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Important functional interactions between the metabotropic glutamate 2 (mGlu2) and 5-hydroxytryptamine2A (5-HT₂A) neurotransmitter receptors have been established based on electrophysiological, biochemical and behavioral evidence. Over the last several years, dimerization between 5-HT₂A and mGlu2 receptors has been proposed to account for the functional cross-talk between these two receptors in the prefrontal cortex. The pros and cons for the existence of a heteromeric complex between 5-HT₂A and mGlu2 receptors will be reviewed here. First, the fundamental criteria needing to establish evidence for heteromeric complexes will be reviewed. Then, the in vitro evidence for and against heteromeric complexes between 5-HT₂A and mGlu2 receptors will be discussed in regard to physical and functional interactions. Finally, the data with native in situ mGlu2 and 5-HT₂A receptors will be discussed with respect to whether heteromeric complexes or a simple functional interaction between two distinct GPCRs based on brain network activity is the more simple explanation for a range of in vivo data., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The capacity of opioids to alleviate inflammatory pain is negatively regulated by the glutamate-binding N-methyl-D-aspartate receptor (NMDAR). Increased activity of this receptor complicates the clinical use of opioids to treat persistent neuropathic pain. Immunohistochemical and ultrastructural studies have demonstrated the coexistence of both receptors within single neurons of the CNS, including those in the mesencephalic periaqueductal gray (PAG), a region that is implicated in the opioid control of nociception. We now report that mu-opioid receptors (MOR) and NMDAR NR1 subunits associate in the postsynaptic structures of PAG neurons. Morphine disrupts this complex by protein kinase-C (PKC)-mediated phosphorylation of the NR1 C1 segment and potentiates the NMDAR-CaMKII, pathway that is implicated in morphine tolerance. Inhibition of PKC, but not PKA or GRK2, restored the MOR-NR1 association and rescued the analgesic effect of morphine as well. The administration of N-methyl-D-aspartic acid separated the MOR-NR1 complex, increased MOR Ser phosphorylation, reduced the association of the MOR with G-proteins, and diminished the antinociceptive capacity of morphine. Inhibition of PKA, but not PKC, CaMKII, or GRK2, blocked these effects and preserved morphine antinociception. Thus, the opposing activities of the MOR and NMDAR in pain control affect their relation within neurons of structures such as the PAG. This finding could be exploited in developing bifunctional drugs that would act exclusively on those NMDARs associated with MORs.

The distribution of postsynaptic glutamate receptors has been shown to be regulated by proimmunocytokine tumor necrosis factor α (TNF-α) signaling. The role of TNF-α receptor subtypes in mediating glutamate receptor expression, trafficking, and function still remains unclear. Here, we report that TNF receptor subtypes (TNFR1 and TNFR2) differentially modulate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) clustering and function in cultured cortical neurons. We find that genetic deletion of TNFR1 decreases surface expression and synaptic localization of the AMPAR GluA1 subunit, reduces the frequency of miniature excitatory postsynaptic current (mEPSC), and reduces AMPA-induced maximal whole-cell current. In addition, these results are not observed in TNFR2-deleted neurons. The decreased AMPAR expression and function in TNFR1-deleted cells are not significantly restored by short (2 h) or long (24 h) term exposure to TNF-α. In TNFR2-deleted cells, TNF-α promotes AMPAR trafficking to the synapse and increases mEPSC frequency. In the present study, we find no significant change in the GluN1 subunit of NMDAR clusters, location, and mEPSC. This includes applying or withholding the TNF-α treatment in both TNFR1- and TNFR2-deleted neurons. Our results indicate that TNF receptor subtype 1 but not 2 plays a critical role in modulating AMPAR clustering, suggesting that targeting TNFR1 gene might be a novel approach to preventing neuronal AMPAR-mediated excitotoxicity.

Fasciculations are characteristic features of amyotrophic lateral sclerosis (ALS), suggesting abnormally increased excitability of motor axons. Previous nerve excitability studies have shown reduced axonal potassium currents in ALS patients that may contribute to the hyperexcitability and thereby generation of fasciculations. To clarify changes in axonal ion channel expression in motor axons of ALS, we performed immunohistochemistry of potassium and sodium channels in the C7 and L5 ventral/dorsal roots obtained from five autopsy cases of sporadic ALS. Compared to controls, the immunoreactivity of potassium channels (Kv1.2) was markedly reduced in the ventral roots, but normal in the dorsal roots of all the ALS patients. Nodal sodium channel expression was not significantly different in ALS patients and control subjects. Our results show prominently reduced expression of axonal potassium channels, and provide the neuropathological and biological basis for decreased accommodative potassium currents in motor axons of ALS patients. The axonal hyperexcitability would lead to generation of fasciculations, and possibly enhances motor neuron death in ALS., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Infection by the human immunodeficiency virus (HIV) can result in debilitating neurological syndromes collectively known as HIV-associated neurocognitive disorders. Although the HIV coat protein gp120 has been identified as a potent neurotoxin that enhances NMDA receptor function, the exact mechanisms for this effect are not known. Here we provide evidence that gp120 activates two separate signaling pathways that converge to enhance NMDA-evoked calcium flux by clustering NMDA receptors in modified membrane microdomains. gp120 enlarged and stabilized the structure of lipid microdomains on dendrites by mechanisms that involved a redox-regulated translocation of a sphingomyelin hydrolase (neutral sphingomyelinase-2) to the plasma membrane. A concurrent pathway was activated that accelerated the forward traffic of NMDA receptors by a PKA-dependent phosphorylation of the NR1 C-terminal serine 897 (masks an ER retention signal), followed by a PKC-dependent phosphorylation of serine 896 (important for surface expression). NMDA receptors were preferentially targeted to synapses and clustered in modified membrane microdomains. In these conditions, NMDA receptors were unable to laterally disperse and did not internalize, even in response to strong agonist induction. Focal NMDA-evoked calcium bursts were enhanced by threefold in these regions. Inhibiting membrane modification or NR1 phosphorylation prevented gp120 from accelerating the surface localization of NMDA receptors. Disrupting the structure of membrane microdomains after gp120 treatments restored the ability of NMDA receptors to disperse and internalize. These findings demonstrate that gp120 contributes to synaptic dysfunction in the setting of HIV infection by interfering with NMDA receptor trafficking.

Nestin is expressed in many different progenitors during development including those of the CNS, heart, skeletal muscle, and kidney. The adult expression is mainly restricted to the subependymal zone and dentate gyrus of the brain, the neuromuscular junction, and renal podocytes. In addition, this intermediate filament protein has served as a marker of neural stem/progenitor cells for close to 20 years. Therefore it is surprising that its function in development and adult physiology is still poorly understood. Here we report that nestin deficiency is compatible with normal development of the CNS. The mutant mice, however, show impaired motor coordination. Furthermore, we found that the number of acetylcholine receptor clusters, the nerve length, and the endplate bandwidth are significantly increased in neuromuscular junction area of nestin-deficient mice. This is similar to the phenotype described for deficiency of cyclin-dependent kinase 5 (Cdk5), a candidate downstream affecter of nestin. Moreover, we demonstrate that nestin deficiency can rescue maintenance of acetylcholine receptor clusters in the absence of agrin, similar to Cdk5/agrin double knock-outs, suggesting that the observed nestin deficiency phenotype is the consequence of aberrant Cdk5 activity.

Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor-ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.

Free κ L chains (FκLCs) are expressed on the surface of myeloma cells and are being assessed as a therapeutic target for the treatment of multiple myeloma. Despite its clinical potential, the mechanism by which FκLCs interact with membranes remains unresolved. In this study, we show that FκLCs associate with sphingomyelin on the plasma membrane of myeloma cells. Moreover, membrane-bound FκLCs are aggregated, suggesting that aggregation is required for intercalation with membranes. Finally, we propose a model where the binding of FκLCs with sphingomyelin on secretory vesicle membranes is stabilized by self-aggregation, with aggregated FκLCs exposed on the plasma membrane after exocytosis. Although it is well known that protein aggregates bind membranes, this is only the second example of an aggregate being found on the surface of cells that also secrete the protein in its native form. We postulate that many other aggregation-prone proteins may associate with cell membranes by similar mechanisms.

At the mammalian skeletal neuromuscular junction, cycling of nicotinic ACh receptors (nAChRs) is critical for the maintenance of a high postsynaptic receptor density. However, the mechanisms that regulate nAChRs recycling in living animals remain unknown. Using in vivo time-lapse imaging, fluorescence recovery after photobleaching, and biochemical pull down assays, we demonstrated that recycling of internalized nAChRs into fully functional and denervated synapses was promoted by both direct muscle stimulation and pharmacologically induced intracellular calcium elevations. Most of internalized nAChRs are recycled directly into synaptic sites. Chelating of intracellular calcium below resting level drastically decreased cycling of nAChRs. Furthermore we found that calcium-dependent AChR recycling is mediated by Ca(2+)/calmodulin-dependent kinase II (CaMKII). Inhibition of CaMKII selectively blocked recycling and caused intracellular accumulation of internalized nAChRs, whereas internalization of surface receptors remained unaffected. Electroporation of CaMKII-GFP isoforms into the sternomastoid muscle showed that muscle-specific CaMKIIβm isoform is highly expressed at the neuromuscular junction (NMJ) and precisely colocalized with nAChRs at crests of synaptic folds while the CaMKIIγ and δ isoforms are poorly expressed in synaptic sites. These results indicate that Ca(2+) along with CaMKII activity are critical for receptor recycling and may provide a mechanism by which the postsynaptic AChR density is maintained at the NMJ in vivo.

The effects of low and high concentrations of cocaine have been studied in vitro on the trafficking of plasma membrane A(2A) and D(2) immunoreactivities in previously characterized A(2A)-D(2) CHO cell lines. Receptor double immunofluorescence staining was performed with D(2) and A(2A) antibodies, planar lipid rafts immunolabeling with biotinylated cholera toxin subunit B and membrane invaginations with an anti-caveolin-1 antibody. A computer-assisted image analysis demonstrated a substantial and highly significant rise of membrane-associated D(2) immunoreactivity (IR) after 8 h of exposure to a low concentration of cocaine (150 nM). At this low concentration of cocaine, there was also an increase of membrane associated A(2A) immunoreactivity but smaller and less significant. However, this increase became considerably larger and highly significant at 150 microM at which concentration the rise of D(2) immunoreactivity had begun to disappear. It may be suggested that an allosteric action of cocaine at 150 nM on the D(2) receptors may primarily increase the insertion of D(2) monomers, homomers and also of a subpopulation of A(2A)-D(2) heteromers from the cytoplasm into the plasma membrane due to the conformational change induced by cocaine in the D(2) receptor. The planar lipid rafts and the caveolae are only affected by the higher concentrations of cocaine. It is proposed that changes in D(2) and A(2A)-D(2) trafficking induced by allosteric actions of cocaine at D(2) receptors may contribute to the alterations of D(2) signaling found in cocaine abusers.

Incubation at 0 degrees C is known to expose b- N -acetyl-D-glucosamine residues on glycoprotein (GP) Ibalpha inducing receptor clustering and alpha(M)beta(2)-mediated platelet destruction by macrophages. Here we show that incubation at 0/37 degrees C (4 hours at 0 degrees C, followed by 1 hour at 37 degrees C to mimic cold-storage and post-transfusion conditions) triggers a conformational change in the N -terminal flank (NTF, amino acids, aa 1-35) but not in aa 36-282 of GPIbalpha as detected by antibody binding. Addition of the sugar N -acetyl-D-glucosamine (GN) inhibits responses induced by 0/37 degrees C. Incubation at 0 degrees C shifts GPIbalpha from the membrane skeleton to the cytoskeleton. Different GPIbalpha conformations have little effect on VWF/ristocetin-induced aggregation, but arrest of NTF change by GN interferes with agglutination and spreading on a VWF-coated surface under flow. Strikingly, incubation at 0/37 degrees C initiates thromboxane A(2) formation through a von Willebrand factor (VWF)-independent and GPIbalpha-dependent mechanism, as confirmed in VWF- and GPIbalpha-deficient platelets. We conclude that the NTF change induced by 0/37 degrees C incubation reflects clustering of GPIbalpha supports VWF/ristocetin-induced agglutination and spreading and is sufficient to initiate platelet activation in the absence of VWF.

Dopaminergic projections to the striatum, crucial for the correct functioning of this brain region in adulthood, are known to be established early in development, but their role is currently uncharacterized. We demonstrate here that dopamine, by activating D(1)- and/or D(2)-dopamine receptors, decreases the number of functional GABAergic synapses formed between the embryonic precursors of the medium spiny neurons, the principal output neurons of the striatum, with associated changes in spontaneous synaptic activity. Activation of these receptors reduces the size of postsynaptic GABA(A) receptor clusters and their overall cell-surface expression, without affecting the total number of clusters or the size or number of GABAergic nerve terminals. These changes result from an increased internalization of GABA(A) receptors, and are mediated by distinct signaling pathways converging at the level of GABA(A) receptors to cause a transient PP2A/PP1-dependent dephosphorylation. Thus, tonic D(1)- and D(2)-receptor activity limits the extent of collateral inhibitory synaptogenesis between medium spiny neurons, revealing a novel role of dopamine in controlling the development of intrinsic striatal microcircuits.

The maturation of neuromuscular junctions (NMJs) requires the topological transformation of postsynaptic acetylcholine receptor (AChR)-containing structures from a simple plaque to an elaborate structure composed of pretzel-like branches. This maturation process results in the precise apposition of the presynaptic and postsynaptic specializations. However, little is known about the molecular mechanisms underlying the plaque-to-pretzel transition of AChR clusters. In this study, we identify an essential role for the RhoGEF ephexin1 in the maturation of AChR clusters. Adult ephexin1(-/-) mice exhibit severe muscle weakness and impaired synaptic transmission at the NMJ. Intriguingly, when ephexin1 expression is deficient in vivo, the NMJ fails to mature into the pretzel-like shape, and such abnormalities can be rescued by re-expression of ephexin1. We further demonstrate that ephexin1 regulates the stability of AChR clusters in a RhoA-dependent manner. Taken together, our findings reveal an indispensible role for ephexin1 in regulating the structural maturation and neurotransmission of NMJs., (Copyright 2010 Elsevier Inc. All rights reserved.)

CollagenQ (ColQ) plays an important structural role at vertebrate neuromuscular junctions (NMJs) by anchoring and accumulating acetylcholinesterase (AChE) in the extracellular matrix (ECM). Moreover, ColQ interacts with perlecan/dystroglycan and the muscle-specific receptor tyrosine kinase (MuSK), key molecules in the NMJ formation. MuSK promotes acetylcholine receptor (AChR) clustering in a process mediated by rapsyn, a cytoplasmic protein that stimulates AChR packing in clusters and regulates synaptic gene transcription. Here, we investigated a regulatory role for ColQ by comparing the clustering and expression of synaptic proteins in wild type and ColQ-deficient muscle cells in culture and at NMJ. We show first that AChR clusters are smaller and more densely packed in the absence of ColQ both in vitro and in vivo. Second, we find that like AChRs and rapsyn, MuSK mRNA levels are increased in cultured cells but not in muscles lacking ColQ. However, membrane-bound MuSK is decreased both in vitro and in vivo suggesting that ColQ controls MuSK sorting or stabilization in the muscle membrane. In line with this, our data show that activation of the MuSK signaling pathway is altered in the absence of ColQ leading to (1) perturbation of AChR clustering and/or beta-AChR subunit phosphorylation and (2) modifications of AChR mRNA level due to the lack of ColQ-MuSK interaction. Together, our results demonstrate that ColQ, in addition to its structural role, has important regulatory functions at the synapse by controlling AChR clustering and synaptic gene expression through its interaction with MuSK.

Acetylcholinesterase (AChE) is well known to process different molecular forms via the distinct interacting partners. Proline-rich membrane anchor (PRiMA)-linked tetrameric globular AChE (G4 AChE) is mainly found in the vertebrate brain; however, recent studies from our laboratory have suggested its existence at neuromuscular junctions (nmjs). Both muscle and motor neuron express AChE at the nmjs. In muscle, the expression of PRiMA-linked AChE is down-regulated during myogenic differentiation and by motor neuron innervation. As compared with muscle, spinal cord possessed higher total AChE activity and contained PRiMA-linked AChE forms. The spinal cord expression of this form increased during development. More importantly, PRiMA-linked G4 AChE identified as aggregates localized at nmjs. These findings suggest that the restricted localization of PRiMA-linked G4 AChE at the nmjs could be contributed by the pre-synaptic motor neuron and/or the post-synaptic muscle fiber.

At the developing vertebrate neuromuscular junction, the acetylcholine receptor becomes aggregated at high density in the postsynaptic muscle membrane. Receptor localization is regulated by the motoneuron-derived factor, agrin, and requires an intracellular, scaffolding protein called rapsyn. However, it remains unclear where rapsyn binds on the acetylcholine receptor and how their interaction is regulated. In this study, we identified rapsyn's binding site on the acetylcholine receptor using chimeric constructs where the intracellular domain of CD4 was substituted for the major intracellular loop of each mouse acetylcholine receptor subunit. When expressed in heterologous cells, we found that rapsyn clustered and cytoskeletally anchored CD4-alpha, beta and epsilon subunit loops but not CD4-delta loop. Rapsyn-mediated clustering and anchoring was highest for beta loop, followed by epsilon and alpha, suggesting that rapsyn interacts with the loops with different affinities. Moreover, by making deletions within the beta subunit intracellular loop, we show that rapsyn interacts with the alpha-helical region, a secondary structural motif present in the carboxyl terminal portion of the subunit loops. When expressed in muscle cells, rapsyn co-immunoprecipitated together with a CD4-alpha helical region chimera, independent of agrin signaling. Together, these findings demonstrate that rapsyn interacts with the acetylcholine receptor via an alpha-helical structural motif conserved between the alpha, beta and epsilon subunits. Binding at this site likely mediates the critical rapsyn interaction involved in localizing the acetylcholine receptor at the neuromuscular junction.

A functional collaboration between growth factor receptors such as platelet derived growth factor receptor (PDGFR) and integrins is required for effective signal transduction in response to soluble growth factors. However, the mechanisms of synergistic PDGFR/integrin signaling remain poorly understood. Our previous work showed that cell surface tissue transglutaminase (tTG) induces clustering of integrins and amplifies integrin signaling by acting as an integrin binding adhesion co-receptor for fibronectin. Here we report that in fibroblasts tTG enhances PDGFR-integrin association by interacting with PDGFR and bridging the two receptors on the cell surface. The interaction between tTG and PDGFR reduces cellular levels of the receptor by accelerating its turnover. Moreover, the association of PDGFR with tTG causes receptor clustering, increases PDGF binding, promotes adhesion-mediated and growth factor-induced PDGFR activation, and up-regulates downstream signaling. Importantly, tTG is required for efficient PDGF-dependent proliferation and migration of fibroblasts. These results reveal a previously unrecognized role for cell surface tTG in the regulation of the joint PDGFR/integrin signaling and PDGFR-dependent cell responses.

Vitamin E is the last of all vitamins whose essentiality is not yet understood. Its widely accepted role as a lipophilic antioxidant has been questioned, since proof of its in vivo relevance remained scarce. The influence of vitamin E on biomarkers of oxidative stress in vivo is inconsistent and metabolites of vitamin E having reacted as an antioxidant are hardly detectable. Novel functions of vitamin E include the regulation of enzymes, most of which are membrane bound or activated by membrane recruitment. Also, expression of genes responds to vitamin E. The search for a transcription factor common to all regulated genes failed so far and a receptor that specifically binds vitamin E has not yet been identified. According to microarray data, pathways preferentially affected by the vitamin E status are the inflammatory response and cellular traffic. A role of vitamin E in cellular trafficking could best explain the neurological symptoms seen in vitamin E deficiency. Emerging knowledge on vitamin E is compiled here with the perspective to unravel the molecular mechanisms that could more likely explain the essentiality of the vitamin than its ability to scavenge free radicals.

Tropoelastin is a 60-72 kDa alternatively spliced extracellular matrix protein and a key component of elastic fibres. It is found in all vertebrates except for cyclostomes. Secreted tropoelastin is tethered to the cell surface, where it aggregates into organised spheres for cross-linking and incorporation into growing elastic fibres. Tropoelastin is characterised by alternating hydrophobic and hydrophilic domains and is highly flexible. The conserved C-terminus is an area of the molecule of particular biological importance in that it is required for both incorporation into elastin and for cellular interactions. Mature cross-linked tropoelastin gives elastin, which confers resilience and elasticity on a diverse range of tissues. Elastin gene disruptions in disease states and knockout mice emphasise the importance of proper tropoelastin production and assembly, particularly in vascular tissue. Tropoelastin constructs hold promise as biomaterials as they mimic many of elastin's physical and biological properties with the capacity to replace damaged elastin-rich tissue.

Aggregation of the high affinity IgE receptor (Fc epsilonRI) activates a cascade of signaling events leading to mast cell activation. Subsequently, inhibitory signals are engaged for turning off activating signals. We identified that regulator of calcineurin (Rcan) 1 serves as a negative regulator for turning off Fc epsilonRI-mediated mast cell activation. Fc epsilonRI-induced Rcan1 expression was identified by suppression subtractive hybridization and verified by real-time quantitative polymerase chain reaction and Western blotting. Deficiency of Rcan1 led to increased calcineurin activity, increased nuclear factor of activated T cells and nuclear factor kappaB activation, increased cytokine production, and enhanced immunoglobulin E-mediated late-phase cutaneous reactions. Forced expression of Rcan1 in wild-type or Rcan1-deficient mast cells reduced Fc epsilonRI-mediated cytokine production. Rcan1 deficiency also led to increased Fc epsilonRI-mediated mast cell degranulation and enhanced passive cutaneous anaphylaxis. Analysis of the Rcan1 promoter identified a functional Egr1 binding site. Biochemical and genetic evidence suggested that Egr1 controls Rcan1 expression. Our results identified Rcan1 as a novel inhibitory signal in Fc epsilonRI-induced mast cell activation and established a new link of Egr1 and Rcan1 in Fc epsilonRI signaling.

Two recent papers reported identification of a long-sought agrin coreceptor, Lrp4 (Kim et al. in Cell and Zhang et al. in Neuron). In this issue of Neuron, Linnoila et al. report the identification of a new player in the agrin-MuSK pathway, Tid1, which directly interacts with MuSK and is responsible for transducing signals from MuSK activation to AChR clustering, culminating in cross-linking to the subsynaptic cytoskeleton. These papers substantially reshape the agrin-MuSK-ACh hypothesis of neuromuscular synaptogenesis.

Motoneuron-derived agrin clusters nicotinic acetylcholine receptors (AChRs) in mammalian muscle cells. We used two-hybrid screens to identify a protein, tumorous imaginal discs (Tid1), that binds to the cytoplasmic domain of muscle-specific kinase (MuSK), a major component of the agrin receptor. Like MuSK, Tid1 colocalizes with AChRs at developing, adult, and denervated motor endplates. Knockdown of Tid1 by short hairpin RNA (shRNA) in skeletal muscle fibers dispersed synaptic AChR clusters and impaired neuromuscular transmission. In cultured myotubes, Tid1 knockdown inhibited AChR clustering, as well as agrin-induced activation of the Rac and Rho small GTPases and tyrosine phosphorylation of the AChR, without affecting MuSK activation. Tid1 knockdown also decreased Dok-7-induced clustering of AChRs. Overexpression of the N-terminal half of Tid1 induced agrin- and MuSK-independent phosphorylation and clustering of AChRs. These results demonstrate that Tid1 is an essential component of the agrin signaling pathway, crucial for synaptic development.

Most G-protein-coupled receptors (GPCRs) form di(oligo)-meric structures that constitute signaling and trafficking units and might be essential for receptor functions. Cell responses to complement C5a receptor (C5aR) are tightly controlled by receptor desensitization and internalization. To examine the implication of dimerization in C5aR regulation, we generated an NH(2)-terminally modified C5aR mutant, unable to bind C5a, and a phosphorylation-deficient mutant. Neither an intact NH(2) terminus nor the presence of COOH-terminal phosphorylation sites appeared to be required for the formation of C5aR dimers. Upon C5a stimulation, mutant receptors did not internalize when individually expressed. C5a stimulation of cells that co-expressed wild type C5aR together with either unliganded or phosphorylation-deficient mutant resulted in co-internalization of mutant receptors with C5aR. Unliganded GPCRs can be cross-phosphorylated within a heterologous receptor dimer or by second messenger-activated kinases. C5a stimulation of (32)P-labeled cells that co-expressed the unliganded mutant with either C5aR or the phosphorylation-deficient mutant did not induce phosphorylation of the unliganded mutant. We can thus postulate that, in the case of C5aR, the stimulation and phosphorylation of one monomer is enough to lead to dimer internalization. The existence and functional implication of di(oligo)mer formation may be important for an accurate C5aR down-regulation in pathological conditions.

Protein O-fucosyltransferase 1 (Pofut1) transfers fucose to serine or threonine on proteins, including Notch receptors, that contain EGF repeats with a particular consensus sequence. Here we demonstrate that agrin is O-fucosylated in a Pofut1-dependent manner, and that this glycosylation can regulate agrin function. Fucosylation of recombinant C45 agrin, both active (neural, z8) and inactive (muscle, z0) splice forms, was eliminated when agrin was overexpressed in Pofut1-deficient cells or by mutation of a consensus site for Pofut1 fucosylation (serine 1726 in the EGF4 domain). Loss of O-fucosylation caused a gain of function for muscle agrin such that it stimulated AChR clustering and MuSK phosphorylation in cultured myotubes at levels normally only found with the neural splice form. Deletion of Pofut1 in cultured primary myotubes and in adult skeletal muscle increased AChR aggregation. In addition, Pofut1 gene and protein expression and Pofut1 activity of the EGF4 domain of agrin were modulated during neuromuscular development. These data are consistent with a role for Pofut1 in AChR aggregation during synaptogenesis via the regulation of the synaptogenic activity of muscle agrin.

Gonadal steroid production is stimulated by gonadotropin binding to G protein-coupled receptors (GPCRs). Although GPCR-mediated increases in intracellular cAMP are known regulators of steroidogenesis, the roles of other signaling pathways in mediating steroid production are not well characterized. Recent studies suggest that luteinizing hormone (LH) receptor activation leads to trans-activation of epidermal growth factor (EGF) receptors in the testes and ovary. This pathway is critical for LH-induced steroid production in ovarian follicles, probably through matrix metalloproteinase (MMP)-mediated release of EGF receptor (EGFR) binding ectodomains. Here we examined LH and EGF receptor cross-talk in testicular steroidogenesis using mouse MLTC-1 Leydig cells. We demonstrated that, similar to the ovary, trans-activation of the EGF receptor was critical for gonadotropin-induced steroid production in Leydig cells. LH-induced increases in cAMP and cAMP-dependent protein kinase (PKA) activity mediated trans-activation of the EGF receptor and subsequent mitogen-activated protein kinase (MAPK) activation, ultimately leading to StAR phosphorylation and mitochondrial translocation. Steroidogenesis in Leydig cells was unaffected by MMP inhibitors, suggesting that cAMP and PKA trans-activated EGF receptors in an intracellular fashion. Interestingly, although cAMP was always needed for steroidogenesis, the EGFR/MAPK pathway was activated and necessary only for early (30-60 min), but not late (120 min or more), LH-induced steroidogenesis in vitro. In contrast, 36-h EGF receptor inhibition in vivo significantly reduced serum testosterone levels in male mice, demonstrating the physiologic importance of this cross-talk. These results suggest that GPCR-EGF receptor cross-talk is a conserved regulator of gonadotropin-induced steroidogenesis in the gonads, although the mechanisms of EGF receptor trans-activation may vary.

Phencyclidine (PCP), a non-competitive NMDA/glutamate receptor antagonist, is a psychotomimetic drug that produces a syndrome in normal humans that resembles schizophrenia. The present study investigated the mechanisms of PCP actions by examining the density of glutamate and muscarinic receptors in the rat brain 4h after a single injection of PCP. We used receptor autoradiography and [3H]MK801, [3H]AMPA, [3H]pirenzepine and [3H]AFDX384 to target glutamate NMDA, glutamate AMPA and muscarinic M1 and M2 receptors, respectively. The major outcome from the present study was an overall decrease in levels of the glutamate AMPA receptor density (F=14.5, d.f.=1, p<0.001) in the PCP treated rats. More specifically, PCP-treated animals displayed decreased AMPA receptor density in hippocampus CA1 (-16%), hippocampus CA2 (-25%), dentate gyrus (-27%), parietal cortex layers III-VI (-19%), central nucleus of the amygdala (-40%), and basolateral amygdala (-19%). Other brain regions examined were unaffected. PCP administration did not significantly affect glutamate NMDA, muscarinic M1 and M2 receptor density. The present study demonstrates the limbic system as the anatomical locus of alterations in AMPA receptor density after acute administration of PCP and may have implications for models of schizophrenia that focus on glutamatergic dysfunction in limbic cortical regions.

Fluorescence resonance energy transfer (FRET) experiments at neuromuscular junctions in the mouse tibialis anterior muscle show that postsynaptic acetylcholine receptors (AChRs) become more tightly packed during the first month of postnatal development. Here, we report that the packing of AChRs into postsynaptic aggregates was reduced in 4-week postnatal mice that had reduced amounts of the AChR-associated protein, rapsyn, in the postsynaptic membrane (rapsyn(+/-) mice). We hypothesize that nerve-derived agrin increases postsynaptic expression and targeting of rapsyn, which then drives the developmental increase in AChR packing. Neural agrin treatment elevated the expression of rapsyn in C2 myotubes by a mechanism that involved slowing of rapsyn protein degradation. Similarly, exposure of synapses in postnatal muscle to exogenous agrin increased rapsyn protein levels and elevated the intensity of anti-rapsyn immunofluorescence, relative to AChR, in the postsynaptic membrane. This increase in the rapsyn-to-AChR immunofluorescence ratio was associated with tighter postsynaptic AChR packing and slowed AChR turnover. Acute blockade of synaptic AChRs with alpha-bungarotoxin lowered the rapsyn-to-AChR immunofluorescence ratio, suggesting that AChR signaling also helps regulate the assembly of extra rapsyn in the postsynaptic membrane. The results suggest that at the postnatal neuromuscular synapse agrin signaling elevates the expression and targeting of rapsyn to the postsynaptic membrane, thereby packing more AChRs into stable, functionally-important AChR aggregates., ((c) 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008.)

The alpha4beta2 subtype is the most abundant nicotinic acetylcholine receptor (nAChR) in the brain and possesses the high-affinity binding site for nicotine. The alpha4 and beta2 nAChR subunits assemble into two alternate stoichiometries, (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2), which differ in their functional properties and sensitivity to chronic exposure to nicotine. Here, we investigated the sensitivity of both receptor stoichiometries to modulation by Zn2+. We show that Zn2+ exerts an inhibitory modulatory effect on (alpha4)(2)(beta2)(3) receptors, whereas it potentiates or inhibits, depending on its concentration, the function of (alpha4)(3)(beta2)(2) receptors. Furthermore, Zn2+ inhibition on (alpha4)(2)(beta2)(3) nAChRs is voltage-dependent, whereas it is not on (alpha4)(3)(beta2)(2) receptors. We used molecular modeling in conjunction with alanine substitution and functional studies to identify two distinct sets of residues that determine these effects and may coordinate Zn(2+). Zn(2+) inhibition is mediated by a site located on the beta2(+)/alpha4(-) subunit interfaces on both receptor stoichiometries. alpha4(H195) and beta2(D218) are key determinants of this site. Zn2+ potentiation on (alpha4)(3)(beta2)(2) nAChRs is exerted by a site that resides on the alpha4(+)/alpha4(-) of this receptor stoichiometry. alpha4(H195) on the (-) side of the ACh-binding alpha4 subunit and alpha4(E224) on the (+) side of the non-ACh-binding alpha4 subunit critically contribute to this site. We also identified residues within the beta2 subunit that confer voltage dependency to Zn2+ inhibition on (alpha4)(2)(beta2)(3), but not on (alpha4)(3)(beta2)(2) nAChRs.

Cell surface receptors mediate the exchange of information between cells and their environment. In the case of adhesion receptors, the spatial distribution and molecular associations of the receptors are critical to their function. Therefore, understanding the mechanisms regulating the distribution and binding associations of these molecules is necessary to understand their functional regulation. Experiments characterizing the lateral mobility of adhesion receptors have revealed a set of common mechanisms that control receptor function and thus cellular behavior. The T cell provides one of the most dynamic examples of cellular adhesion. An individual T cell makes innumerable intercellular contacts with antigen presenting cells, the vascular endothelium, and many other cell types. We review here the mechanisms that regulate T cell adhesion receptor lateral mobility as a window into the molecular regulation of these systems, and we present a general framework for understanding the principles and mechanisms that are likely to be common among these and other cellular adhesion systems. We suggest that receptor lateral mobility is regulated via four major mechanisms-reorganization, recruitment, dispersion, and anchoring-and we review specific examples of T cell adhesion receptor systems that utilize one or more of these mechanisms.

Repeated administration of psychostimulants produces a behavioural sensitization. Amphetamine-sensitized animals are known to have a higher proportion of high-affinity states of dopamine D2 receptors (D2(High) receptors) in the striatum. We recently reported that repeated administration of a dopamine D1 receptor agonist, R-(+)-SKF38393, reverses the established behavioural sensitization to methamphetamine (MAP). To investigate the mechanisms for reversal of behavioural sensitization, we examined the effect of repeated administration of the dopamine D1 receptor agonist on the proportions of D2(High) receptors and the high-affinity states of dopamine D1 receptors (D1(High) receptors) in the striatum. In the striatum from the MAP-sensitized rats, the proportions of D1(High) and D2(High) receptors (28.5 +/- 1.96 and 57.5 +/- 3.58%) were higher than those in the saline-control rats (12.0 +/- 1.01 and 21.9 +/- 1.60%, respectively). Repeated administration of R-(+)-SKF38393 to the MAP-sensitized rats reduced the increased proportions of D1(High) and D2(High) receptors to 12.4 +/- 1.57 and 31.0 +/- 2.14%, respectively, which were similar to the proportions in the saline-control rats. The total densities of dopamine D1 and D2 receptors were not altered in each treatment condition. The results demonstrate that the proportions of D1(High) and D2(High) receptors in the striatum are elevated in MAP-sensitized rats, and that repeated administration of the dopamine D1 receptor agonist to the MAP-sensitized rats reverses the increased proportions of D1(High) and D2(High) receptors. The findings reveal postsynaptic mechanisms for the development of behavioural sensitization to MAP and the reversal of established sensitization by repeated administration of the dopamine D1 receptor agonist.

Neuromuscular junctions (NMJs) normally form in the central region of developing muscle. In this process, agrin released from motor neurons has been considered to initiate the formation of synaptic acetylcholine receptor (AChR) clusters (neurocentric model). However, in muscle developing in the absence of nerves and thus of agrin, AChR clusters still form in the muscle center. This raises the possibility that the region of NMJ formation is determined by muscle-derived cues that spatially restrict the nerve to form synapses from aneural AChR clusters, e.g., by patterned expression of the agrin receptor MuSK (muscle-specific kinase) (myocentric model). Here we examine at initial stages of synaptogenesis whether the responsiveness of myotubes to agrin is spatially restricted, whether the regions of NMJ formation in wild-type muscle and of aneural AChR cluster formation in agrin-deficient animals correlate, and whether AChR cluster growth depends on the presence of agrin. We show that primary myotubes form AChR clusters in response to exogenous agrin in their central region only, a pattern that can spatially restrict NMJ formation. However, the nerve also makes synapses in regions in which aneural AChR clusters do not form, and agrin promotes synaptic cluster growth from the first stages of neuromuscular contact formation. These data indicate that aneural AChR clusters per se are not required for NMJ formation. A model is proposed that explains either the neurocentric or the myocentric mode of NMJ formation depending on a balance between the levels of MuSK expression and the availability of nerve-released agrin.

Dimerization or oligomerization of G-protein-coupled receptors (GPCRs) is a novel concept, which may lead to the reevaluation of the actions of pharmacological ligands, hormones, neurotransmitters, and other mediators acting on GPCRs. Although a large number of data obtained using different biophysical, biochemical and structural methods, and functional approaches argue for dimerization or oligomerization of these receptors, several publications criticized the applied methods and challenged the concept. The aim of this paper is to review the data that support the concept of receptor oligomerization, and the most important arguments against it. We conclude that it will require major methodical improvements to obtain decisive proof, whether GPCRs exist in their native membrane environments as homo- or heterodimeric or oligomeric complexes, in which receptor monomers have stable direct interactions. However, overwhelming amounts of data suggest that many GPCRs exhibit functional properties that require direct or indirect interactions between clustered receptors. Although it is difficult to conclude, about the exact nature of these interactions, dimerization or oligomerization of GPCRs is a useful paradigm for pharmacologists to study properties of receptors, which require functionally important clustering of receptors, such as trafficking of newly synthesized receptors to the cell surface, allosteric modulation of ligand binding, signaling specificity, co-internalization, or cross-inhibition of GPCRs.

Most animal cells move cross-linked surface antigens to one pole of the cell, a phenomenon called 'capping'. It is closely related to the rearward movement of particles attached to their surface. Cap formation is one of the most accessible dynamic properties of cells and is closely related to how they move. Yet, how this occurs is unknown.

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although recent work has shown that receptor recycling plays an important role in NCC motility on laminin, the molecular mechanisms regulating NCC motility on fibronectin remain unclear. One mechanism by which cells regulate motility is by modulating the affinity of integrin receptors. Here, we provide evidence that cranial and trunk NCCs rely on functional regulation of integrins to migrate efficiently on fibronectin (FN) in vitro. For NCCs cultured on fibronectin, velocity decreases after Mn2+ application (a treatment that activates all surface integrins) while velocity on laminin (LM) is not affected. The distribution of activated integrin beta 1 receptors on the surface of NCCs is also substratum-dependent. Integrin activation affects cranial and trunk NCCs differently when cultured on different concentrations of FN substrata; only cranial NCCs slow in a FN concentration-dependent manner. Furthermore, Mn2+ treatment alters the distribution and number of activated integrin beta 1 receptors on the surface of cranial and trunk NCCs in different ways. We provide a hypothesis whereby a combination of activated surface integrin levels and the degree to which those receptors are clustered determines NCC motility on fibronectin.

Activation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs) can lead to the release of Ca(2+) from intracellular stores and propagating Ca(2+) waves. Previous studies of these proteins in neurons have focused on their distribution in adult tissue, whereas, recent functional studies have examined neural tissue extracted from prenatal and young postnatal animals. In this study we examined the distribution of InsP(3)R isotypes 1-3 and RyR isotypes 1-3 in rat hippocampus during postnatal maturation, with a focus on InsP(3)R1 because it is most prominent in the hippocampus. InsP(3)R1 was observed in pyramidal cells and granule cells, InsP(3)R2 immunoreactivity was observed in perivascular astrocytes and endothelial cells, and InsP(3)R3 immunoreactivity was detected in axon terminals located in stratum pyramidale of CA1 and microvessels in stratum radiatum. RyR1 immunolabeling was enriched in CA1, RyR2 was most intense in CA3 and the dentate gyrus, and RyR3 immunolabeling was detected in all subfields of the hippocampus, but was most intense in stratum lacunosum-moleculare. During maturation from 2 to 10 weeks of age there was a shift in InsP(3)R1 immunoreactivity from a high density in the proximal apical dendrites to a uniform distribution along the dendrites. Independent of age, InsP(3)R1 immunoreactivity was observed to form clusters within the primary apical dendrite and at dendritic bifurcations of pyramidal neurons. As CA1 pyramidal neurons matured, InsP(3)R1 was often co-localized with the Ca(2+) binding protein calbindin D-28k. In contrast, InsP(3)R1 immunolabel was never co-localized with calbindin D-28k immunopositive interneurons located outside of stratum pyramidale or with parvalbumin, typically found in hippocampal basket cells, suggesting that InsP(3)R1s do not play a role in internal Ca(2+) release in these interneurons. These findings should help to interpret past functional studies and inform future studies examining the characteristics and consequences of InsP(3)R-mediated internal Ca(2+) release and Ca(2+) waves in hippocampal neurons.

The organochlorine pesticide dieldrin is a persistent organic pollutant that accumulates in the fatty tissue of living organisms. In mammals, it antagonizes the GABA(A) receptor, producing convulsions after acute exposure. Although accumulation in human brain has been reported, little is known about the effects of long-term exposure to dieldrin in the nervous system. Homeostatic control of the balance between excitation and inhibition has been reported when neuronal activity is chronically altered. We hypothesized that noncytotoxic concentrations of dieldrin could decrease glutamatergic neurotransmission as a consequence of a prolonged reduction in GABA(A) receptor function. Long-term exposure of primary cerebellar granule cell cultures to 3 microM dieldrin reduced the GABA(A) receptor function to 55% of control, as measured by the GABA-induced (36)Cl(-) uptake. This exposure produced a significant reduction (approximately 35%) of the NMDA-induced increase in [Ca(2+)](i) and of the [(3)H]MK-801 binding, which was not accompanied by a reduction in the NMDA receptor subunit NR1, as determined by Western blot. Consistent with the decreased NMDA receptor function, dieldrin-treated cultures were insensitive to an excitotoxic stimulus induced by exposure to high potassium. In summary, we report that the chronic reduction of GABA(A) receptor function induced by dieldrin decreases the number of functional NMDA receptors, which may be attributable to a mechanism of synaptic scaling. These effects could underlie neural mechanisms involved in cognitive impairment produced by low-level exposure to dieldrin., ((c) 2007 Wiley-Liss, Inc.)

Synapses between parallel fibres and Purkinje cells in the cerebellum exhibit unique forms of synaptic plasticity thought to be associated with the refinement of motor skills. Since the discovery of Long Term Depression (LTD), more than twenty years ago, many molecular signalling pathways potentially underlying LTD have been explored. These have revealed a surprisingly diverse array of cellular and molecular mechanisms. Foremost has been the now well-established discovery that LTD is the electrophysiological manifestation of a reduced density of AMPA receptors at the synapse, following induction. Although LTD is primarily an electrophysiologically defined phenomenon, recent studies have increasingly combined electrophysiological, imaging, proteomic and biochemical approaches to probe its mechanisms. The challenge is now to integrate data from different modalities into a unified formalism that can deal with the complexity of the system, as well as generate experimental predictions. Here, we use particle-based stochastic modelling as a prototype to explore the feasibility of building realistic model of synaptic plasticity, at the molecular level.

This study was focused on the relationship between the plasma-membrane localization of neurokinin-1 receptor (NK1-R) and its endocytic and signaling properties. First, we employed electron paramagnetic resonance (EPR) to study the domain structure of HEK-293 cells and NK1-R microlocalization. EPR spectra and the GHOST condensation routine demonstrated that NK1-R was distributed in a well-ordered domain of HEK-293 cells possibly representing lipid raft/caveolae microdomains, whereas the impairment of caveolae changed the NK1-R plasma-membrane distribution. Internalization and second messenger assays combined with bioluminescence resonance energy transfer were employed subsequently to evaluate the functional importance of the NK1-R microlocalization in lipid raft/caveolae microdomains. The internalization pattern was delineated through the use of dominant-negative mutants (DNM) of caveolin-1 S80E (Cav1 S80E), dynamin-1 K44A (Dyn K44A), and beta-arrestin (beta-arr 319-418) and by means of cell lines that expressed various endogenous levels of beta-arrestins. NK1-R displayed rapid internalization that was substantially reduced by DNMs of dynamin-1 and beta-arrestin and even more profoundly in cells lacking both beta-arrestin1 and beta-arrestin2. These internalization data were highly suggestive of the predominant use of the clathrin-mediated pathway by NK1-R, even though NK1-R tended to reside constitutively in lipid raft/caveolae microdomains. Evidence was also obtained that the proper clustering of the receptor in these microdomains was important for effective agonist-induced NK1-R signaling and for its interaction with beta-arrestin2.

We evaluated the effect of haloperidol (HP) and its metabolites on [(3)H](+)-pentazocine binding to sigma(1) receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P(1), P(2) and P(3) subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other sigma(1) antagonists or (-)-sulpiride), [(3)H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of sigma(1) receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-alpha-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked sigma(1) receptors in guinea pig brain homogenate and P(2) fraction in vitro. We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated sigma(1) receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P(2) fraction membranes, which suggests that HP is metabolized to inactivate sigma(1) receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of sigma(1) receptors in brain homogenates. These results suggest that HP may irreversibly inactivate sigma(1) receptors in guinea pig and human cells, probably after metabolism to reduced HP.

Agrin induces, whereas acetylcholine (ACh) disperses, ACh receptor (AChR) clusters during neuromuscular synaptogenesis. Such counteractive interaction leads to eventual dispersal of nonsynaptic AChR-rich sites and formation of receptor clusters at the postjunctional membrane. However, the underlying mechanisms are not well understood. Here we show that calpain, a calcium-dependent protease, is activated by the cholinergic stimulation and is required for induced dispersion of AChR clusters. Interestingly, the AChR-associated protein rapsyn interacted with calpain in an agrin-dependent manner, and this interaction inhibited the protease activity of calpain. Disrupting the endogenous rapsyn/calpain interaction enhanced CCh-induced dispersion of AChR clusters. Moreover, the loss of AChR clusters in agrin mutant mice was partially rescued by the inhibition of calpain via overexpressing calpastatin, an endogenous calpain inhibitor, or injecting calpeptin, a cell-permeable calpain inhibitor. These results demonstrate that calpain participates in ACh-induced dispersion of AChR clusters, and rapsyn stabilizes AChR clusters by suppressing calpain activity.

Background: Development of neural networks requires that synapses are formed, eliminated and stabilized. At the neuromuscular junction (NMJ), agrin/MuSK signaling, by triggering downstream pathways, causes clustering and phosphorylation of postsynaptic acetylcholine receptors (AChRs). Postnatally, AChR aggregates are stabilized by molecular pathways that are poorly characterized. Gain or loss of function of Src-family kinases (SFKs) disassembles AChR clusters at adult NMJs in vivo, whereas AChR aggregates disperse rapidly upon withdrawal of agrin from cultured src-/-;fyn-/- myotubes. This suggests that a balance between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) such as those of the Src-family may be essential in stabilizing clusters of AChRs., Results: We have analyzed the role of PTPs in maintenance of AChR aggregates, by adding and then withdrawing agrin from cultured myotubes in the presence of PTP or PTK inhibitors and quantitating remaining AChR clusters. In wild-type myotubes, blocking PTPs with pervanadate caused enhanced disassembly of AChR clusters after agrin withdrawal. When added at the time of agrin withdrawal, SFK inhibitors destabilized AChR aggregates but concomitant addition of pervanadate rescued cluster stability. Likewise in src-/-;fyn-/- myotubes, in which agrin-induced AChR clusters form normally but rapidly disintegrate after agrin withdrawal, pervanadate addition stabilized AChR clusters. The PTP SHP-2, known to be enriched at the NMJ, associated and colocalized with MuSK, and agrin increased this interaction. Specific SHP-2 knockdown by RNA interference reduced the stability of AChR clusters in wild-type myotubes. Similarly, knockdown of SHP-2 in adult mouse soleus muscle by electroporation of RNA interference constructs caused disassembly of pretzel-shaped AChR-rich areas in vivo. Finally, we found that src-/-;fyn-/- myotubes contained elevated levels of SHP-2 protein., Conclusion: Our data are the first to show that the fine balance between PTPs and SFKs is a key aspect in stabilization of postsynaptic AChR clusters. One phosphatase that acts in this equilibrium is SHP-2. Thus, PTPs such as SHP-2 stabilize AChR clusters under normal circumstances, but when these PTPs are not balanced by SFKs, they render clusters unstable.

CEA functions as an intercellular adhesion molecule and is up-regulated in a wide variety of human cancers, including colon, breast and lung. Its over-expression inhibits cellular differentiation, blocks cell polarization, distorts tissue architecture, and inhibits anoikis of many different cell types. Here we report results concerning the molecular mechanism involved in these biological effects, where relatively rapid molecular changes not requiring alterations in gene expression were emphasized. Confocal microscopy experiments showed that antibody-mediated clustering of a deletion mutant of CEA (DeltaNCEA), normally incapable of self binding and clustering, led to the co-localization of integrin alpha5beta1 with patches of DeltaNCEA on the cell surface. Activation of alpha5, as defined by an anti-alpha5 mAb-sensitive increase in cell adhesion to immobilized fibronectin, and an increased binding of soluble fibronectin to cells, was also observed. This was accompanied by the recruitment of integrin-linked kinase (ILK), protein kinase B (PKB/Akt), and the mitogen-activated protein kinase (MAPK) to membrane microdomains and the phosphorylation of Akt and MAPK. Inhibition of PI3-K and ILK, but not MAPK, prevented the alpha5beta1 integrin activation. Conversely, anti-alpha5 antibody inhibited the PI3-K-mediated activation of Akt, implying the involvement of outside-in and inside-out signaling in integrin activation. Therefore we propose that CEA-mediated signaling involves clustering of CEA and co-clustering and activation of the alpha5beta1 and associated specific signaling elements on the internal surfaces of membrane microdomains. These changes may represent a molecular mechanism for the biological effects of CEA.

Efficient internalization of cell surface receptors requires actin polymerization mediated by Arp2/3 complex and cortactin, a prominent substrate of the protein-tyrosine kinase Src. However, the significance of cortactin tyrosine phosphorylation in endocytosis is unknown. We found that overexpression of a cortactin mutant deficient in tyrosine phosphorylation decreased transferrin uptake. Suppression of cortactin expression by RNA interference also reduced transferrin internalization. Such inhibition was effectively rescued by overexpressing wild-type cortactin but not a cortactin mutant deficient in tyrosine phosphorylation or a mutant with deletion of the Src homology 3 domain. Likewise, purified phosphorylation-null cortactin failed to restore the formation of clathrin-coated vesicles in a cortactin-depleted cell extract. In vitro analysis revealed that Src-mediated phosphorylation enhanced the association of cortactin with dynamin-2 in a tyrosine phosphorylation-dependent manner. Quantitative analysis demonstrated that Src enhances the affinity of cortactin for dynamin-2 by more than 3-fold. On the other hand, Src-treated dynamin-2 had no effect on its interaction with cortactin. These data indicate that Src kinase is implicated in clathrin-mediated endocytosis by phosphorylation of cortactin.

Neuromuscular synaptic transmission depends upon tight packing of acetylcholine receptors (AChRs) into postsynaptic AChR aggregates, but not all postsynaptic AChRs are aggregated. Here we describe a new confocal Fluorescence Resonance Energy Transfer (FRET) assay for semi-quantitative comparison of the degree to which AChRs are aggregated at synapses. During the first month of postnatal life the mouse tibialis anterior muscle showed increases both in the number of postsynaptic AChRs and the efficiency with which AChR was aggregated (by FRET). There was a concurrent two-fold increase in immunofluorescent labeling for the AChR-associated cytoplasmic protein, rapsyn. When 1-month old muscle was denervated, postsynaptic rapsyn immunostaining was reduced, as was the efficiency of AChR aggregation. In vivo electroporation of rapsyn-EGFP into muscle fibers increased postsynaptic rapsyn levels. Those synapses with higher ratios of rapsyn-EGFP to AChR displayed a slower metabolic turnover of AChR. Conversely, the reduction of postsynaptic rapsyn after denervation was accompanied by an acceleration of AChR turnover. Thus, a developmental increase in the amount of rapsyn targeted to the postsynaptic membrane may drive enhanced postsynaptic AChRs aggregation and AChR stability within the postsynaptic membrane.

Agrin is believed to be a factor used by motoneurons to direct acetylcholine receptor (AChR) clustering at the neuromuscular junction. However, exactly how agrin mediates this effect remains unclear. Here we demonstrate that the beta-catenin interacts with rapsyn, a molecule key for AChR clustering. Agrin stimulation increases the association of beta-catenin with surface AChRs. Suppression of beta-catenin expression inhibited agrin-induced AChR clustering, suggesting a necessary role of beta-catenin in this event. The beta-catenin action did not appear to require the function of T-cell factors (TCFs), suggesting a mechanism independent of TCF-mediated transcription. In contrast, prevention of beta-catenin from interacting with alpha-catenin attenuated agrin-induced AChR clustering. These results suggest that beta-catenin may serve as a link between AChRs and alpha-catenin-associated cytoskeleton, revealing a novel function of beta-catenin in synaptogenesis.

Rapsyn, a cytoplasmic receptor-associated protein, is required for the clustering of acetylcholine receptors (AChRs). Although AChR dynamics have been extensively studied, little is known about the dynamics of rapsyn. Here, we used a rapsyn-green fluorescent protein (GFP) fusion protein and quantitative fluorescent imaging to study the dynamics of rapsyn in transfected C2C12 myotubes. First, we found that rapsyn-GFP expression at clusters did not alter AChR aggregation, function, or turnover. Quantification of rapsyn immunofluorescence indicated that the expression of rapsyn-GFP proteins at clusters does not increase the overall rapsyn density compared with untransfected myotube clusters. Using time lapse imaging and fluorescence recovery after photobleaching, we demonstrated that the recovery of rapsyn-GFP fluorescence at clusters was very fast, with a halftime of about approximately 1.5 h (approximately 3 times faster than AChRs). Inhibition of protein kinase C significantly altered receptor insertion, but it had no effect on rapsyn insertion. When cells were treated with the broad spectrum kinase inhibitor staurosporine, receptor insertion was decreased even further. However, inhibition of protein kinase A had no effect on insertion of either rapsyn or receptors. Finally, when cells were treated with neural agrin, rapsyn and AChRs were both directed away from preexisting clusters and accumulated together in new small clusters. These results demonstrate the remarkable dynamism of rapsyn, which may underlie the stability and maintenance of the postsynaptic scaffold and suggest that the insertion of different postsynaptic proteins may be operating independently.

Accumulation of AMPA receptors at synapses is a fundamental feature of glutamatergic synaptic transmission. Stargazin, a member of the TARP family, is an AMPAR auxiliary subunit allowing interaction of the receptor with scaffold proteins of the postsynaptic density, such as PSD-95. How PSD-95 and Stargazin regulate AMPAR number in synaptic membranes remains elusive. We show, using single quantum dot and FRAP imaging in live hippocampal neurons, that exchange of AMPAR by lateral diffusion between extrasynaptic and synaptic sites mostly depends on the interaction of Stargazin with PSD-95 and not upon the GluR2 AMPAR subunit C terminus. Disruption of interactions between Stargazin and PSD-95 strongly increases AMPAR surface diffusion, preventing AMPAR accumulation at postsynaptic sites. Furthermore, AMPARs and Stargazin diffuse as complexes in and out synapses. These results propose a model in which the Stargazin-PSD-95 interaction plays a key role to trap and transiently stabilize diffusing AMPARs in the postsynaptic density.

Ampakines, positive AMPA receptor modulators, can improve cognitive function in schizophrenia, and enhancement of AMPA receptor-mediated currents by them potentiates the activity of antipsychotics. In vitro studies have revealed that trafficking of AMPA receptors is mediated by specific interactions of a complex network of proteins that also target and anchor them at the postsynaptic density (PSD). The aim of this study was to determine whether there are abnormalities of the molecules associated with trafficking and localization of AMPA receptors at the PSD in the dorsolateral prefrontal cortex (DLPFC) in schizophrenia. We analyzed AMPA receptor expression in DLPFC in schizophrenia, major depression, bipolar disorder, and a control group, by examining transcript levels of all four AMPA receptor subunits by in situ hybridization. We found decreased GluR2 subunit expression in all three illnesses, decreased GluR3 in major depression, and decreased GluR4 in schizophrenia. However, autoradiography experiments showed no changes in AMPA receptor binding; thus, we hypothesized that these changes in receptor subunit stoichiometry do not alter binding to the assembled receptor, but rather intracellular processing. In situ hybridization for AMPA-trafficking molecules showed decreased expression of PICK1 and increased expression of stargazin in DLPFC in schizophrenia, both restricted to large cells of cortical layer III. These data suggest that AMPA-mediated glutamatergic neurotransmission is compromised in schizophrenia, particularly at the level of AMPA-related PSD proteins that mediate AMPA receptor trafficking, synaptic surface expression, and intracellular signaling.