A novel protein specifically interacting with Homer2 regulates ubiquitin-proteasome systems

Homer family proteins are encoded by three genes, homer1, 2 and 3. Most of these proteins are expressed constitutively in nervous systems and accumulated in postsynaptic regions. However, the functional significance of these proteins, especially the significance of the distinction among the proteins encoded by homer1, 2 and 3, is still obscure. In the present study, we isolated a cDNA clone encoding a novel protein by two-hybrid system screening using the C-terminal half of Homer2b as the bait. This protein, termed 2B28, has 297 amino acid residues and contains three major domains: a UBA domain, a coiled-coil region, and a UBX domain. When expressed in HEK293T cells, 2B28 showed colocalization with uniquitin and enhanced the expression levels of IkappaB or Homer1a proteins, which are known to be degraded by proteasomes, indicating that 2B28 is involved in ubiquitin-proteasome functions. 2B28 specifically interacted and colocalized with Homer2 proteins, but not with Homer1 proteins. So far, we have identified no counterpart of 2B28 for Homer1 experimentally or in the protein databases. These results suggest that the specific interaction of 2B28 with Homer2 may play a role in regulation of protein degradation by ubiquitin-proteasome systems and that this function may be specific to Homer2 proteins among Homer family proteins.     

Homer proteins shape Xenopus optic tectal cell dendritic arbor development in vivo

Considerable evidence suggests that the Homer family of scaffolding proteins contributes to synaptic organization and function. We investigated the role of both Homer 1b, the constitutively expressed, and developmentally regulated form of Homer, and Homer 1a, the activity-induced immediate early gene, in dendritic arbor elaboration and synaptic function of developing Xenopus optic tectal neurons. We expressed exogenous Homer 1a or Homer 1b in developing Xenopus tectal neurons. By collecting in vivo time lapse images of individual, EGFP-labeled and Homer-expressing neurons over 3 days, we found that Homer 1b leads to a significant decrease in dendritic arbor growth rate and arbor size. Synaptic transmission was also altered in developing neurons transfected with Homer 1b. Cells expressing exogenous Homer 1b over 3 days had a significantly greater AMPA to NMDA ratios, and increased AMPA mEPSC frequency. These data suggest that increasing Homer 1b expression increases excitatory synaptic inputs, increases synaptic maturation, and slows dendritic arbor growth rate. Exogenous Homer 1a expression increases AMPA mEPSC frequency, but did not significantly affect tectal cell dendritic arbor development. Changes in the ratio of Homer 1a to Homer 1b may signal the neuron that overall activity levels in the cell have changed, and this in turn could affect protein interactions at the synapse, synaptic transmission, and structural development of the dendritic arbor.     

Homer 1b/c expression correlates with zebrafish olfactory system development

The zebrafish, (Danio rerio) is an important model organism for the analysis of molecular mechanisms that govern neuronal circuit development. The neuronal circuitry that mediates olfaction is crucial for the development and survival of all teleost fishes. In concert with other sensory systems, olfaction is functional at early stages in zebrafish development and mediates important behavioral and survival strategies in the developing larva. Odorant cues are transduced by an array of signaling molecules from receptors in olfactory sensory neurons. The scaffolding protein family known as Homer is well placed to orchestrate this signaling cascade by interacting with and coupling membrane bound receptors to cytosolic signaling partners. To date, Homer has not been demonstrated in the zebrafish. Here we report that the Homer 1b/c isoform was prominent in the olfactory system from the earliest stages of differentiation. We describe the spatial and temporal distribution of Homer in the zebrafish olfactory system. At 24 hours post fertilization (hpf), Homer expression delineated the boundary of the presumptive olfactory placode. Subsequent expression steadily increased throughout the developing olfactory placode, with a prominent localization to the dendritic knobs of the olfactory sensory neurons. Homer expression in the developing olfactory bulb was punctate and prominent in the glomeruli, displaying an apparent synaptic localization. This work supports the hypothesis that Homer is an important molecule in neuronal circuit development, necessary for crucial behaviors required for development and survival.     

The Homer family proteins

The Homer family of adaptor proteins consists of three members in mammals, and homologs are also known in other animals but not elsewhere. They are predominantly localized at the postsynaptic density in mammalian neurons and act as adaptor proteins for many postsynaptic density proteins. As a result of alternative splicing each member has several variants, which are classified primarily into the long and short forms. The long Homer forms are constitutively expressed and consist of two major domains: the amino-terminal target-binding domain, which includes an Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) domain, and the carboxy-terminal self-assembly domain containing a coiled-coil structure and leucine zipper motif. Multimers of long Homer proteins, coupled through their carboxy-terminal domains, are thought to form protein clusters with other postsynaptic density proteins, which are bound through the amino-terminal domains. Such Homer-mediated clustering probably regulates or facilitates signal transduction or cross-talk between target proteins. The short Homer forms lack the carboxy-terminal domain; they are expressed in an activity-dependent manner as immediate-early gene products, possibly disrupting Homer clusters by competitive binding to target proteins. Homer proteins are also involved in diverse non-neural physiological functions.     

Homer/vesl proteins and their roles in CNS neurons

Since their initial discovery in 1997, Homer/Vesl proteins have become increasingly investigated as putative regulators of receptor and ion-channel function in the central nervous system. Within a relatively brief period, numerous research reports have described manifold effects of Homer proteins, including the modulation of the trafficking of type I metabotropic glutamate receptors (mGluRs), axonal pathfinding, mGluR coupling to calcium and potassium channels, agonist-independent mGluR activity, ryanodine receptor regulation, locomotor activity, and behavioral plasticity. This review summarizes our current knowledge on the induction, expression, and structure of the various forms of Homer proteins, as well as their roles in neuronal function. In addition, we provide an outlook on novel developments with regard to the involvement of Homer-1a in hippocampal synaptic function.     

Down-regulation of Homer1b/c attenuates glutamate-mediated excitotoxicity through endoplasmic reticulum and mitochondria pathways in rat cortical neurons

Glutamate-mediated excitotoxicity is involved in many acute and chronic brain diseases. Homer proteins, a new member of the postsynaptic scaffolding proteins, regulate glutamatergic signaling and intracellular calcium mobilization in the central nervous system. Here we investigated the effects of down-regulating Homer1b/c, a constitutively expressed long form of Homer proteins, on glutamate excitotoxicity-induced neuronal injury. In our in vitro excitotoxic models, we demonstrated that glutamate insults led to a dose-dependent neuronal injury, which was mediated by the intracellular calcium-dependent reactive oxygen species (ROS) production. We found that down-regulation of Homer1b/c with specific small interfering RNA (siRNA) improved neuronal survival, inhibited intracellular ROS production, and reduced apoptotic cell death after neurotoxicity. Homer1b/c knockdown decreased the intracellular calcium overload through inhibition of the group I metabotropic glutamate receptor (mGluR)/inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the endoplasmic reticulum (ER) in injured neurons. In addition, Homer1b/c siRNA transfection attenuated the activation of eukaryotic initiation factor 2α (eIF2α), RNA-dependent protein kinase-like ER kinase (PERK) and caspase-12, and inhibited the up-regulation of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) after glutamate treatment. Homer1b/c knockdown also preserved the mitochondrial membrane potential (MMP), reduced cytochrome c (Cyt. c) release, and partly blocked the increase of capase-9 activity and Bax/Bcl-2 ratio. Taken together, these results suggest that down-regulation of Homer1b/c protects cortical neurons against glutamate-induced excitatory damage, and this neuroprotection may be dependent at least in part on the inhibition of calcium-dependent ROS production and the preservation of the ER and mitochondrial function.     

Ena/VASP: proteins at the tip of the nervous system

The emergence of neurites from a symmetrical cell body is an essential feature of nervous system development. Neurites are the precursors of axons and dendrites and are tipped by growth cones, motile structures that guide elongating axons in the developing nervous system. Growth cones steer the axon along a defined path to its appropriate target in response to guidance cues. This navigation involves the dynamic extension and withdrawal of actin-filled finger-like protrusions called filopodia that continuously sample their environment. Ena/VASP proteins, a conserved family of actin-regulatory proteins, are crucial for filopodia formation and function downstream of several guidance cues. Here we review recent findings into Ena/VASP function in neurite initiation, axon outgrowth and guidance.     

Homer regulation of native plasma membrane calcium channels in A431 cells

Homers are adapter proteins that play a significant role in the organization of calcium signaling protein complexes. Previous functional studies linked Homer proteins to calcium influx in nonexcitable cells. These studies utilized calcium imaging or whole-cell current recordings. Because of limited resolution of these methods, an identity of Homer-modulated ion channels remained unclear. There are several types of plasma membrane calcium influx channels in A431 cells. In the present study, we demonstrated that Homer dissociation resulted in specific activation of I(min) channels but not of I(max) channels in inside-out patches taken from A431 cells. In contrast, inositol 1,4,5-trisphosphate activated both I(min) and I(max) channels in inside-out patches. Short (1a) and long (1c) forms of Homer had different effects on I(min) channel activity. Homer 1a but not Homer 1c activated I(min) in the patches. This study indicates that I(min) channels are specifically regulated by Homer proteins in A431 cells.     

Scaffold protein Homer 1: Implications for neurological diseases

Homer proteins are commonly known as scaffold proteins at postsynaptic density. Homer 1 is a widely studied member of the Homer protein family, comprising both synaptic structure and mediating postsynaptic signaling transduction. Both an immediate-early gene encoding a Homer 1 variant and a constitutively expressed Homer 1 variant regulate receptor clustering and trafficking, intracellular calcium homeostasis, and intracellular molecule complex formation. Substantial preclinical investigations have implicated that each of these Homer 1 variants are associated with the etiology of many neurological diseases, such as pain, mental retardation syndromes, Alzheimer's disease, schizophrenia, drug-induced addiction, and traumatic brain injury.     

Mice lacking Homer 1 exhibit a skeletal myopathy characterized by abnormal transient receptor potential channel activity

Transient receptor potential (TRP) channels are nonselective cation channels, several of which are expressed in striated muscle. Because the scaffolding protein Homer 1 has been implicated in TRP channel regulation, we hypothesized that Homer proteins play a significant role in skeletal muscle function. Mice lacking Homer 1 exhibited a myopathy characterized by decreased muscle fiber cross-sectional area and decreased skeletal muscle force generation. Homer 1 knockout myotubes displayed increased basal current density and spontaneous cation influx. This spontaneous cation influx in Homer 1 knockout myotubes was blocked by reexpression of Homer 1b, but not Homer 1a, and by gene silencing of TRPC1. Moreover, diminished Homer 1 expression in mouse models of Duchenne's muscular dystrophy suggests that loss of Homer 1 scaffolding of TRP channels may contribute to the increased stretch-activated channel activity observed in mdx myofibers. These findings provide direct evidence that Homer 1 functions as an important scaffold for TRP channels and regulates mechanotransduction in skeletal muscle.     

Homer 2 tunes G protein-coupled receptors stimulus intensity by regulating RGS proteins and PLCbeta GAP activities

Homers are scaffolding proteins that bind G protein-coupled receptors (GPCRs), inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), ryanodine receptors, and TRP channels. However, their role in Ca2+ signaling in vivo is not known. Characterization of Ca2+ signaling in pancreatic acinar cells from Homer2-/- and Homer3-/- mice showed that Homer 3 has no discernible role in Ca2+ signaling in these cells. In contrast, we found that Homer 2 tunes intensity of Ca2+ signaling by GPCRs to regulate the frequency of [Ca2+]i oscillations. Thus, deletion of Homer 2 increased stimulus intensity by increasing the potency for agonists acting on various GPCRs to activate PLCbeta and evoke Ca2+ release and oscillations. This was not due to aberrant localization of IP3Rs in cellular microdomains or IP3R channel activity. Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit Ca2+ signaling in vivo. Moreover, Homer 2 preferentially bound to PLCbeta in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLCbeta in an in vitro reconstitution system, with minimal effect on PLCbeta-mediated PIP2 hydrolysis. These findings describe a novel, unexpected function of Homer proteins, demonstrate that RGS proteins and PLCbeta GAP activities are regulated functions, and provide a molecular mechanism for tuning signal intensity generated by GPCRs and, thus, the characteristics of [Ca2+]i oscillations.     

Live imaging of endogenous Collapsin response mediator protein-1 expression at subcellular resolution during zebrafish nervous system development

Collapsin response mediator proteins (CRMPs) are cytosolic phosphoproteins that are functionally important during vertebrate development. We have generated a zebrafish gene trap line that produces fluorescently tagged Crmp1 protein, which can be dynamically tracked in living fish at subcellular resolution. The results show that Crmp1 is expressed in numerous sites in the developing nervous system. Early expression is apparent in the forebrain, epiphysis, optic tectum and the developing spinal cord. In the larval brain, Crmp1 is expressed in several distinct brain regions, such as the telencephalon, habenula and cerebellum. In addition, it is expressed in the spinal cord in a manner that persists in the larva. The results suggest that this Crmp1 protein trap line offers a powerful tool to track selected neuronal populations at high resolution.     

Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain

Glutamatergic signaling and intracellular calcium mobilization in the spinal cord are crucial for the development of nociceptive plasticity, which is associated with chronic pathological pain. Long-form Homer proteins anchor glutamatergic receptors to sources of calcium influx and release at synapses, which is antagonized by the short, activity-dependent splice variant Homer1a. We show here that Homer1a operates in a negative feedback loop to regulate the excitability of the pain pathway in an activity-dependent manner. Homer1a is rapidly and selectively upregulated in spinal cord neurons after peripheral inflammation in an NMDA receptor-dependent manner. Homer1a strongly attenuates calcium mobilization as well as MAP kinase activation induced by glutamate receptors and reduces synaptic contacts on spinal cord neurons that process pain inputs. Preventing activity-induced upregulation of Homer1a using shRNAs in mice in vivo exacerbates inflammatory pain. Thus, activity-dependent uncoupling of glutamate receptors from intracellular signaling mediators is a novel, endogenous physiological mechanism for counteracting sensitization at the first, crucial synapse in the pain pathway. Furthermore, we observed that targeted gene transfer of Homer1a to specific spinal segments in vivo reduces inflammatory hyperalgesia. Thus, Homer1 function is crucially involved in pain plasticity and constitutes a promising therapeutic target for the treatment of chronic inflammatory pain.     

Homer proteins and InsP(3) receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres

Striated muscle represents one of the best models for studies on Ca(2+) signalling. However, although much is known on the localisation and molecular interactions of the ryanodine receptors (RyRs), far less is known on the localisation and on the molecular interactions of the inositol trisphosphate receptors (InsP(3)Rs) in striated muscle cells. Recently, members of the Homer protein family have been shown to cluster type 1 metabotropic glutamate receptors (mGluR1) in the plasma membrane and to interact with InsP(3)R in the endoplasmic reticulum of neurons. Thus, these scaffolding proteins are good candidates for organising plasma membrane receptors and intracellular effector proteins in signalosomes involved in intracellular Ca(2+) signalling. Homer proteins are also expressed in skeletal muscle, and the type 1 ryanodine receptor (RyR1) contains a specific Homer-binding motif. We report here on the relative sub-cellular localisation of InsP(3)Rs and Homer proteins in skeletal muscle cells with respect to the localisation of RyRs. Immunofluorescence analysis showed that both Homer and InsP(3)R proteins present a staining pattern indicative of a localisation at the Z-line, clearly distinct from that of RyR1. Consistent herewith, in sub-cellular fractionation experiments, Homer proteins and InsP(3)R were both found in the fractions enriched in longitudinal sarcoplasmic reticulum (LSR) but not in fractions of terminal cisternae that are enriched in RyRs. Thus, in skeletal muscle, Homer proteins may play a role in the organisation of a second Ca(2+) signalling compartment containing the InsP(3)R, but are apparently not involved in the organisation of RyRs at triads.