonists. Furthermore, the relative affinity of the series of MC4R ligands for displacement of radiolabeled antagonist 125I-AgRP (86-132) versus radiolabeled agonist 125I-NDP-MSH did not correlate with ligand efficacy, which is more consistent with an induced-fit model than a simple two-state model of MC4R activation. These data shed new light on the determinants and mechanism of inverse agonism at the MC4R.

Agouti Signaling Protein↗

PubMed2003

Diabetic neuropathy: inhibitory G protein dysfunction involves PKC-dependent phosphorylation of Goalpha.

We examined the hypothesis that decreased inhibitory G protein function in diabetic neuropathy is associated with increased protein kinase C (PKC)-dependent phosphorylation of the Goalpha subunit. Streptozotocin-induced diabetic rats were studied between 4 and 8 weeks after onset of diabetes and compared with aged-matched healthy animals as controls. Opioid-mediated inhibition of forskolin-stimulated cyclic AMP was significantly less in dorsal root ganglia (DRGs) from diabetic rats compared with controls. Activation of PKC in DRGs from control rats was associated with a significant decrease in opioid-mediated inhibition of forskolin-stimulated cyclic AMP that was similar to the decrease in inhibition observed in DRGs from diabetic rats. Both basal and PKC-mediated labeling of Goalpha with 32Pi was significantly less in DRGs from diabetic rats, supporting increased endogenous PKC-dependent phosphorylation of Goalpha. Probing of immunoprecipitated Goalpha with an anti-phospho-serine/threonine specific antibody revealed a significant increase in baseline phosphorylation in diabetic DRGs. Activation of PKC produced a significant increase in phosphorylation in control DRGs but no significant increase in Goalpha in diabetic DRGs. Phosphorylation of PKC-alpha was increased, PKC-betaII was unchanged and PKC-delta decreased in diabetic DRGs. These results suggest that diminished inhibitory G protein function observed in DRGs neurons from diabetic rats involves an isoform-specific PKC-dependent pathway.

Animals↗

PubMed2003

Ligand-receptor-G-protein molecular assemblies on beads for mechanistic studies and screening by flow cytometry.

G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Gialpha2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein. Streptavidin-coated polystyrene beads used biotinylated anti-FLAG antibodies to bind FLAG-tagged G proteins for ternary complex assembly. Validation was achieved by showing time and concentration dependence of ternary complex formation. Affinity measurements of ligand for receptor on particles, of the ligand-receptor complex for G protein on the particles, and receptor-Gialpha2 fusion protein for Gbetagamma, were consistent with comparable assemblies in detergent suspension. Performance was assessed in applications representing the potential of these assemblies for ternary complex mechanisms. We showed the relationship for a family of ligands between LR and LRG affinity and characterized the affinity of both the wild-type and GFP fusion receptors with G protein. We also showed the potential of kinetic measurements to allow observation of individual steps of GTP-induced ternary complex disassembly and discriminated a fast step caused by RG disassembly compared with the slower step of Galphabetagamma disassembly.

Flow Cytometry↗

PubMed2003

International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology.

The recommendations that follow have been updated from the proposals of a Technical Subcommittee set up by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (Jenkinson DH, Barnard EA, Hoyer D, Humphrey PPA, Leff P, and Shankley NP (1995) International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. IX. Recommendations on terms and symbols in quantitative pharmacology. Pharmacol Rev 47:255-266).

Pharmaceutical Preparations↗

PubMed2002

A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals.

Animals↗

PubMed2002

Fluorescence analysis of receptor-G protein interactions in cell membranes.

The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluorescein-labeled myristoylated Galpha(i1) (F-alpha(i1)) was used as the ligand bound to Gbetagamma in competition binding studies with differently modified Galpha subunit isoforms. In detergent solutions, the binding affinity of Galpha(i) to betagamma was 2 orders of magnitude higher than for Galpha(o) and Galpha(s) (IC50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in alpha(2A)AR receptor expressing CHO cell membranes into which purified betagamma subunits and F-alpha(i1) were reconstituted. These cell membrane studies led to the following observations: (1) binding of alpha subunit to the betagamma was not enhanced by receptor in the presence or absence of agonist, indicating that betagamma contributed essentially all of the binding energy for alpha(i1) interaction with the membrane; (2) activation of the receptor facilitated GTPgammaS-stimulated detachment of F-alpha(i1) from betagamma and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.

Adrenergic alpha-2 Receptor Agonists↗

PubMed2002

Expression of novel splice variants of the G protein subunit, Go alpha, is tissue-specific and age-dependent in the rat.

Heterotrimeric G proteins play an essential role in coupling numerous surface membrane receptors to intracellular signal transduction pathways. Relatively little is known about the splice variants of G proteins, including whether they undergo differential expression as a function of aging. We screened for splice variants of the alpha subunit of the dominant inhibitory G protein family member G(o) in a variety of tissues from rat and examined the expression of these splice variants during development. The splice variants were characterized using specific primers for G(o)alpha1 and G(o)alpha2 in conjunction with reverse transcription-polymerase chain reaction, and subsequently sequenced. G(o)alpha1 expression dominated over G(o)alpha2 in all neuronal tissues screened, including cerebral cortex, pituitary, spinal cord, colon myenteric plexus, dorsal root ganglion, and prenatal cortex. The sequence data of G(o)alpha1 supports the presence of three splice variants: G(o)alpha1a, G(o)alpha1b, G(o)alpha1c. The G(o)alpha1a variant was reported previously [J. Biol. Chem. 262 (1987) 14241], whereas G(o)alpha1b and G(o)alpha1c represent novel variants. The G(o)alpha1b splice variant demonstrates a 94 bp deletion using a cryptic donor site in exon 10. The G(o)alpha1c variant demonstrates a complete deletion of exon 10. A protein product with a molecular weight of approximately 34 kDa consistent with that expected for G(o)alpha1c was identified using Western blot analysis and two-dimensional gel electrophoresis. The expression of G(o)alpha1a decreased postnatally, supporting a potential physiological role during fetal development, whereas G(o)alpha1c expression increased postnatally. The age-dependent and tissue-specific expression of the G(o)alpha1 splice variants presage a broader functional role than has been observed historically with G(o).

Aging↗

PubMed2002

Mutagenesis and peptide analysis of the DRY motif in the alpha2A adrenergic receptor: evidence for alternate mechanisms in G protein-coupled receptors.

In G protein-coupled receptors (GPCRs), a conserved aspartic acid in the DRY motif at the cytoplasmic end of helix 3 regulates the transition to the active state, while the adjacent arginine is crucial for G protein activation. To examine the functions of these two residues, we made D130I and R131Q mutations in the alpha2A adrenergic receptor (AR). We demonstrate that, unlike other GPCRs, the alpha2A AR is not constitutively activated by the D130I mutation, although the mutation increases agonist affinity. While the R131Q mutation severely disrupts function, it decreases rather than increasing agonist affinity as seen in other GPCRs. We then investigated the molecular effects of the same mutations in a peptide model and showed that Arg131 is not required for peptide-mediated G protein activation. These results indicate that the alpha2A AR does not follow the conventional GPCR mechanistic paradigm with respect to the function of the DRY motif.

Adrenergic alpha-Agonists↗

PubMed2002

Receptor-selective effects of endogenous RGS3 and RGS5 to regulate mitogen-activated protein kinase activation in rat vascular smooth muscle cells.

Regulators of G protein signaling (RGS) proteins compose a highly diverse protein family best known for inhibition of G protein signaling by enhancing GTP hydrolysis by Galpha subunits. Little is known about the function of endogenous RGS proteins. In this study, we used synthetic ribozymes targeted to RGS2, RGS3, RGS5, and RGS7 to assess their function. After demonstrating the specificity of in vitro cleavage by the RGS ribozymes, rat aorta smooth muscle cells were used for transient transfection with the RGS-specific ribozymes. RGS3 and RGS5 ribozymes differentially enhanced carbachol- and angiotensin II-induced MAP kinase activity, respectively, whereas RGS2 and RGS7 ribozymes had no effect. This enhancement was pertussis toxin-insensitive. Thus RGS3 is a negative modulator of muscarinic m3 receptor signaling, and RGS5 is a negative modulator of angiotensin AT1a receptor signaling through G(q/11). Also, RGS5 ribozyme enhanced angiotensin-stimulated inositol phosphate release. These results indicate the feasibility of using the ribozyme technology to determine the functional role of endogenous RGS proteins in signaling pathways and to define novel receptor-selective roles of endogenous RGS3 and RGS5 in modulating MAP kinase responses to either carbachol or angiotensin.

Animals↗

PubMed2002

NMR structure of the second intracellular loop of the alpha 2A adrenergic receptor: evidence for a novel cytoplasmic helix.

A major, unresolved question in signal transduction by G protein coupled receptors (GPCRs) is to understand how, at atomic resolution, a GPCR activates a G protein. A step toward answering this question was made with the determination of the high-resolution structure of rhodopsin; we now know the intramolecular interactions that characterize the resting conformation of a GPCR. To what degree does this structure represent a structural paradigm for other GPCRs, especially at the cytoplasmic surface where GPCR-G protein interaction occurs and where the sequence homology is low among GPCRs? To address this question, we performed NMR studies on approximately 35-residue-long peptides including the critical second intracellular loop (i2) of the alpha 2A adrenergic receptor (AR) and of rhodopsin. To stabilize the secondary structure of the peptide termini, 4-12 residues from the adjacent transmembrane helices were included and structures determined in dodecylphosphocholine micelles. We also characterized the effects on an alpha 2A AR peptide of a D130I mutation in the conserved DRY motif. Our results show that in contrast to the L-shaped loop in the i2 of rhodopsin, the i2 of the alpha 2A AR is predominantly helical, supporting the hypothesis that there is structural diversity within GPCR intracellular loops. The D130I mutation subtly modulates the helical structure. The spacing of nonpolar residues in i2 with helical periodicity is a predictor of helical versus loop structure. These data should lead to more accurate models of the intracellular surface of GPCRs and of receptor-mediated G protein activation.

Amino Acid Sequence↗