Dendritic spine morphology determines membrane-associated protein exchange between dendritic shafts and spine heads.

The purpose of this study was to examine whether variability in the shape of dendritic spines affects protein movement within the plasma membrane. Using a combination of confocal microscopy and the fluorescence loss in photobleaching technique in living hippocampal CA1 pyramidal neurons expressing membrane-linked GFP, we observed a clear correlation between spine shape parameters and the diffusion and compartmentalization of membrane-associated proteins. The kinetics of membrane-linked GFP exchange between the dendritic shaft and the spine head compartment were slower in dendritic spines with long necks and/or large heads than in those with short necks and/or small heads. Furthermore, when the spine area was reduced by eliciting epileptiform activity, the kinetics of protein exchange between the spine compartments exhibited a concomitant decrease. As synaptic plasticity is considered to involve the dynamic flux by lateral diffusion of membrane-bound proteins into and out of the synapse, our data suggest that spine shape represents an important parameter in the susceptibility of synapses to undergo plastic change.

Investigating the Role of cAMP-Signaling in the Regulation of Vascular Endothelial Cell Adaptive Responses to Fluid Shear Stress

The circulating blood exerts a force on the vascular endothelium, termed fluid shear stress (FSS), which directly impacts numerous vascular endothelial cell (VEC) functions. For example, high rates of linear and undisturbed (i.e. laminar) blood flow maintains a protective and quiescent VEC phenotype. Meanwhile, deviations in blood flow, which can occur at vascular branchpoints and large curvatures, create areas of low, and/or oscillatory FSS, and promote a pro-inflammatory, pro-thrombotic and hyperpermeable phenotype. Indeed, it is known that these areas are prone to the development of atherosclerotic lesions. Herein, we show that cyclic nucleotide phosphodiesterase (PDE) 4D (PDE4D) activity is increased by FSS in human arterial endothelial cells (HAECs) and that this activation regulates the activity of cAMP-effector protein, Exchange Protein-activated by cAMP-1 (EPAC1), in these cells. Importantly, we also show that these events directly and critically impact HAEC responses to FSS, especially when FSS levels are low. Both morphological events induced by FSS, as measured by changes in cell alignment and elongation in the direction of FSS, and the expression of critical FSS-regulated genes, including Krüppel-like factor 2 (KLF2), endothelial nitric oxide synthase (eNOS) and thrombomodlin (TM), are mediated by EPAC1/PDE4D signaling. At a mechanistic level, we show that EPAC1/PDE4D acts through the vascular endothelial-cadherin (VECAD)/ platelet-cell adhesion molecule-1 (PECAM1)/vascular endothelial growth factor receptor 2 (VEGFR2) mechanosensor to activate downstream signaling though Akt. Given the critical role of PDE4D in mediating these effects, we also investigated the impact of various patterns of FSS on the expression of individual PDE genes in HAECs. Notably, PDE2A was significantly upregulated in response to high, laminar FSS, while PDE3A was upregulated under low, oscillatory FSS conditions only. These data may provide novel therapeutic targets to limit FSS-dependent endothelial cell dysfunction (ECD) and atherosclerotic development.

Ric-8A, a G alpha protein guanine nucleotide exchange factor potentiates taste receptor signaling

Taste receptors for sweet, bitter and umami tastants are G-protein-coupled receptors (GPCRs). While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS), RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs) are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of G alpha subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with G alpha-gustducin and G alpha i2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

Protein interaction data curation: the International Molecular Exchange (IMEx) consortium.

The International Molecular Exchange (IMEx) consortium is an international collaboration between major public interaction data providers to share literature-curation efforts and make a nonredundant set of protein interactions available in a single search interface on a common website (http://www.imexconsortium.org/). Common curation rules have been developed, and a central registry is used to manage the selection of articles to enter into the dataset. We discuss the advantages of such a service to the user, our quality-control measures and our data-distribution practices.

Insight into the strand exchange reaction promoted by the RecA protein of {\it Escherichia coli\/}

In vitro, RecA protein catalyses the exchange of single strands of DNA between different DNA molecules with sequence complementarity. In order to gain insight into this complex reaction and the roles of ATP binding and hydrolysis, two different approaches have been taken. The first is to use short single-stranded deoxyoligonucleotides as the ssDNA in strand exchange. These were used to determine the signal for hydrolysis and the structure of the RecA-DNA complex that hydrolyses ATP. I present a defined kinetic analysis of the nucleotide triphosphatase activity of RecA protein using short oligonucleotides as ssDNA cofactor. I compare the effects of both homopolymers and mixed base composition oligomers on the ATPase activity of RecA protein. I examine the steady state kinetic parameters of the ATPase reaction using these oligonucleotides as ssDNA cofactor, and show that although RecA can both bind to, and utilise, oligonucleotides 7 to 20 residues in length to support the repressor cleavage activity of RecA, these oligonucleotides are unable to efficiently stimulate the ATPase activity of RecA protein. I show that the K$\sb{\rm m}\sp{\rm ATP}$, the Hill coefficient for ATP binding, the extent of reaction, and k$\sb{\rm cat}$ are all a function of ssDNA chain length and that secondary structure may also play a role in determining the effects of a particular chain length on the ATPase activity of RecA protein.^ The second approach is to utilise one of the many mutants of RecA to gain insight into this complex reaction. The mutant selected was RecA1332. Surprisingly, in vitro, this mutant possesses a DNA-dependent ATPase activity. The K$\sb{\rm m}\sp{\rm ATP}$, Hill coefficient for ATP binding, and K$\sb{\rm m}\sp{\rm DNA}$ are similar to that of wild type. k$\sb{\rm cat}$ for the ATPase activity is reduced 3 to 12-fold, however. RecA1332 is unable to use deoxyoligonucleotides as DNA cofactors in the ATPase reaction, and demonstrates an increased sensitivity to inhibition by monovalent ions. It is able to perform strand exchange with ATP and ATP$\lbrack\gamma\rbrack$S but not with UTP, whereas the wild type protein is able to use all three nucleotide triphosphates. RecA1332 appears to be slowed in its ability to form intermediates and to convert these intermediates to products. (Abstract shortened by UMI.) ^

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales, Phaeophyceae) under variable pH

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO3-) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO3- by the surface-bound enzyme carbonic anhydrase (CAext). Here, we examined other putative HCO3- uptake mechanisms in M. pyrifera under pHT 9.00 (HCO3-: CO2 = 940:1) and pHT 7.65 (HCO3-: CO2 = 51:1). Rates of photosynthesis, and internal CA (CAint) and CAext activity were measured following the application of AZ which inhibits CAext, and DIDS which inhibits a different HCO3- uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO3- uptake by M. pyrifera is via an AE protein, regardless of the HCO3-: CO2 ratio, with CAext making little contribution. Inhibiting the AE protein led to a 55%-65% decrease in photosynthetic rates. Inhibiting both the AE protein and CAext at pHT 9.00 led to 80%-100% inhibition of photosynthesis, whereas at pHT 7.65, passive CO2 diffusion supported 33% of photosynthesis. CAint was active at pHT 7.65 and 9.00, and activity was always higher than CAext, because of its role in dehydrating HCO3- to supply CO2 to RuBisCO. Interestingly, the main mechanism of HCO3- uptake in M. pyrifera was different than that in other Laminariales studied (CAext-catalyzed reaction) and we suggest that species-specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO3-:CO2 due to ocean acidification.

Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein α subunit Gα13

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein α subunits were characterized in transfection systems. Gαq, Gα12, and Gα13, but not Gαi, activate SRF through RhoA. When Gαq, α12, or α13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Gα13, but not Gαq or Gα12, showed synergistic activation of SRF with GEF115. The synergy between Gα13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between Gα13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited Gα13- and Gα12-induced, but not GEF115 itself- or Gαq-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Gα12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Gα12 and Gα13. Thus, the inhibition of Gα12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Gα13 and GEF115 indicates that GEF115 mediates Gα13-induced activation of Rho and SRF.

Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain

Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor (ARF)-bound GDP for GTP by a Golgi-associated guanine nucleotide-exchange protein (GEP) [Helms, J. B. & Rothman, J. E. (1992) Nature (London) 360, 352–354; Donaldson, J. G., Finazzi, D. & Klausner, R. D. (1992) Nature (London) 360, 350–352]. Cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, it was no longer BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 305–309]. We describe here purification from bovine brain cytosol of a BFA-inhibited GEP. After chromatography on DEAE–Sephacel, hydroxylapatite, and Mono Q and precipitation at pH 5.8, GEP was eluted from Superose 6 as a large molecular weight complex at the position of thyroglobulin (≈670 kDa). After SDS/PAGE of samples from column fractions, silver-stained protein bands of ≈190 and 200 kDa correlated with activity. BFA-inhibited GEP activity of the 200-kDa protein was demonstrated following electroelution from the gel and renaturation by dialysis. Four tryptic peptides from the 200-kDa protein had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae (total of 51 amino acids), consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7 that plays a similar role in cellular vesicular transport and Sec7 may be a GEP for one or more yeast ARFs.

Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor

A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from Saccharomyces cerevisiae, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (≈208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a ≈7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.

Remodeling of the Actin Cytoskeleton Is Coordinately Regulated by Protein Kinase C and the ADP-Ribosylation Factor Nucleotide Exchange Factor ARNO

ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.

The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H+ and Na+(K+)/H+ exchange

Recent work has suggested that the chromosomally encoded TetA(L) transporter of Bacillus subtilis, for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both Na+- and K+-dependent pH homeostasis and in Na+ resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)–hexahistidine proteoliposomes exhibit high activities of tetracycline–cobalt/H+, Na+/H+, and K+/H+ antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt and vice versa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline–cobalt/H+ and Na+/H+ antiports is presented. K+ and Li+ inhibit Na+ uptake, but there is little cross-inhibition between Na+ and tetracycline–cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.

Binding site of brefeldin A at the interface between the small G protein ADP-ribosylation factor 1 (ARF1) and the nucleotide-exchange factor Sec7 domain

Sec7 domains (Sec7d) catalyze the exchange of guanine nucleotide on ARFs. Recent studies indicated that brefeldin A (BFA) inhibits Sec7d-catalyzed nucleotide exchange on ARF1 in an uncompetitive manner by trapping an early intermediate of the reaction: a complex between GDP-bound ARF1 and Sec7d. Using 3H-labeled BFA, we show that BFA binds to neither isolated Sec7d nor isolated ARF1–GDP, but binds to the transitory Sec7d–ARF1–GDP complex and stabilizes it. Two pairs of residues at positions 190–191 and 198–208 (Arno numbering) in Sec7d contribute equally to the stability of BFA binding, which is also sensitive to mutation of H80 in ARF1. The catalytic glutamic (E156) residue of Sec7d is not necessary for BFA binding. In contrast, BFA does not bind to the intermediate catalytic complex between nucleotide-free ARF1 and Sec7d. These results suggest that, on initial docking steps between ARF1–GDP and Sec7d, BFA inserts like a wedge between the switch II region of ARF1–GDP and a surface encompassing residues 190–208, at the border of the characteristic hydrophobic groove of Sec7d. Bound BFA would prevent the switch regions of ARF1–GDP from reorganizing and forming tighter contacts with Sec7d and thereby would maintain the bound GDP of ARF1 at a distance from the catalytic glutamic finger of Sec7d.

ARF-GEP100, a guanine nucleotide-exchange protein for ADP-ribosylation factor 6

A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP100, which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a ≈8-kb mRNA that hybridized with an ARF-GEP100 cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP100 accelerated [35S]GTPγS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP100 Sec7 domain contains Asp543 and Met555, corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe535 and Ala536, associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP100 activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP100, a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP100 in those areas. No similar coincidence of ARF-GEP100 with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.

Role of Rab3 GDP/GTP Exchange Protein in Synaptic Vesicle Trafficking at the Mouse Neuromuscular Junction

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca2+-dependent neurotransmitter release. These small G proteins are activated by Rab3 GDP/GTP exchange protein (Rab3 GEP). To determine the function of Rab3 GEP during neurotransmitter release, we have knocked out Rab3 GEP in mice. Rab3 GEP−/− mice developed normally but died immediately after birth. Embryos at E18.5 showed no evoked action potentials of the diaphragm and gastrocnemius muscles in response to electrical stimulation of the phrenic and sciatic nerves, respectively. In contrast, axonal conduction of the spinal cord and the phrenic nerve was not impaired. Total numbers of synaptic vesicles, especially those docked at the presynaptic plasma membrane, were reduced at the neuromuscular junction ∼10-fold compared with controls, whereas postsynaptic structures and functions appeared normal. Thus, Rab3 GEP is essential for neurotransmitter release and probably for formation and trafficking of the synaptic vesicles.

Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchange

Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. As previously shown, Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Here we demonstrate that Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depended on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins.