Wild-type Rho Research Articles

The Binding of C10 Oligomers to Escherichia coli Transcription Termination Factor Rho

The binding of C10 RNA oligomers to wild type and mutant Escherichia coli transcription termination factor Rho provides a model for the enzyme-RNA interactions that lead to transcription termination. One surprising finding is that wild type Rho binds between five and six C10 oligomers per hexamer with KD = 0.3 microm, and five to six additional C10 molecules with KD = 7 microm. Previously, approximately half this number of oligomer-binding sites was reported (Wang, Y., and von Hippel, P. H. (1993) J. Biol. Chem. 268, 13947-13955); however, the E155K mutant form of Rho, thought at the time to be wild type, was used in that work. The present results with E155K Rho agree with the earlier work. C10 binding with mutant forms of Rho that are altered in RNA interactions, bearing amino acid changes F62S, G99V, F232C, T286A, or K352E, indicate that the higher affinity binding sites constitute what has been termed the primary RNA site, and the lower affinity sites constitute the secondary sites. The binding data together with the crystal structures for wild type Rho (Skordalakes, E., and Berger, J. M. (2003) Cell 114, 135-146) support structurally distinct locations on Rho for the two classes of C10-binding sites. The results are consistent with participation of residues 33 A apart in secondary site RNA interactions. The data further indicate that not all RNA sites on Rho must be filled for full ATPase and transcription termination activity, and suggest a model in which RNA binding to the higher affinity sites leads to a protein conformation change that exposes the previously hidden lower affinity sites.

Alsin is a Rab5 and Rac1 guanine nucleotide exchange factor.

ALS2 is the gene mutated in a recessive juvenile form of amyotrophic lateral sclerosis (ALS2). ALS2 encodes a large protein termed alsin, which contains a number of predicted cell signaling and protein trafficking sequence motifs. To gain insight into the overall function of alsin and to begin to evaluate its role in motor neuron maintenance, we examined the subcellular localization of alsin and the biochemical activities associated with its individual subdomains. We found that the Vps9p domain of alsin has Rab5 guanine nucleotide exchange activity. In addition, alsin interacted specifically with and acted as a guanine nucleotide exchange factor for Rac1. Immunofluorescence and fractionation experiments in both fibroblasts and neurons revealed that alsin is a cytosolic protein, with a significant portion associated with small, punctate membrane structures. Many of these membrane structures also contained Rab5 or Rac1. Upon overexpression of full-length alsin, the overexpressed material was largely cytosolic, indicating that the association with membrane structures could be saturated. We also found that alsin was present in membrane ruffles and lamellipodia. These data suggest that alsin is involved in membrane transport events, potentially linking endocytic processes and actin cytoskeleton remodeling.

Bicyclomycin fluorescent probes: synthesis and biochemical, biophysical, and biological properties.

Bicyclomycin (1) is a commercially available antibiotic whose primary site of action in Escherichia coli is the transcription termination factor rho. Key aspects of the 1.rho interaction-K(d), stoichiometry for 1.rho binding, and whether 1 and ATP binding induce conformational changes in rho-remain unknown. In this study, the design, synthesis, and characterization of a series of bicyclomycin fluorescent probes (BFP) constructed to sense the 1.rho interaction are described and their use documented. We show that dihydrobicyclomycins with medium-to-large C(5a)-substituents afforded excellent inhibitory activities exceeding those of 1 in the poly(C)-dependent ATPase assay. The utility of BFP in bicyclomycin-rho binding studies was documented through the use of 5a-(phenazin-2-ylmethylsulfanyl)dihydrobicyclomycin (15). Excitation (290 nm) of W381 in wild-type rho in the presence of 15 and ATP led to fluorescence resonance energy transfer (FRET) and gave a K(d) (15) of 9.9 microM. Using ADP in place of ATP or excluding nucleotide did not result in energy transfer, which suggests that ATP binding induced a conformational change in rho. FRET measurements provided an approximate weighted average distance (23 A) between W381 and 15 in the presence of bound ATP. The K(d) value for 15.rho was correlated with ATP binding at the 3 tight ATP binding (K(d)(ATP) = 95 nM) sites in wild-type rho.

Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA.

The role of RhoA in myosin light-chain (MLC)(20) dephosphorylation and smooth muscle relaxation by PKA and PKG was examined in freshly dispersed and cultured smooth muscle cells expressing wild-type RhoA, constitutively active Rho(V14), and phosphorylation site-deficient Rho(A188). Activators of PKA (5,6-dichloro-1-beta-ribofuranosyl benzimidazole 3',5'-cyclic monophosphothionate, Sp-isomer; cBIMPS) or PKG [8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP), sodium nitroprusside (SNP)] or both PKA and PKG (VIP) induced phosphorylation of constitutively active Rho(V14) and agonist (ACh)- or GTPgammaS-stimulated wild-type RhoA but not Rho(A188). Phosphorylation was accompanied by translocation of membrane-bound wild-type RhoA and Rho(V14) to the cytosol and complete inhibition of ACh-stimulated Rho kinase and phospholipase D activities, RhoA/Rho kinase association, MLC(20) phosphorylation, and sustained muscle contraction. Each of these events was blocked depending on the agent used, by the PKG inhibitor KT5823 or the PKA inhibitor myristoylated PKI. Inhibitors were used at a concentration (1 microM) previously shown by direct measurement of kinase activity to selectively inhibit the corresponding kinase. In muscle cells overexpressing the active phosphorylation site-deficient mutant Rho(A188), MLC(20) phosphorylation was partly inhibited by SNP, VIP, cBIMPS, and 8-pCPT-cGMP, suggesting the existence of an independent inhibitory mechanism downstream of RhoA. Results demonstrate that dephosphorylation of MLC(20) and smooth muscle relaxation are preferentially mediated by PKG- and PKA-dependent phosphorylation and inactivation of RhoA.

ATP Binding to Rho Transcription Termination Factor: MUTANT F355W ATP-INDUCED FLUORESCENCE QUENCHING REVEALS DYNAMIC ATP BINDING

Rho transcription termination factor mutant, F355W, showed tryptophan fluorescence intensity approximately twice that of wild-type Rho at equivalent protein concentrations and underwent a decrease in relative fluorescence intensity at 350 nm when 100 microm ATP was added in the presence or absence of RNA. Titration of this fluorescence quenching with varying concentrations of ATP (0-600 microm), where Rho is shown to exist as a hexamer (400 nm Rho), revealed tight and loose ATP-binding sites. Bicyclomycin, a specific inhibitor of Rho, increased the tight ATP binding and was used to calibrate ATP-induced fluorescence quenching by using [gamma-(32)P]ATP filter binding. For the Rho mutant F355W, three tight (K(d)(1) = 3 +/- 0.3 microm) and three loose (K(d)(2) = 58 +/- 3 microm) ATP-binding sites per hexamer were seen on Scatchard analysis in the absence of bicyclomycin and poly(C). In the presence of bicyclomycin, the K(d)(1) changed from 3.0 to 1.4 microm, but K(d)(2) underwent a lesser change. The non-hydrolyzable ATP analogue, gamma-S-ATP, gave a similar profile with three tight (K(d)(1) = 0.2 microm) and three loose (K(d)(2) = 70 microm) ATP-binding sites per hexamer. Adding poly(C) to F355W did not alter the K(d)(1) or K(d)(2) for ATP or for gamma-S-ATP. ADP-induced quenching produced 5.5 loose (K(d) = 92 microm) binding sites in the absence of poly(C), and the binding became weaker (K(d) = 175 microm) in the presence of poly(C). The data suggest that in the presence of ADP Rho has six equivalent nucleotide-binding sites. When ATP was added these sites converted to three tight and three loose binding loci. We propose an alternating ATP site mechanism where ATP binding creates heterogeneity in the ATP binding in adjacent subunits, and we suggest that ATP binding to a neighboring loose site stimulates hydrolysis at a neighboring tight binding site such that all six subunits can be potential "active" sites for ATP hydrolysis. The dynamic nature of the ATP binding to Rho is discussed in the terms of the mechanism of RNA tracking driven by ATP hydrolysis.

Mutations in the Rho Transcription Termination Factor That Affect RNA Tracking

Model studies have identified 16 conserved positively charged amino acids that form a positive strip pointing toward the center hole of Rho. Fourteen residues were individually changed to either an alanine or a glycine and one to a glutamate. Residues Arg(269), Arg(272), Lys(283), Arg(296), Lys(298), and Arg(299) form a subdomain (locus) located N-terminal to (above) the ATP hydrolysis domain (P-loop) and mutations in these residues led to either inactive Rho or to proteins displaying decreased k(cat) for poly(C)-dependent ATP hydrolysis, increased K(m) for ribo(C)(10) activation, and decreased transcription termination efficiencies (57-77%) compared with wild-type Rho. Residues Arg(347), Lys(348), Lys(352), and Arg(353) form a subdomain (locus) C-terminal to (below) the ATP hydrolysis domain, and mutations in these residues also show a decreased k(cat) for poly(C)-dependent ATP hydrolysis, an increased K(m) for ribo(C)(10) activation, and a 50-70% decrease in transcription termination, compared with wild-type Rho. Residues Arg(212) and Lys(336) surround the ATP hydrolysis domain, and mutations in these residues also altered the kinetic properties of Rho. We conclude that the secondary RNA-tracking site consists of amino acids whose putative orientation faces the central hole in Rho and in part reside in two clusters of positively charged residues located above and below the ATP hydrolysis domain.

Involvement of Rho GTPases and Their Effectors in the Secretory Process of PC12 Cells

We investigated the involvement of Rho GTPases in the secretory process of PC12 cells. Overexpression of wild-type RhoA, Rac1, or Cdc42 did affect exocytosis. In contrast, secretion elicited by depolarizing K+ concentrations was enhanced by the dominant negative mutants RhoAN19, Rac1N17, and Cdc42N17 and was diminished by the constitutively active mutants RhoAV14, Rac1V12, and Cdc42V12. The inhibition observed in the presence of RhoAV14 was likely a result of the activation of ROKα, since the catalytic domain of this kinase was able to mimic both the reorganization of the actin cytoskeleton and the decrease in exocytosis induced by the RhoA mutant. Part of the effect of Rac1V12 may be due to POR1 activation. Thus, overexpression of full-length POR1 diminished K+-stimulated exocytosis, and a point mutation in the effector domain of Rac1V12 that prevents the interaction with POR1 abolished the inhibitory effect of the GTPase. We also searched for the Cdc42V12 target but overexpression of the Cdc42 effector WASP did not mimic the inhibition of exocytosis observed in cells transfected with the activated GTPase. Our findings indicate that different signaling cascades resulting in the activation of RhoA, Rac1, or Cdc42 can modulate the exocytotic process of neuroendocrine cells.

Rho-dependent Agonist-induced Spatio-temporal Change in Myosin Phosphorylation in Smooth Muscle Cells

Agonist-induced translocation of RhoA and the spatio-temporal change in myosin regulatory light chain (MLC20) phosphorylation in smooth muscle was clarified at the single cell level. We expressed green fluorescent protein-tagged RhoA in the differentiated tracheal smooth muscle cells and visualized the translocation of RhoA in a living cell with three-dimensional digital imaging analysis. The stimulation of the cells by carbachol initiated the translocation of green fluorescent protein-tagged wild type RhoA to the plasma membrane within a minute. The change in MLC20 phosphorylation level after carbachol stimulation was monitored by using phospho-Ser-19-specific antibody recognizing the phosphorylated MLC20 in single cells. Cells expressing the dominant negative form (T19N) of RhoA significantly suppressed sustained MLC20 phosphorylation during the prolonged phase (>300 s), whereas the maximum phosphorylation level (reached at 10 s after stimulation) of these cells was not significantly different from the control cells. The kinetics of RhoA translocation was consistent with that of sustained myosin phosphorylation, suggesting the involvement of a RhoA pathway. Carbachol stimulation increased myosin phosphorylation within a minute both at the cortical and the central region. On the other hand, during prolonged phase, myosin phosphorylation was sustained at the cortical region of the cells but not at the central fibers. A myosin light chain kinase-specific inhibitor, ML-9, diminished myosin phosphorylation at the central region of the cells after the stimulation but not at the cortical area. On the other hand, Y-27632, a Rho kinase-specific inhibitor, diminished myosin phosphorylation at the cortical region but not the central region. The results clearly show that the myosin light chain kinase pathway and the Rho pathway distinctly change myosin phosphorylation in smooth muscle cells in both a temporal and spatial manner.

Na,K-ATPase activity is required for formation of tight junctions, desmosomes, and induction of polarity in epithelial cells.

Na,K-ATPase is a key enzyme that regulates a variety of transport functions in epithelial cells. In this study, we demonstrate a role for Na,K-ATPase in the formation of tight junctions, desmosomes, and epithelial polarity with the use of the calcium switch model in Madin-Darby canine kidney cells. Inhibition of Na,K-ATPase either by ouabain or potassium depletion prevented the formation of tight junctions and desmosomes and the cells remained nonpolarized. The formation of bundled stress fibers that appeared transiently in control cells was largely inhibited in ouabain-treated or potassium-depleted cells. Failure to form stress fibers correlated with a large reduction of RhoA GTPase activity in Na,K-ATPase-inhibited cells. In cells overexpressing wild-type RhoA GTPase, Na,K-ATPase inhibition did not affect the formation of stress fibers, tight junctions, or desmosomes, and epithelial polarity developed normally, suggesting that RhoA GTPase is an essential component downstream of Na,K-ATPase-mediated regulation of these junctions. The effects of Na,K-ATPase inhibition were mimicked by treatment with the sodium ionophore gramicidin and were correlated with the increased intracellular sodium levels. Furthermore, ouabain treatment under sodium-free condition did not affect the formation of junctions and epithelial polarity, suggesting that the intracellular Na(+) homeostasis plays a crucial role in generation of the polarized phenotype of epithelial cells. These results thus demonstrate that the Na,K-ATPase activity plays an important role in regulating both the structure and function of polarized epithelial cells.

Interactions between Rho GTPases and Rho GDP dissociation inhibitor (Rho-GDI)

The Rho-GDP dissociation inhibitor (Rho-GDI) was used as bait in a two-hybrid screen of a human leucocyte cDNA library. Most of the isolated cDNAs encoded GTPases of the Rho subfamily: RhoA, B, C, Rac1, 2, CDC42 and RhoG. The newly discovered RhoH interacted very poorly with Rho-GDI. Another protein partner shared a homology with RhoA that points to Asp67 RhoA-Arg68 RhoA-Leu69 RhoA as critical for interaction with Rho-GDI. A second screen with RhoA as bait led to the isolation of GDI only. In order to investigate the relative role of protein-protein and protein-lipid interactions between Rho GTPases and Rho-GDI, CAAX box mutants of RhoA were produced. They were found to interact with Rho-GDI as efficiently as wild type RhoA, indicating that protein-protein interactions alone lead to strong binding of the two proteins. The C-terminal polybasic region of RhoA was also shown to be a site of protein-protein interaction with Rho-GDI.

Expression of gamma -aminobutyric acid rho 1 and rho 1Delta 450 as gene fusions with the green fluorescent protein

The functional characteristics and cellular localization of the gamma aminobutyric acid (GABA) rho 1 receptor and its nonfunctional isoform rho 1 Delta 450 were investigated by expressing them as gene fusions with the enhanced version of the green fluorescent protein (GFP). Oocytes injected with rho 1-GFP had receptors that gated chloride channels when activated by GABA. The functional characteristics of these receptors were the same as for those of wild-type rho 1 receptors. Fluorescence, because of the chimeric receptors expressed, was over the whole oocyte but was more intense near the cell surface and more abundant in the animal hemisphere. Similar to the wild type, rho 1 Delta 450-GFP did not lead to the expression of functional GABA receptors, and injected oocytes failed to generate currents even after exposure to high concentrations of GABA. Nonetheless, the fluorescence displayed by oocytes expressing rho 1 Delta 450-GFP was distributed similarly to that of rho 1-GFP. Mammalian cells transfected with the rho 1-GFP or rho 1 Delta 450-GFP constructs showed mostly intracellularly distributed fluorescence in confocal microscope images. A sparse localization of fluorescence was observed in the plasma membrane regardless of the cell line used. We conclude that rho 1 Delta 450 is expressed and transported close to, and perhaps incorporated into, the plasma membrane. Thus, rho 1- and rho 1 Delta 450-GFP fusions provide a powerful tool to visualize the traffic of GABA type C receptors.

5a-formylbicyclomycin: studies on the bicyclomycin-Rho interaction.

Bicyclomycin (1) is a commercial antibiotic whose primary site of action is the rho transcription termination factor. A new bicyclomycin irreversible inactivator, 5a-formylbicyclomycin (3), was prepared to provide information concerning the bicyclomycin-rho inactivation process and the drug's binding pocket within rho. The apparent I(50) value for 3 was 35 microM, showing that 3 was a more effective inhibitor of rho poly C-dependent ATPase activity than 1 (I(50) = 60 microM). Mechanistic studies demonstrated that 3 inhibited poly C-dependent ATP hydrolysis, in part, by a reversible, noncompetitive pathway with respect to ATP (K(i) = 62 microM). Incubation of 3 with rho led to efficient imine formation. Adding excess 1 to solutions containing 3 and rho prevented imine formation, demonstrating that 1 and 3 bind to the same active site in the protein. The 3-rho imine was stabilized by either ATP or ADP or by both, and was converted to the nonreversible 3-rho amine adduct upon treatment with NaBH(4). Mass spectrometric analysis of the amine provided a stoichiometry of approximately five bound 3 per rho hexamer indicating the number of bicyclomycin binding sites for the rho hexamer is between five and six. Monomer exchange experiments using modified 3-rho amine and wild type rho demonstrated that no more than two modified subunits per rho hexamer are sufficient to halt poly C-dependent rho ATPase activity.

Unique in Vivo Associations with SmgGDS and RhoGDI and Different Guanine Nucleotide Exchange Activities Exhibited by RhoA, Dominant Negative RhoAAsn-19, and Activated RhoAVal-14

We compared the in vivo characteristics of hemagglutinin (HA)-tagged RhoA, dominant negative RhoA(Asn-19), and activated RhoA(Val-14) stably expressed in Chinese hamster ovary (CHO) cells. Proteins co-precipitating with these HA-tagged GTPases were identified by peptide sequencing or by Western blotting. Dominant negative RhoA(Asn-19) co-precipitates with the guanine nucleotide exchange factor (GEF) SmgGDS but does not detectably interact with other expressed GEFs, such as Ost or Dbl. SmgGDS co-precipitates minimally with wild-type RhoA and does not detectably associate with RhoA(Val-14). The guanine nucleotide dissociation inhibitor RhoGDI co-precipitates with RhoA, and to a lesser extent with RhoA(Val-14), but does not detectably co-precipitate with RhoA(Asn-19). Wild-type RhoA is predominantly in the [(32)P]GDP-bound form, RhoA(Val-14) is predominantly in the [(32)P]GTP-bound form, and negligible levels of [(32)P]GDP or [(32)P]GTP are bound to RhoA(Asn-19) in (32)P-labeled cells. Immunofluorescence analyses indicate that HA-RhoA(Asn-19) is excluded from the nucleus and cell junctions. Microinjection of SmgGDS cDNA into CHO cells stably expressing HA-RhoA causes HA-RhoA to be excluded from the nucleus and cell junctions, similar to the distribution of RhoA(Asn-19). Our findings indicate that the expression of RhoA(Asn-19) may specifically inhibit signaling pathways that rely upon the SmgGDS-dependent activation of RhoA.

Overexpression of wild-type RhoA produces growth arrest by disrupting actin cytoskeleton and microtubules.

We have investigated the role of Rho GTPase in cell growth by generating stable cells that express the wild-type RhoA (RhoA(wt)) under the control of an inducible promoter. Induction of RhoA(wt) had a biphasic effect on the actin cytoskeleton. At low levels of expression, RhoA(wt) stimulated the assembly of actin stress fibers without affecting cell growth. At high levels, there was a paradoxical disruption of the actin cytoskeleton accompanied by a growth arrest. Cell cycle analysis revealed a dual block at the G(1)/S and G(2)/M checkpoints. The G(1)/S arrest correlated with the accumulation of p21(Cip1), resulting in the inhibition of cdk2 activity, whereas the G(2)/M block correlated with the loss of microtubules. The cyclin B level and the cdc2 kinase activity, however, were increased, suggesting that the progression through mitosis rather than entry into the G(2)/M is defective when RhoA(wt) is overexpressed. Similar cell cycle defects and the loss of microtubules were observed after a cytochalasin D treatment, indicating that the ability of RhoA to regulate the integrity of actin cytoskeleton may be critical for the cell cycle transition through both the G(1)/S and M phase checkpoints.

Activated Rac1 Selectively Up-regulates the Expression of Integrin α6β4 and Induces Cell Adhesion and Membrane Ruffles of Nonadherent Colon Cancer Colo201 Cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin β4 dependent since an antibody for integrin β4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin α6β4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of α6β4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated α6β4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin α6β4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.

Modulation of endocytic traffic in polarized Madin-Darby canine kidney cells by the small GTPase RhoA.

Efficient postendocytic membrane traffic in polarized epithelial cells is thought to be regulated in part by the actin cytoskeleton. RhoA modulates assemblies of actin in the cell, and it has been shown to regulate pinocytosis and phagocytosis; however, its effects on postendocytic traffic are largely unexplored. To this end, we expressed wild-type RhoA (RhoAWT), dominant active RhoA (RhoAV14), and dominant inactive RhoA (RhoAN19) in Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. RhoAV14 expression stimulated the rate of apical and basolateral endocytosis, whereas RhoAN19 expression decreased the rate from both membrane domains. Polarized basolateral recycling of transferrin was disrupted in RhoAV14-expressing cells as a result of increased ligand release at the apical pole of the cell. Degradation of basolaterally internalized epidermal growth factor was slowed in RhoAV14-expressing cells. Although apical recycling of immunoglobulin A (IgA) was largely unaffected in cells expressing RhoAV14, transcytosis of basolaterally internalized IgA was severely impaired. Morphological and biochemical analyses demonstrated that a large proportion of IgA internalized from the basolateral pole of RhoAV14-expressing cells remained within basolateral early endosomes and was slow to exit these compartments. RhoAN19 and RhoAWT expression had little effect on these postendocytic pathways. These results indicate that in polarized MDCK cells activated RhoA may modulate endocytosis from both membrane domains and postendocytic traffic at the basolateral pole of the cell.

Cardiac-specific overexpression of RhoA results in sinus and atrioventricular nodal dysfunction and contractile failure.

RhoA is a low-molecular-weight GTPase that has been implicated in the regulation of hypertrophic cardiac muscle cell growth. To study the role of RhoA in control of cardiac function in vivo, transgenic mice expressing wild-type and constitutively activated forms of RhoA under the control of the cardiac-specific alpha-myosin heavy chain promoter were generated. Transgene-positive mice expressing high levels of either wild-type or activated RhoA showed pronounced atrial enlargement and manifested a lethal phenotype, often preceded by generalized edema, with most animals dying over the course of a few weeks. Echocardiographic analysis of visibly healthy wild-type RhoA transgenic mice revealed no significant change in left ventricular function. As their condition deteriorated, significant dilation of the left ventricular chamber and associated decreases in left ventricular contractility were detected. Heart rate was grossly depressed in both wild-type and activated RhoA-expressing mice, even prior to the onset of ventricular failure. Electrocardiography showed evidence of atrial fibrillation and atrioventricular block. Interestingly, muscarinic receptor blockade with atropine did not elicit a positive chronotropic response in the transgenic mice. We suggest that RhoA regulates cardiac sinus and atrioventricular nodal function and that its overexpression results in bradycardia and development of ventricular failure.

A single amino acid confers barbiturate sensitivity upon the GABA rho 1 receptor.

Many structurally diverse general anaesthetics enhance inhibitory neurotransmission in the central nervous system by interacting with the GABAA receptor. By contrast, GABA receptors composed of the rho 1 subunit are anaesthetic-insensitive. Here, we demonstrate that both delta-hexachlorocyclohexane (delta-HCH; 1-100 microM), a positive allosteric modulator of the GABAA receptor, and the anaesthetic pentobarbitone (10-600 microM) have no effect on GABA-evoked currents mediated by wild-type rho 1 recombinant receptors (expressed in Xenopus laevis oocytes). By contrast, these agents produce up to a 10 fold enhancement of GABA responses transduced by a rho 1 receptor in which a transmembrane located isoleucine residue is replaced by serine. However, not all general anaesthetics were similarly influenced by this mutation, because propofol and 5 beta-pregnan-3 alpha-ol-20-one (5 beta 3 alpha) remained ineffective. These data are discussed in relation to the specificity of general anaesthetic action.

Identifying the Bicyclomycin Binding Domain through Biochemical Analysis of Antibiotic-resistant Rho Proteins

Mutations M219K, S266A, and G337S in transcription termination factor Rho have been shown to confer resistance to the antibiotic bicyclomycin (BCM). All three His-tagged mutant Rho proteins exhibited similar Km values for ATP; however, the Vmax values at infinite ATP concentrations were one-fourth to one-third that for the His-tagged wild-type enzyme. BCM inhibition kinetics of poly(C)-dependent ATPase activity for the mutant proteins were non-competitive with respect to ATP (altering catalytic function but not ATP binding) and showed increased Ki values compared with His-tagged wild-type Rho. M219K and G337S exhibited increased ratios of poly(U)/poly(C)-stimulated ATPase activity and lower apparent Km values for ribo(C)10 in the poly(dC).ribo(C)10-dependent ATPase assay compared with His-tagged wild-type Rho. The S266A mutation did not show an increased poly(U)/poly(C) ATPase activity ratio and maintained approximately the same Km for ribo(C)10 in the poly(dC). ribo(C)10-dependent ATPase assay. The kinetic studies indicated that M219K and G337S altered the secondary RNA binding domain in Rho whereas the S266A mutation did not. Transcription termination assays for each mutant showed different patterns of Rho-terminated transcripts. Tyrosine substitution of Ser-266 led to BCM sensitivity intimating that an OH (hydroxyl) moiety at this position is needed for BCM (binding) inhibition. Our results suggest BCM binds to Rho at a site distinct from both the ATP and the primary RNA binding domains but close to the secondary RNA-binding (tracking) site and the ATP hydrolysis pocket.

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

The commercial antibiotic bicyclomycin (Bcm) has been shown to target the essential transcription termination factor Rho in Escherichia coli. Little is known about the Bcm binding domain in Rho. A recent structure-activity relationship study led us to evaluate the reductive amination probe, 5a-(3-formylanilino)dihydrobicyclomycin (FD-Bcm). Biochemical studies showed that FD-Bcm possessed inhibitory activities comparable to Bcm in Rho-dependent ATPase and transcription termination assays. Incubation of Rho with FD-Bcm, ATP, and poly(C) followed by NaBH4 reduction and dialysis led to an appreciable loss of ATPase activity. Inclusion of Bcm with FD-Bcm in the reductive amination reaction protected Rho, indicating that Bcm and FD-Bcm competed for the same binding site in Rho. Incubation of Rho with FD-Bcm and poly(C) followed by NaBH4 reduction provided a sample with residual ATPase activity (12%). Mass spectrometric analysis indicated the presence of two proteins in an approximate 1.2:1 ratio, whose masses corresponded to wild-type Rho (47,010 Da) and lysine-modified Rho (47,417 Da), respectively. Trypsin digestion of the Rho sample followed by high performance liquid chromatography separation and tandem mass spectrometry analysis identified the site of modification as Lys181 within the combined tryptic fragment, Gly-Leu-Ile-Val-Ala-Pro-Pro-Lys-Ala-Gly-Lys (residues 174-184). Similar analysis of a lesser modified sample (following incubation with inclusion of ATP) showed that addition had again occurred at Lys181. These findings provide the first structural information concerning the site of Bcm binding in Rho.