Rab proteins and endocytic trafficking: potential targets for therapeutic intervention

Rab GTPases serve as master regulators of vesicular membrane transport on both the exo- and endocytic pathways. In their active forms, rab proteins serve in cargo selection and as scaffolds for the sequential assembly of effectors requisite for vesicle budding, cytoskeletal transport, and target membrane fusion. Rab protein function is in turn tightly regulated at the level of protein expression, localization, membrane association, and activation. Alterations in the rab GTPases and associated regulatory proteins or effectors have increasingly been implicated in causing human disease. Some diseases such as those resulting in bleeding and pigmentation disorders (Griscelli syndrome), mental retardation, neuropathy (Charcot-Marie-Tooth), kidney disease (tuberous sclerosis), and blindness (choroideremia) arise from direct loss of function mutations of rab GTPases or associated regulatory molecules. In contrast, in a number of cancers (prostate, liver, breast) as well as vascular, lung, and thyroid diseases, the overexpression of select rab GTPases have been tightly correlated with disease pathogenesis. Unique therapeutic opportunities lie ahead in developing strategies that target rab proteins and modulate the endocytic pathway.     

Core proteins of the secretory machinery

Members of the Rab, SM- and SNARE-protein families play key roles in all intracellular membrane trafficking steps. While SM- and SNARE-proteins become directly involved in the fusion reaction at a late stage, Rabs and their effectors mediate upstream steps such as vesicle budding, delivery, tethering, and transport. Exocytosis of synaptic vesicles and regulated secretory granules are among the best-studied fusion events and involve the Rab3 isoforms Rab3A-D, the SM protein munc18-1, and the SNAREs syntaxin 1A, SNAP-25, and synaptobrevin 2. According to the current view, syntaxin 1A and SNAP-25 at the presynaptic membrane form a complex with synaptic vesicle-associated synaptobrevin 2. As complex formation proceeds, the opposed membranes are pulled tightly together, enforcing the fusion reaction. Munc18-1 is essential for regulated exocytosis and interacts with syntaxin 1A alone or with SNARE complexes, suggesting a role for munc18-1 in controlling the SNARE-assembly reaction. Compared to other intracellular fusion steps, special adaptations evolved in the synapse to allow for the tight regulation and high membrane turnover rates required for synaptic transmission. Synaptic vesicle fusion is triggered by the intracellular second messenger calcium, with members of the synaptotagmin protein family being prime candidates for linking calcium influx to fusion in the fast phase of exocytosis. To compensate for the massive incorporation of synaptic vesicles into the plasma membrane during exocytosis, special adaptations to endocytic mechanisms have evolved at the synapse to allow for efficient vesicle recycling.     

The role of RIM1alpha in BDNF-enhanced glutamate release

Brain-derived neurotrophic factor (BDNF) is known to activate proline-directed Ser/Thr protein kinases and to enhance glutamatergic transmission via a Rab3a-dependent molecular pathway. The identity of molecular targets in BDNF's action on Rab3a pathway, a synaptic vesicle protein involved in vesicle trafficking and synaptic plasticity, is not fully known. Here we demonstrate that BDNF enhances depolarization-evoked efflux of [(3)H]-glutamate from nerve terminals isolated from the CA1 region of the hippocampus. BDNF also potentiated hyperosmotic shock-evoked [(3)H]-glutamate efflux, indicating an effect on the size of the readily releasable pool. This effect of BDNF was completely abolished in nerve terminals derived from Rim1alphaKO (Rab3 interacting molecule 1alpha null mutant) mice. Using in vitro phosphorylation assays we identified two novel phosphorylation sites, Ser447 and Ser745 that were substrates for ERK2, a proline-directed kinase known to be activated by BDNF. The pSer447 site was phosphorylated under resting conditions in hippocampal CA1 nerve terminals and its phosphorylation was enhanced by BDNF treatment, as indicated by the use of a pSer447-RIM1alpha antibody we have developed. Together these findings identify RIM1alpha, a component of the Rab3a molecular pathway in mediating presynaptic plasticity, as a necessary factor in BDNF's enhancement of [(3)H]-glutamate efflux from hippocampal CA1 nerve terminals and indicate a possible role for RIM1alpha phosphorylation in BDNF-dependent presynaptic plasticity.     

Munc18-dependent regulation of synaptic vesicle exocytosis by syntaxin-1A in hippocampal neurons

The fusion of secretory vesicles with the plasma membrane requires the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes between the vesicle-SNARE vesicle-associated membrane protein present on the vesicular membrane and the target-SNAREs SNAP-25 and syntaxin-1A. Syntaxin-1A fluctuates between an open and closed form allowing it to selectively bind to different biological effectors in different conformations. In the open form, it can participate in SNARE complex formation, however, in the closed form it negatively regulates N- and P/Q-type voltage-dependent calcium channels, and is capable of inhibiting calcium influx. Thus paradoxically, syntaxin appears to have both positive and negative roles in controlling calcium-driven synaptic vesicle fusion at synaptic terminals. We show here that overexpression of syntaxin-1A inhibited exocytosis, in a manner that could be rescued by either elevating or reducing external calcium, or increasing action potential firing frequency. Elevating the level of Munc18 by coexpression with syntaxin-1A also abolished this inhibition, suggesting that Munc18 serves to limit the negative regulatory role of syntaxin by binding to, and thereby buffering, its closed form. Our results also indicate that syntaxin can control the frequency-response characteristics of the presynaptic fusion machinery.     

Enhanced long-term depression and impaired reversal learning in phosphodiesterase 4B-knockout (PDE4B-/-) mice

3'-5'-Cyclic adenosine monophosphate (cAMP) is known to be an important regulator of synaptic plasticity. The effects of cAMP are mediated through downstream effectors such as protein kinase A?(PKA), Ca(2+) and cAMP-response element binding protein (CREB). The phosphodiesterase 4 (PDE4) family of enzymes, which is comprised of four genes and at least 25 protein isoforms, mediates the hydrolysis of cAMP, yet little is presently known about the contribution of specific PDE4 isoforms to synaptic plasticity and cognitive behavior. The purpose of the present studies was to determine the contribution of the PDE4B gene in mediating synaptic plasticity and cognitive behavior. Electrophysiological recordings from hippocampal slice preparations of mice deficient in the PDE4B gene (PDE4B(-/-)) showed that knockout animals displayed markedly enhanced basal postsynaptic responses to stimulation and long-term depression as compared to wild-type littermates. Interestingly, no genotypic differences were noted in long-term potentiation experiments following several different induction protocols. On the behavioral level PDE4B(-/-) mice displayed impaired reversal learning in the Morris water maze compared to wild-type littermates, but no differences in acquisition and retention of spatial memory and fear conditioning. Taken together, these results suggest that the PDE4B gene may play a role in synaptic activity and long-term depression and is involved in spatial reversal memory. Our findings support the view that various PDE4 isoforms are non-redundant and have distinct neurological roles.     

Effect of reboxetine treatment on brain cAMP- and calcium/calmodulin-dependent protein kinases

Previous studies showed that the type II Ca(2+)/calmodulin- and cAMP-dependent protein kinases (CaMKII and PKA) are affected by long-term antidepressant treatment in presynaptic and somatodendritic compartments, respectively. This study describes the long-term effects of the selective noradrenaline reuptake inhibitor reboxetine on PKA and CaMKII, in both the microtubule and subsynaptosomal fractions of rat brain. Unlike other antidepressants, chronic reboxetine induced in the cerebrocortical soluble and microtubule fractions a decrease in the [(32)P]cAMP binding to the type II PKA regulatory subunit. No change in the cAMP-dependent endogenous phosphorylation of the protein substrate, microtubule-associated protein 2 was observed. In the hippocampal subsynaptosomal fractions (synaptic vesicles and synaptosomal membranes) reboxetine induced a robust increase in the activity but not in the expression of CaMKII. An increase in the calcium/calmodulin-dependent phosphorylation of presynaptic substrates was also detected. These findings showed that reboxetine modulates post-receptor signal transduction systems in rat brain.     

Chronic morphine treatment increases the expression of vesicular glutamate transporter 1 in the mouse spinal cord

Long-term exposure to morphine results in tolerance to morphine-induced antinociception. Here, we found that mice tolerant to morphine exhibited the significant increase in the protein levels of the vesicular glutamate transporter 1 and the synaptic vesicle-specific small G protein Rab3A, but not vesicular glutamate transporter 2 and vesicular gamma-aminobutyric acid transporter. These findings suggest that repeated treatment with morphine enhances excitatory synaptic transmission in the spinal cord, and in turn suppresses the morphine-induced antinociception.     

Construction of lentiviral vector carrying Rab9 gene and its expression in mouse brain

Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. Rab9 mediates late endosome to trans-Golgi-network trafficking. To explore the possibility of Rab9-related gene therapy for neurodegenerative diseases, we packed lentivirus encoding Rab9 cDNA. The expressing plasmid pCDH1-MCF1-Rab9-EF1-copGFP was constructed by using molecular biological techniques. The lentivirus encoding Rab9 cDNA was packed by Lifectamine-2000 mediated co-transfection of the plasmid pPACKH1-GAG, pPACKH1-REV and pVSV-G into 293T cells. DNA sequencing proved the successful construction of pCDH1-MCF1-Rab9-EF1-copGFP. After 72 h, the expression of GFP could be detected in BV-2 cells. Western blotting revealed that the Rab9 gene expression in BALB/c mice brain was up-regulated significantly 4 weeks after injection with lentivirus encoding Rab9 cDNA, which evidenced a satisfactory increasing effect of this virus. Administration of lenti-Rab9 to postnatal day 3 Niemann-Pick disease type C (NPC) mice reduced motor defects and prevented the weight loss associated with female NPC mice, as well as modulated the death rate of Purkinje neurons. It is concluded that the packaging of lentivirus encoding Rab9 cDNA was successful. lentivirus encoding Rab9 can increase the expression of Rab9 cDNA gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.     

Effect of sucralfate on gastric mucosal calcium channels activity.

1. The effect of anti-ulcer agent, sucralfate, on the activity of the gastric mucosal calcium channel was investigated using calcium channels purified from rat gastric epithelial cell membranes. 2. The channels on reconstitution into phosphatidylcholine vesicles responded in a concentration-dependent manner to a calcium channel activator, BAY K8644, as well as to a calcium channel antagonist, PN200-110. The 45Ca2+ uptake was inhibited by sucralfate. Maximum inhibitory effect was attained at 100 micrograms/ml sucralfate, at which point a 52% decrease in the uptake occurred. 3. EGF-induced channel protein phosphorylation showed an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the vesicles containing the phosphorylated channels displayed a 48% greater 45Ca2+ uptake. This phosphorylation process was inhibited by sucralfate. Furthermore, sucralfate also interfered with the binding of EGF to calcium channel protein. 4. The results indicate that sucralfate protects the cellular integrity from calcium imbalance by modulating the EGF-stimulated gastric mucosal calcium channel phosphorylation.     

Rab35与肿瘤关系的研究现状及展望

摘要：Rab蛋白是小分子GTP结合蛋白Ras超家族中最大的亚家族,在囊泡的形成、运输、融合等过程中发挥着 重要的作用。Rab35作为Rab家族的新成员,在人体组织中的分布比较广泛。已有研究证实Rab35与肿瘤迁移和侵 袭等生物学特性有关,可能为潜在的肿瘤预后标志物,可为临床治疗提供新的靶点。本文就Rab35的来源、结构、功 能及其在肿瘤中的作用进行综述。     

Phosphorylation of protein phosphatase-1 inhibitors, inhibitor-1 and DARPP-32, in renal medulla

Inhibitor-1 and DARPP-32 (dopamine and cAMP-regulated phosphoprotein, Mr 32 kDa) are each phosphorylated by cAMP-dependent protein kinase, resulting in their conversion to potent inhibitors of protein phosphatase-1. Protein phosphatase-1 is involved in the regulation of Na(+) reabsorption from renal tubule by modulating the activity of Na(+),K(+)-ATPase. In this study, we have investigated the regulation of inhibitor-1 and DARPP-32 phosphorylation in slices of renal medulla. Activation of cAMP-dependent protein kinase by forskolin and 8-bromo-cAMP increased the level of phosphorylated inhibitor-1. Okadaic acid (1 microM), used to inhibit protein phosphatase-2A, increased the level of phosphorylated inhibitor-1, but cyclosporin A had no effect. DARPP-32, like inhibitor-1, was phosphorylated by cAMP-dependent protein kinase and dephosphorylated only by protein phosphatase-2A. These data demonstrate that the phosphorylation of inhibitor-1 and DARPP-32 is regulated by the balance of phosphorylation by cAMP-dependent protein kinase and dephosphorylation by protein phosphatase-2A in renal medulla. Furthermore, the phosphorylation step is regulated by pharmacological stimuli such as activation of beta(1)-adrenoceptors and dopamine D1 receptors.     

New aspects of neurotransmitter release and exocytosis: dynamic and differential regulation of synapsin I phosphorylation by acute neuronal excitation in vivo

Synapsin I is a synaptic vesicle-associated protein that is phosphorylated at multiple sites by various protein kinases. It has been proposed to play an important role in the regulation of neurotransmitter release and the organization of cytoskeletal architecture in the presynaptic terminal. In the present minireview, I describe the dynamic changes in synapsin I phosphorylation induced by acute neuronal excitation in vivo, and discuss its regulation by protein kinases and phosphatases and its functional significance in vivo. When acute neuronal excitation was induced by electroconvulsive treatment (ECT) in rats, phosphorylation of synapsin I at multiple sites was decreased during brief seizure activity in hippocampal and parieto-cortical homogenates. After termination of the seizure activity, phosphorylation at mitogen-activated protein kinase-dependent sites was increased dramatically. Phosphorylation at a Ca(2+)/calmodulin-dependent protein kinase II-dependent site was also increased moderately afterwards. The dynamic and differential changes in synapsin I phosphorylation induced by acute neuronal excitation may be involved in plastic changes induced by ECT and may have some role in its effectiveness for the treatment of psychiatric diseases in humans.     

Chronic antidepressants induce redistribution and differential activation of alphaCaM kinase II between presynaptic compartments.

Changes in synaptic plasticity are involved in pathophysiology of depression and in the mechanism of antidepressants. Ca(2+)/calmodulin (CaM) kinase II, a protein kinase involved in synaptic plasticity, has been previously shown to be a target of antidepressants. We previously found that antidepressants activate the kinase in hippocampal neuronal cell bodies by increasing phosphorylation at Thr(286), reduce the kinase phosphorylation in synaptic membranes, and in turn its phosphorylation-dependent interaction with syntaxin-1 and the release of glutamate from hippocampal synaptosomes. Here, we investigated the chronic effect of different antidepressants (fluoxetine, desipramine, and reboxetine) on the expression and function of the kinase in distinct subcellular compartments in order to dissect the different kinase pools affected. Acute treatments did not induce any change in the kinase. In total tissue extracts chronic drug treatments induced activation of the kinase; in hippocampus (HC), but not in prefrontal/frontal cortex, this was partially accounted for by increased Thr(286) phosphorylation, suggesting the involvement of different mechanisms of activation. In synaptosomes, all drugs reduced the kinase phosphorylation, particularly in HC where, upon fractionation of the synaptosomal particulate into synaptic vesicles and membranes, we found that the drugs induced a redistribution and differential activation of the kinase between membranes and vesicles. Furthermore, a large decrease in the level and phosphorylation of synapsin I located at synaptic membranes was consistent with the observed decrease of CaM kinase II. Overall, antidepressants induce a complex pattern of modifications in distinct subcellular compartments; at presynaptic level, these changes are in line with a dampening of glutamate release.     

SNAP-25 Ser187 does not mediate phorbol ester enhancement of hippocampal synaptic transmission

Phorbol esters, activators of protein kinase C (PKC), have been shown to enhance synaptic transmission. One potential downstream target of PKC in the presynaptic terminal is the soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) SNAP-25, which has a PKC phosphorylation site in its C-terminal coil centered at serine 187 (S187/Ser187). We examined the role of S187 in hippocampal synaptic transmission. After proteolytic cleavage of native SNAP-25 by botulinum neurotoxin E (BoNT/E), synaptic transmission was restored in a subset of transfected CA3 pyramidal cells with a toxin-resistant form of SNAP-25 containing unaltered S187 (Swt), S187 mutated to alanine (SA) or S187 mutated to glutamate (SE). We observed that phorbol-12,13-diacetate (PDAc, 10 microM) induced potentiation of neurotransmission to a similar degree for both Swt and SA (2.4-fold and 3.1-fold increase, respectively). Furthermore, basal levels of transmission mediated by SE were reduced relative to that of Swt (failure rates of 72% and 41%, respectively). Together, these data suggest that phosphorylation of SNAP-25 S187 does not mediate the observed enhancement of neurotransmission by phorbol esters at hippocampal synapses.     

The elusive alpha(1D)-adrenoceptor: molecular and cellular characteristics and integrative roles

alpha(1)-Adrenoceptors seem to play key roles in cardiovascular, genitourinary, and central nervous system functions. This review will be focused on alpha(1D)-adrenoceptors. These receptors have intrinsic activity, and many of the more commonly used antagonists are in reality inverse agonists. alpha(1D)-Adrenoceptors are phosphorylated in the basal state, and the natural agonists, adrenaline and noradrenaline, increase their phosphorylation; similar effects are induced by direct activation of protein kinase C and through activation of nonadrenergic receptors. Interestingly, a large proportion of alpha(1D)-adrenoceptors are located in intracellular vesicles. Such intracellular location can be changed to surface expression through the use of inverse agonists and coexpression of alpha(1B)-adrenoceptors, which seem to act as pharmacological chaperons for proper plasma membrane insertion. The alpha(1D)-adrenoceptor amino terminus seems to contain a signal that keeps the receptor intracellularly, but interaction with other proteins may also contribute. The precise relationship between the intrinsic activity, phosphorylation, and intracellular location is currently unknown. alpha(1D)-Adrenoceptor activation induces contraction in a variety of vessels, and a role in the control of blood pressure has been suggested. Studies using young prehypertensive and adult spontaneously hypertensive rats as well as knockout mice suggest that vascular alpha(1D)-adrenoceptors are involved in the genesis/maintenance of hypertension.     

A Ser/Thr cluster within the C-terminal domain of the rat prostaglandin receptor EP3alpha is essential for agonist-induced phosphorylation, desensitization and internalization

Two isoforms of the rat prostaglandin E(2) receptor, rEP3alpha-R and rEP3beta-R, differ only in their C-terminal domain. To analyze the function of the rEP3-R C-terminal domain in agonist induced desensitization, a cluster of Ser/Thr residues in the C-terminal domain of the rEP3alpha-R was mutated to Ala and both isoforms and the receptor mutant (rEP3alpha-ST341-349A-R) were stably expressed in HEK293 cells. All rEP3-R receptors showed a similar ligand-binding profile. They were functionally coupled to Gi and reduced forskolin-induced cAMP-formation. Repeated exposure of cells expressing the rEP3alpha-R isoform to PGE(2) reduced the agonist induced inhibition of forskolin-stimulated cAMP-formation by 50% and led to internalization of the receptor to intracellular endocytotic vesicles. By contrast, Gi-response as well as plasma membrane localization of the rEP3beta-R and the rEP3alpha-ST341-349A-R were not affected by prior agonist-stimulation. Agonist-stimulation of HEK293-rEP3alpha-R cells induced a time- and dose-dependent phosphorylation of the receptor most likely by G protein-coupled receptor kinases and not by protein kinase A or protein kinase C. By contrast, upon agonist-stimulation the rEP3beta-R was not phosphorylated and the rEP3alpha-ST341-349A-R was phosphorylated only weakly. These results led to the hypothesis that agonist-induced desensitization of the rEP3alpha-R isoform is mediated most likely by a GRK-dependent phosphorylation of Ser/Thr residues 341-349. Phosphorylation then initiates uncoupling of the receptor from Gi protein and receptor internalization.     

General anesthetics selectively modulate glutamatergic and dopaminergic signaling via site-specific phosphorylation in vivo

Isoflurane, propofol and ketamine are representative general anesthetics with distinct molecular mechanisms of action that have neuroprotective properties in models of excitotoxic ischemic damage. We characterized the effects of these agents on neuronal glutamate and dopamine signaling by profiling drug-induced changes in brain intracellular protein phosphorylation in vivo to test the hypothesis that they affect common downstream effectors. Anesthetic-treated and control mice were killed instantly by focused microwave irradiation, frontal cortex and striatum were removed, and the phosphorylation profile of specific neuronal signaling proteins was analyzed by immunoblotting with a panel of phospho-specific antibodies. At anesthetic doses that produced loss of righting reflex, isoflurane, propofol, and ketamine all reduced phosphorylation of the activating residue T183 of ERK2 (but not of ERK1); S897 of the NR1 NMDA receptor subunit; and S831 (but not S845) of the GluR1 AMPA receptor subunit in cerebral cortex. At sub-anesthetic doses, these drugs only reduced phosphorylation of ERK2. Isoflurane and ketamine also reduced phosphorylation of spinophilin at S94, but oppositely regulated phosphorylation of presynaptic (tyrosine hydroxylase) and postsynaptic (DARPP-32) markers of dopaminergic neurotransmission in striatum. These data reveal both shared and agent-specific actions of CNS depressant drugs on critical intracellular protein phosphorylation signaling pathways that integrate multiple second messenger systems. Reduced phosphorylation of ionotropic glutamate receptors by all three anesthetics indicates depression of normal glutamatergic synaptic transmission and reduced potential excitotoxicity. This novel approach indicates a role for phosphorylation-mediated down-regulation of glutamatergic synaptic transmission by general anesthetics and identifies specific in vivo targets for focused evaluation of anesthetic mechanisms.     

New aspects of neurotransmitter release and exocytosis: Rho-kinase-dependent myristoylated alanine-rich C-kinase substrate phosphorylation and regulation of neurofilament structure in neuronal cells

Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin-binding protein whose function may be regulated by the phosphorylation of multiple sites, in which the phosphorylation site domain (PSD) is recognized to have three or four PKC-dependent sites. Recently, it is considered that MARCKS is implicated in some neuronal functions, such as synaptic vesicle trafficking and neurotransmitter release, through regulation of the actin-containing cytoskeletal structure; this is based on the experimental results with short-term or prolonged pretreatment with phorbol esters and treatment by protein kinase C (PKC) inhibitor. However, the precise molecular mechanism is yet obscure. Recently, we have demonstrated that MARCKS is phosphorylated at Ser159 in PSD by Rho-kinase in vitro and that the phosphorylation occurred in neuronal cells upon stimulation with lysophosphatidic acid (LPA), and its phosphorylation was inhibited by a novel and specific Rho-kinase inhibitor, H-1152. Our results allow us to speculate that a preinflammatory substance, such as LPA, interleukin 1-beta, and bradykinin, augments MARCKS phosphorylation in a novel signal transduction pathway besides the PKC-involved one, and thereby induces the release of a neurotransmitter through a reorganization of actin-containing microfilaments at the cell periphery, the so-called "active zone". In this section, I address a novel mechanism for MARCKS phosphorylation and its related cellular function.     

GM1-ganglioside regulation of EGF-induced gastric mucosal calcium channel activation.

1. Calcium channels, isolated from gastric epithelial cell membranes when reconstituted into phosphatidylcholine vesicles exhibited active 45Ca2+ uptake as evidenced by a dose dependent response to calcium channel activator, BAY K8644, and antagonist, PN200-110. 2. The channels on epidermal growth factor (EGF) binding in the presence of ATP showed an increase in tyrosine phosphorylation of 55 and 170 kDa calcium channel proteins. Such phosphorylated channels following reconstitution into the vesicles displayed a 48% greater 45Ca2+ uptake than that of the controls. 3. The binding of EGF to calcium channel protein was inhibited by GM1-ganglioside reaching maximum inhibition of 65% at 40 nM GM1. In contrast, calcium channel antagonist, PN200-110, had no effect on EGF binding. 4. The EGF-stimulated calcium channel protein phosphorylation was inhibited by GM1. This inhibitory effect was mainly reflected in the decrease of tyrosine phosphorylation of 55 and 170 kDa proteins. 5. The results suggest the participation of GM1-ganglioside in the regulation of EGF-stimulated gastric mucosal calcium channel activation.