Molecular characterization of the GTPase-activating domain of ADP-ribosylation factor domain protein 1 (ARD1)

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins recognized as critical components in intracellular vesicular transport and phospholipase D activation. Both guanine nucleotide-exchange proteins and GTPase-activating proteins (GAPs) for ARFs have been cloned recently. A zinc finger motif near the amino terminus of the ARF1 GAP was required for stimulation of GTP hydrolysis. ARD1 is an ARF family member that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We had reported that the ARF domain of ARD1 binds specifically GDP and GTP and that the amino-terminal extension acts as a GAP for the ARF domain of ARD1 but not for ARF proteins. The GAP domain of ARD1, synthesized in Escherichia coli, stimulated hydrolysis of GTP bound to the ARF domain of ARD1. Using ARD1 truncations, it appears that amino acids 101-190 are critical for GAP activity, whereas residues 190-333 are involved in physical interaction between the two domains of ARD1 and are required for GTP hydrolysis. The GAP function of the amino-terminal extension of ARD1 required two arginines, an intact zinc finger motif, and a group of residues which resembles a sequence present in Rho/Rac GAPs. Interaction between the two domains of ARD1 required two negatively charged residues (Asp427 and Glu428) located in the effector region of the ARF domain and two basic amino acids (Arg249 and Lys250) found in the amino-terminal extension. The GAP domain of ARD1 thus is similar to ARF GAPs but differs from other GAPs in its covalent association with the GTP-binding domain.     

ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP ribosylation factor 1, is involved in the control of Golgi structure and function

Budding of transport vesicles in the Golgi apparatus requires the recruitment of coat proteins and is regulated by ADP ribosylation factor (ARF) 1. ARF1 activation is promoted by guanine nucleotide exchange factors (GEFs), which catalyze the transition to GTP-bound ARF1. We recently have identified a human protein, ARNO (ARF nucleotide-binding-site opener), as an ARF1-GEF that shares a conserved domain with the yeast Sec7 protein. We now describe a human Sec7 domain-containing GEF referred to as ARNO3. ARNO and ARNO3, as well as a third GEF called cytohesin-1, form a family of highly related proteins with identical structural organization that consists of a central Sec7 domain and a carboxy-terminal pleckstrin homology domain. We show that all three proteins act as ARF1 GEF in vitro, whereas they have no effect on ARF6, an ARF protein implicated in the early endocytic pathway. Substrate specificity of ARNO-like GEFs for ARF1 depends solely on the Sec7 domain. Overexpression of ARNO3 in mammalian cells results in (i) fragmentation of the Golgi apparatus, (ii) redistribution of Golgi resident proteins as well as the coat component beta-COP, and (iii) inhibition of SEAP transport (secreted form of alkaline phosphatase). In contrast, the distribution of endocytic markers is not affected. This study indicates that Sec7 domain-containing GEFs control intracellular membrane compartment structure and function through the regulation of specific ARF proteins in mammalian cells.     

The role of ADP-ribosylation factor and phospholipase D in adaptor recruitment

AP-1 and AP-2 adaptors are recruited onto the TGN and plasma membrane, respectively. GTPgammaS stimulates the recruitment of AP-1 onto the TGN but causes AP-2 to bind to an endosomal compartment (Seaman, M.N.J., C.L. Ball, and M.S. Robinson. 1993. J. Cell Biol. 123:1093-1105). We have used subcellular fractionation followed by Western blotting, as well as immunofluorescence and immunogold electron microscopy, to investigate both the recruitment of AP-2 adaptors onto the plasma membrane and their targeting to endosomes, and we have also examined the recruitment of AP-1 under the same conditions. Two lines of evidence indicate that the GTPgammaS-induced targeting of AP-2 to endosomes is mediated by ADP-ribosylation factor-1 (ARF1). First, GTPgammaS loses its effect when added to ARF-depleted cytosol, but this effect is restored by the addition of recombinant myristoylated ARF1. Second, adding constitutively active Q71L ARF1 to the cytosol has the same effect as adding GTPgammaS. The endosomal membranes that recruit AP-2 adaptors have little ARF1 or any of the other ARFs associated with them, suggesting that ARF may be acting catalytically. The ARFs have been shown to activate phospholipase D (PLD), and we find that addition of exogenous PLD has the same effect as GTPgammaS or Q71L ARF1. Neomycin, which inhibits endogenous PLD by binding to its cofactor phosphatidylinositol 4,5-bisphosphate, prevents the recruitment of AP-2 not only onto endosomes but also onto the plasma membrane, suggesting that both events are mediated by PLD. Surprisingly, however, neither PLD nor neomycin has any effect on the recruitment of AP-1 adaptors onto the TGN, even though AP-1 recruitment is ARF mediated. These results indicate that different mechanisms are used for the recruitment of AP-1 and AP-2.     

Properties of ATP receptor-mediated synaptic transmission in the rat medial habenula

The properties of central ATP-mediated synaptic currents were studied using whole-cell patch-clamp recording in rat medial habenula slices. Release was shown to be calcium dependent with a Hill coefficient of approximately 2. The voltage dependence of synaptic current amplitudes was approximately linear. Some reduction of the synaptic current amplitudes was observed at 10 mM extracellular calcium, suggesting calcium block/permeability of the channels. This was confirmed by observation of current-voltage reversal potentials in different calcium concentrations. We estimate that the channels underlying half the synapses showed a negligible calcium permeability. In the other four out of eight synapses the results suggest a very high calcium permeability with an estimated PCa/PCs of > 10. Thus, at -70 mV, in 1 mM calcium, more than 15% of the ATP-mediated synaptic current is estimated to be carried by calcium, but only at synapses with calcium-permeable channels. Net current through these synaptic channels is also controlled by the voltage dependence of synaptic current decay time constants (increasing e-fold for 158 mV depolarization) and by a strong dependence of transmitter release on the frequency of stimulation of the presynaptic neurone, with failure rates increasing 3-fold as stimulation rates were increased from 1 to 10 Hz.     

beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced beta2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating beta2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.     

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.     

Purification and characterization of a guanine nucleotide-exchange protein for ADP-ribosylation factor from spleen cytosol

ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins and are active in the GTP-bound state and inactive with GDP bound. ARF-GTP has a critical role in vesicular transport in several cellular compartments. Conversion of ARF-GDP to ARF-GTP is promoted by a guanine nucleotide-exchange protein (GEP). We earlier reported the isolation from bovine brain cytosol of a 700-kDa protein complex containing GEP activity that was inhibited by brefeldin A (BFA). Partial purification yielded an approximately 60-kDa BFA-insensitive GEP that enhanced binding of ARF1 and ARF3 to Golgi membranes. GEP has now been purified extensively from rat spleen cytosol in a BFA-insensitive, approximately 55-kDa form. It activated class I ARFs (ARFs 1 and 3) that were N-terminally myristoylated, but not nonmyristoylated ARFs from class-I, II, or III. GEP activity required MgCl2. In the presence of 0.6-0.8 mM MgCl2 and 1 mM EDTA, binding of guanosine 5'-[gamma[35S]thio]triphosphate ([35S]GTP gamma S) by ARF1 and ARF3 was equally high without and with GEP. At higher Mg2+ concentrations, binding without GEP was much lower; with 2-5 mM MgCl2, GEP-stimulated binding was maximal. The rate of GDP binding was much less than that of GTP gamma S with and without GEP. Phospholipids were necessary for GEP activity; phosphatidylinositol was more effective than phosphatidylserine, and phosphatidic acid was less so. Other phospholipids tested were ineffective. Maximal effects required approximately 200 microM phospholipid, with half-maximal activation at 15-20 microM. Release of bound [35S]GTP gamma S from ARF3 required the presence of both GEP and unlabeled GTP or GTP gamma S; GDP was much less effective. This characterization of the striking effects of Mg2+ concentration and specific phospholipids on the purified BFA-insensitive ARF GEP should facilitate experiments to define its function in vesicular transport.     

Effects of arfaptin 1 on guanine nucleotide-dependent activation of phospholipase D and cholera toxin by ADP-ribosylation factor

Arfaptin 1, a approximately 39-kDa protein based on the deduced amino acid sequence, had been initially identified in a yeast two-hybrid screen using dominant active ARF3 (Q71L) as bait with an HL-60 cDNA library. It was suggested that arfaptin 1 may be involved in Golgi functions, since the FLAG-tagged protein was associated with Golgi membranes when expressed in COS-7 cells and could be bound to Golgi in vitro in an ADP-ribosylation factor (ARF)- and GTPgammaS-dependent, brefeldin A-inhibited fashion. Arfaptin 2, found in the same two-hybrid screen as arfaptin 1, is 60% identical in amino acid sequence and may or may not have an analogous function. We now report some effects of arfaptin 1 on ARF activation of phospholipase D and cholera toxin ADP-ribosyltransferase. Arfaptin 1 inhibited activation of both enzymes in a concentration-dependent manner and was without effect in the absence of ARF. Two ARF1 mutants that activated the toxin, one lacking 13 N-terminal amino acids and the other, in which 73 residues at the N terminus were replaced with the analogous sequence from ARL1, were not inhibited by arfaptin, consistent with the conclusion that arfaptin interaction requires the N terminus of ARF. This region has also been implicated in phospholipase D activation, but whether the two proteins interact with the same structural elements in ARF remains to be determined. Arfaptin inhibition of the action of ARF5 and ARF6 was less than that of ARF1 and ARF3; its effects were less on nonmyristoylated than myristoylated ARFs. Arfaptin effects on guanine nucleotide binding by ARFs were minimal whether or not a purified ARF guanine nucleotide-exchange protein was present. These findings indicate that arfaptin acts as an inhibitor of ARF actions in vitro, raising the possibility that it has a similar role in vivo.     

Presynaptic modulation of synaptic transmission and plasticity by brain-derived neurotrophic factor in the developing hippocampus

In addition to the regulation of neuronal survival and differentiation, neurotrophins may play a role in synapse development and plasticity. Application of brain-derived neurotrophic factor (BDNF) promotes long-term potentiation (LTP) in CA1 synapses of neonatal hippocampus, which otherwise exhibit only short-term potentiation. This is attributable, at least in part, to an attenuation of the synaptic fatigue induced by high-frequency stimulation (HFS). However, the prevention of synaptic fatigue by BDNF could be mediated by an attenuation of synaptic vesicle depletion from presynaptic terminals and/or a reduction of the desensitization of postsynaptic receptors. Here we provide evidence supporting a presynaptic effect of BDNF. The effect of BDNF on synaptic fatigue depended on the stimulation frequency, not on the stimulus duration nor on the number of stimulation pulses. BDNF was only effective when the synapses were stimulated at frequencies >50 Hz. Treatment with BDNF also potentiated paired-pulse facilitation (PPF), a parameter reflecting changes in the properties of presynaptic terminals. This effect of BDNF was restricted only to PPF elicited with interpulse intervals 相似文献   

Very short-term plasticity in hippocampal synapses

Hippocampal pyramidal neurons often fire in bursts of action potentials with short interspike intervals (2-10 msec). These high-frequency bursts may play a critical role in the functional behavior of hippocampal neurons, but synaptic plasticity at such short times has not been carefully studied. To study synaptic modulation at very short time intervals, we applied pairs of stimuli with interpulse intervals ranging from 7 to 50 msec to CA1 synapses isolated by the method of minimal stimulation in hippocampal slices. We have identified three components of short-term paired-pulse modulation, including (i) a form of synaptic depression manifested after a prior exocytotic event, (ii) a form of synaptic depression that does not depend on a prior exocytotic event and that we postulate is based on inactivation of presynaptic N-type Ca2+ channels, and (iii) a dependence of paired-pulse facilitation on the exocytotic history of the synapse.     

ADP-ribosylation factor functions synergistically with a 50-kDa cytosolic factor in cell-free activation of human neutrophil phospholipase D

Proteins in both the cytosol and plasma membrane are needed to reconstitute cell-free phospholipase D activity from phagocytes (Olson, S., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242); membrane factors include a small GTP-binding protein in the Rho family (Bowman, E., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512). ADP-ribosylation factor (ARF) was recently implicated as the cytosolic factor, as it activates phospholipase D in HL-60 membranes. Herein, we show that ion exchange chromatography separates ARF from the major phospholipase D-stimulating cytosolic factor. Both bovine brain ARF and recombinant human ARF-1 stimulated a small amount of phospholipase D activity in the absence of cytosol (about 10% of the response seen with cytosol). With a high concentration of ARF-depleted cytosol, ARF did not further activate. However, at low cytosol, ARF caused marked activation. Thus, ARF synergizes with the cytosolic factor in phospholipase D activation.     

Arfaptin 1, an ARF-binding protein, inhibits phospholipase D and endoplasmic reticulum/Golgi protein transport

Class I ADP-ribosylation factors (ARFs) are essential for coatomer and clathrin coat assembly and vesicular transport in the Golgi apparatus. However, little is known about the in vivo regulation of ARF actions. Recently we cloned arfaptin 1, a 39 kDa protein that binds active, GTPgammaS-liganded ARF and translocates with it to Golgi membranes. Here we show that phorbol ester-stimulated phospholipase D (PLD) activity is inhibited in arfaptin 1-overexpressing NIH 3T3 cells and that arfaptin 1 inhibits ARF activation of Golgi-associated PLD. Since PLD activity is thought to play a role in regulating vesicular transport in the secretory pathway, we determined the rate of glycosylation of vesicular stomatitis virus glycoprotein as a measure of protein transport from the endoplasmic reticulum through the Golgi apparatus. Arfaptin 1 overexpression was found to decrease the rate of this reaction approximately two-fold. These data suggest that arfaptin 1 is a regulator of ARF action in the Golgi apparatus.     

Identification of a brefeldin A-insensitive guanine nucleotide-exchange protein for ADP-ribosylation factor in bovine brain

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that participate in vesicular transport in the Golgi and other intracellular compartments and stimulate cholera toxin ADP-ribosyltransferase activity. ARFs are active in the GTP-bound form; hydrolysis of bound GTP to GDP, possibly with the assistance of a GTP hydrolysis (GTPase)-activating protein results in inactivation. Exchange of GDP for GTP and reactivation were shown by other workers to be enhanced by Golgi membranes in a brefeldin A-sensitive reaction, leading to the proposal that the guanine nucleotide-exchange protein (GEP) was a target of brefeldin A. In the studies reported here, a soluble GEP was partially purified from bovine brain. Exchange of nucleotide on ARFs 1 and 3, based on increased ARF activity in a toxin assay and stimulation of binding of guanosine 5'-[gamma-[35S]thio]triphosphate, was dependent on phospholipids, with phosphatidylserine being more effective than cardiolipin. GEP appeared to increase the rate of nucleotide exchange but did not affect the affinity of ARF for GTP. Whereas the crude GEP had a size of approximately 700 kDa, the partially purified GEP behaved on Ultrogel AcA 54 as a protein of 60 kDa. With purification, the GEP activity became insensitive to brefeldin A, consistent with the conclusion that, in contrast to earlier inferences, the exchange protein is not itself the target of brefeldin A.     

A requirement for ARF6 in Fcgamma receptor-mediated phagocytosis in macrophages

Phagocytosis requires extension of F-actin-rich pseudopods and is accompanied by membrane fusion events. Members of the ARF family of GTPases are essential for many aspects of membrane trafficking. To test a role for this family of proteins in Fcgamma receptor-mediated phagocytosis, we utilized the fungal metabolite brefeldin A (BFA). The addition of 100 microM BFA to a subclone of RAW 264.7 macrophages disrupted the appearance and function of the Golgi apparatus as indicated by altered immunofluorescent distribution of beta-COP and reduced efflux of BODIPY C5-ceramide, a phospholipid that normally accumulates in the Golgi apparatus. In contrast, BFA had no effect on phagocytosis of IgG-coated erythrocytes. These results suggested that activation of BFA-sensitive ARFs is not required for phagocytosis. ARF6 is unique among members of the ARF family in that its membrane association is unaffected by BFA. Expression of ARF6 mutants defective in either GTP hydrolysis (Q67L) or binding (T27N) inhibited phagocytosis of IgG-coated erythrocytes and attenuated the focal accumulation of F-actin beneath the test particles. These results indicate a requirement for ARF6 in Fcgamma receptor-mediated phagocytosis and suggest that ARF6 is an important mediator of cytoskeletal alterations after Fcgamma receptor activation.     

Evidence for NMDA and mGlu receptor-dependent long-term potentiation of mossy fiber-granule cell transmission in rat cerebellum

Long-term potentiation (LTP) is a form of synaptic plasticity that can be revealed at numerous hippocampal and neocortical synapses following high-frequency activation of N-methyl--aspartate (NMDA) receptors. However, it was not known whether LTP could be induced at the mossy fiber-granule cell relay of cerebellum. This is a particularly interesting issue because theories of the cerebellum do not consider or even explicitly negate the existence of mossy fiber-granule cell synaptic plasticity. Here we show that high-frequency mossy fiber stimulation paired with granule cell membrane depolarization (-40 mV) leads to LTP of granule cell excitatory postsynaptic currents (EPSCs). Pairing with a relatively hyperpolarized potential (-60 mV) or in the presence of NMDA receptor blockers [5-amino--phosphonovaleric acid (APV) and 7-chloro-kynurenic acid (7-Cl-Kyn)] prevented LTP, suggesting that the induction process involves a voltage-dependent NMDA receptor activation. Metabotropic glutamate receptors were also involved because blocking them with (+)-alpha-methyl-4-carboxyphenyl-glycine (MCPG) prevented potentiation. At the cytoplasmic level, EPSC potentiation required a Ca2+ increase and protein kinase C (PKC) activation. Potentiation was expressed through an increase in both the NMDA and non-NMDA receptor-mediated current and by an NMDA current slowdown, suggesting that complex mechanisms control synaptic efficacy during LTP. LTP at the mossy fiber-granule cell synapse provides the cerebellar network with a large reservoir for memory storage, which may be needed to optimize pattern recognition and, ultimately, cerebellar learning and computation.     

Nerve terminal currents induced by autoreception of acetylcholine release

The activation of autoreceptors is known to be important in the modulation of presynaptic transmitter secretion in peripheral and central neurons. Using whole-cell recordings made from the free growth cone of myocyte-contact motoneurons of Xenopus cell cultures, we have observed spontaneous nerve terminal currents (NTCs). These spontaneous NTCs are blocked by d-tubocurarine (d-TC) and alpha-bungarotoxin (alpha-BuTx), indicating that endogenously released acetylcholine (ACh) can produce substantial membrane depolarization in the nerve terminals. Local application of NMDA to the growth cone increased the frequency of spontaneous NTCs. When the electrical stimulations were applied at the soma to initiate evoked-release of ACh, evoked ACh-induced potentials were recorded in the nerve terminals, which were inhibited by d-TC and hexamethonium but not by atropine. Replacement of normal Ringer's solution with high-Mg2+, low-Ca2+ solution also reversibly inhibited evoked ACh-induced potentials. The possible regulatory role of presynaptic nicotinic autoreceptors on the synaptic transmission was also examined. When the innervated myocyte was whole-cell voltage-clamped to record synaptic currents, application of hexamethonium inhibited the amplitude of evoked synaptic currents at a higher degree than that of iontophoretic ACh-induced currents. Furthermore, hexamethonium markedly reduced the frequency of spontaneous synaptic currents at high-activity synapses. Pretreatment of neurons with alpha-BuTx also inhibited the evoked synaptic currents in manipulated synapses. These results suggest that ACh released spontaneously or by electrical stimulation may act on the presynaptic nicotinic autoreceptors of the same nerve terminals to produce membrane potential change and to regulate synaptic transmission.     

NMDA EPSCs at glutamatergic synapses in the spinal cord dorsal horn of the postnatal rat

In rat dorsal horn, little is known about the properties of synaptic NMDA receptors during the first two postnatal weeks, a period of intense synaptogenesis. Using transverse spinal cord slices from postnatal day 0-15 rats, we show that 20% of glutamatergic synapses tested at low-stimulation intensity in spinal cord laminae I and II were mediated exclusively by NMDA receptors. Essentially all of the remaining glutamatergic EPSCs studied were attributable to the activation of both NMDA and AMPA receptors. Synaptic NMDA receptors at pure and mixed synapses showed similar sensitivity to membrane potential, independent of age, indicating similar Mg2+ sensitivity. Kinetic properties of NMDA EPSCs from pure and mixed synapses were measured at +50 mV. The 10-90% rise times of the pure NMDA EPSCs were slower (16 vs 10 msec), and the decay tau values were faster (tau1, 24 vs 42 msec; tau2, 267 vs 357 msec) than NMDA EPSCs at mixed synapses. Our results indicate that NMDA receptors are expressed at glutamatergic synapses at a high frequency, either alone or together with AMPA receptors, consistent with the prominent role of NMDA receptors in central sensitization (McMahon et al., 1993).     

Phosphoinositide 3-OH kinase activates the beta2 integrin adhesion pathway and induces membrane recruitment of cytohesin-1

Signal transduction through phosphoinositide 3-OH kinase (PI 3-kinase) has been implicated in the regulation of lymphocyte adhesion mediated by integrin receptors. Cellular phosphorylation products of PI 3-kinases interact with a subset of pleckstrin homology (PH) domains, a module that has been shown to recruit proteins to cellular membranes. We have recently identified cytohesin-1, a cytoplasmic regulator of beta2 integrin adhesion to intercellular adhesion molecule 1. We describe here that expression of a constitutively active PI 3-kinase is sufficient for the activation of Jurkat cell adhesion to intercellular adhesion molecule 1, and for enhanced membrane association of cytohesin-1. Up-regulation of cell adhesion by PI 3-kinase and membrane association of endogenous cytohesin-1 is abrogated by overexpression of the isolated cytohesin-1 PH domain, but not by a mutant of the PH domain which fails to associate with the plasma membrane. The PH domain of Bruton's tyrosine kinase (Btk), although strongly associated with the plasma membrane, had no effect on either membrane recruitment of cytohesin-1 or on induction of adhesion by PI 3-kinase. Having delineated the critical steps of the beta2 integrin activation pathway by biochemical and functional analyses, we conclude that PI 3-kinase activates inside-out signaling of beta2 integrins at least partially through cytohesin-1.     

Activity-dependent modulation of synaptic AMPA receptor accumulation

Both theoretical and experimental work have suggested that central neurons compensate for changes in excitatory synaptic input in order to maintain a relatively constant output. We report here that inhibition of excitatory synaptic transmission in cultured spinal neurons leads to an increase in mEPSC amplitudes, accompanied by an equivalent increase in the accumulation of AMPA receptors at synapses. Conversely, increasing excitatory synaptic activity leads to a decrease in synaptic AMPA receptors and a decline in mEPSC amplitude. The time course of this synaptic remodeling is slow, similar to the metabolic half-life of neuronal AMPA receptors. Moreover, inhibiting excitatory synaptic transmission significantly prolongs the half-life of the AMPA receptor subunit GluR1, suggesting that synaptic activity modulates the size of the mEPSC by regulating the turnover of postsynaptic AMPA receptors.