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. Author manuscript; available in PMC: 2022 Feb 27.
Published in final edited form as: J Neurochem. 2012 Apr 27;122(1):24–37. doi: 10.1111/j.1471-4159.2012.07752.x

Proteasomal degradation of the metabotropic glutamate receptor 1α is mediated by Homer-3 via the proteasomal S8 ATPase

Khosrow Rezvani *, Kelli Baalman †, Yanfen Teng †, Maureen P Mee †, Simon P Dawson ‡, Hongmin Wang *, Mariella De Biasi †, R John Mayer ‡
PMCID: PMC8882371  NIHMSID: NIHMS1758471  PMID: 22486777

Abstract

The metabotropic glutamate receptors (mGluRs) fine-tune the efficacy of synaptic transmission. This unique feature makes mGluRs potential targets for the treatment of various CNS disorders. There is ample evidence to show that the ubiquitin proteasome system mediates changes in synaptic strength leading to multiple forms of synaptic plasticity. The present study describes a novel interaction between post-synaptic adaptors, long Homer-3 proteins, and one of the 26S proteasome regulatory subunits, the S8 ATPase, that influences the degradation of the metabotropic glutamate receptor 1α (mGluR1α). We have shown that the two human long Homer-3 proteins specifically interact with human proteasomal S8 ATPase. We identified that mGluR1α and long Homer-3s immunoprecipitate with the 26S proteasome both in vitro and in vivo. We further found that the mGluR1α receptor can be ubiquitinated and degraded by the 26S proteasome and that Homer-3A facilitates this process. Furthermore, the siRNA mediated silencing of Homer-3 led to increased levels of total and plasma membrane-associated mGluR1α receptors. These results suggest that long Homer-3 proteins control the degradation of mGluR1α receptors by shuttling ubiquitinated mGluR-1α receptors to the 26S proteasome via the S8 ATPase which may modulate synaptic transmission.

Keywords: 26S proteasome, degradation, Homer protein, metabotropic glutamate receptor, protein trafficking, ubiquitin

Members of mGluRs (mGluR1–8) are involved in brain development as well as learning, injury, and neuroprotection processes through modulation of synaptic transmission, synaptic plasticity, neuronal excitability, and glial functions. Both mGluR1 and mGluR5 receptors are located in the neuronal soma and dendrites at the periphery of post-synaptic densities and they have a key role in some forms of synaptic plasticity, including long-term potentiation and long-term depression (Benarroch 2008). Group 1 mGluR receptors are associated with several accessory proteins including Homer proteins which are important in regulating receptor stability, localization and function (Xiao et al. 2000; Foa and Gasperini 2009). The Homer family of proteins has 17 isoforms alternatively spliced into long and short forms (Soloviev et al. 2000a). The first short Homer protein (Homer-1A) was discovered as an immediate early gene expressed during intense neuronal activity (Brakeman et al. 1997) and homeostatic scaling (Hu et al. 2010). In contrast to Homer-1A, long Homers are expressed constitutively. Both long and short Homer proteins contain an enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) domain at the N-terminus. In addition to EVH1 domain, only long Homer proteins have a coiled-coil structure and leucine zipper motif at the C-terminus (Renfranz and Beckerle 2002). The EVH1-like domains of Homer proteins bind to proline-rich motifs in the C-termini of metabotropic glutamate receptors. Initially, Homer proteins were described as adaptor proteins that control the interaction of G protein-coupled metabotropic glutamate receptors (mGluRs) with intracellular calcium stores, thereby contributing to the regulation of brain functions such as cognition, motor control, and neural development (Tu et al. 1998). Further reports suggest that besides regulation of intracellular calcium homeostasis, Homer proteins are involved in other cellular functions such as receptor trafficking, clustering activity, and cytoskeletal organization (Foa and Gasperini 2009).

Group 1 mGluR receptors are exclusively ubiquitinated by Siah1A a RING-finger-containing E3 ubiquitin ligase. Consequently, poly-ubiquitinated forms of mGluR1α receptors are degraded by the 26S proteasome complex (Moriyoshi et al. 2004). The 26S proteasome recognizes and binds proteins (mainly polyubiquitinated), unfolds these substrates, and feeds them into the proteolytic chambers of the 20S cylindrical core. The 19S regulator of the 26S proteasome consists of a base and a lid: the base contains six related non-redundant ATPases of the AAA superfamily (S4, S6, S6′, S7, S8, and S10b) plus two non-ATPases (Finley 2009). These regulatory subunits possess ATPase and coiled-coil domains without a predicted proline-rich domain (Fu et al. 2001). Recent studies indicate that the ubiquitin-proteasome system (UPS) is able to control a wide-range of functions in the nervous system (Hegde 2010) through its interaction with a variety of adaptor proteins (Guerrero et al. 2008). More interestingly, the 26S proteasome-interacting proteins are different in the cytosolic and synaptic proteome and their associations with the proteasome complex are altered by neuronal activity resulting in changes necessary for fine-tuning of synaptic connections during development and synaptic plasticity (Tai et al. 2010).

Regulation of mGluR’s activities by different Homer family proteins suggests that Homer proteins can use different mechanisms, including the UPS to temporally and spatially fine-tune these receptors. Therefore, it is important to understand the mechanisms and role of Homer proteins when interacting with these receptors. In the present paper, we demonstrate that long Homer-3 proteins can regulate the proteasomal degradation of mGluR1α receptors through a novel pathway which involves the S8 ATPase of the 26S proteasome. Over-expression and siRNA studies show that Homer-3 regulates the total and plasma membrane (PM) level of mGluR1α. This newly functional role of Homer-3 will promote a better understanding of the neuronal physiological and pathological functions of this protein family. A preliminary report of these studies has been presented orally (Rezvani et al. 2003).

Materials and methods

Yeast two-hybrid screens and antibodies

Identification of Homer-3A11 as an S8 ATPase interaction partner was performed with the Matchmarker two-hybrid system (Clontech) essentially as described by the manufacturer (for details, see Appendix S1). A list of antibodies used for Western blot and immunofluorescence experiments are summarized in Table S1.

Cell culture, transfection, and immunofluorescence microscopy

HEK293 cells and PC12 cells (dPC12) differentiated with 50 ng/mL nerve growth factor (Sigma, St Louis, MO, USA) as well as rat hippocampal and cortical primary neuronal cells were cultured and transfected as described previously (Rezvani et al. 2009; Schafer et al. 2009). For degradation assays, the proteasomal inhibitor MG132 (10 μM; Enzo Life Science, Farmingdale, NY, USA) or PS-341 (Velcade, generous gift from Dr John F. DeGroot, M.D. Anderson Cancer Center, Houston) was used in the presence of the protein synthesis blocker emetine (150 μM; Calbiochem San Diego, CA, USA). A Tris–HCl buffer (pH 7.5) containing 0.025% digitonin was used to prepare cytosolic extracts (Kisselev and Goldberg 2005).

siRNA transfections

A predesigned siRNA against Homer-3 was obtained from Qiagen Inc (Valencia, CA, USA). The siRNA targets the CAGCTGGAA GCTCTGGTGCAA oligonucleotide sequence (clone 3–1), and has been characterized previously (Parisiadou et al. 2008). siRNAs (clone 3–1 or control) were transfected into cells using the HiPerFect Transfection Reagent according to the manufacturer’s protocol (Qiagen). Differentiated PC12 cells and rat primary cortical neurons (Figure S4) were transfected with siRNA.

Densitometric quantification of western blot bands

Digitalized western blot bands were quantified by measuring pixel numbers with an automated digitizing system (UN-Scan-it gel, version 6.1). The total measured pixels of each band were normalized by the corresponding loading control.

Statistical analysis of data

Atleast three biological repeats were performed for all experiments with bar graph. The rest of experiments were repeated twice with similar results. Statistical values were analysed with either the Student’s t test or by one-way anova and Tukey’s multiple comparison post-tests, when appropriate. The means were compared considering a p-value ≤ 0.05 as a significant difference (mean ± SEM).

Full methods and any associated references are available in Appendix S1.

Results

Homer-3A11 binds exclusively to the proteasomal S8 (rpt6) ATPase

We screened a HeLa cDNA library with the human S8 ATPase cDNA in a yeast two-hybrid (Y2H) screen and detected the protein Homer-3A11 (Fig. 1a). The N-terminal half (1–171 amino acids) of the S8 ATPase with a coiled-coil domain is sufficient to interact with the C-terminal half (191–358 amino acids) of Homer-3A11 containing a coiled-coil domain. Further Y2H experiments showed that only long Homer-3 subfamily (3A and 3B), but not short Homer-3D, Homer-1A and -1B, and Homer-2A family, members interact with the S8 ATPase of the 19S proteasomal regulator (Tables S2–S4,). Our data strongly suggest that the long Homer-3 proteins (3A and 3B) use their coiled-coil domains to bind to the coiled-coil domain of the S8 ATPase as has been described for several other protein-protein interactions (Newman et al. 2000). Because the coiled-coil interactions do not occur indiscriminately and instead are quite specific (Zhang et al. 2009), we believe that only the coiled-coil domain of the long Homer-3 proteins (table S3) is capable of binding to the coiled-coli domain of the S8 ATPase (Tables S2 and S4). [35S] Homer-3A11 and [35S] Homer-3B11 proteins interacted with the (His)6-S8 ATPase in vitro, thus confirming the genetic interactions in the Y2H screen biochemically (Fig. 1b). Of course, genetic and biochemical analyses of all the Homer isoforms were not completed because of their large number (Shiraishi-Yamaguchi and Furuichi 2007), but the overall analysis confirmed that only the long Homer-3 (Homer-3A and -3B) subfamily of proteins, and not Homer-1 and -2, bind to the S8 ATPase of the proteasome complex.

Fig. 1.

Fig. 1

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The S8 ATPase interacts selectively with long Homer-3 proteins. (a) This is a schematic representation of the modulatory domains in the human S8 ATPase subunit of the 26S proteasome (upper panel), Homer-3A11 (middle panel) and Homer-3B11 (lower panel). The S8 ATPase has a coiled-coil domain located at the N-terminus and an AAA domain (for ATPase associated with alternative cellular activities) at the C-terminus. Homer-3A11 and -3B11 protein splice variants have an EVH1-like (Enabled/Vasp homology) domain in the N-terminus and a coiled-coil domain at the C-terminus. Homer-3B11 lacks 36 amino acids at the N-terminus. Arrowheads indicate other splice variants in Homer-3A and -3B which are generated by the presence or the absence of amino acids located at 817–825aa (no. 1) and 895–903aa (no. 2). (b) In vitro co-immunoprecipitation analyses of the interaction of the S8 ATPase and Homer-3 subfamily confirmed the Y2H screen because [35S] Homer-3A11 and [35S] Homer-3B11 (arrows in lane I and II) interact with the (His)6-S8 ATPase using Nickel-NTA Sepharose. Left to right lanes are [35S] Homer-3A11 and (His)6 -S8 ATPase, [35S] Homer-3B11 and (His)6 -S8 ATPase, [35S] Homer-3C and (His)6-S8 ATPase and [35S] Homer-3A11 and (His)6-S6 ATPase.

Homer-3 co-fractionates with the S8 ATPase and mGluR1α in rat hippocampal HC2S2 cells

Cell lysates prepared from differentiated HC2S2 cells (Hoshimaru et al. 1996) were subjected to a linear iodixanol gradient fractionation (Rezvani et al. 2010), enabling an optimized separation of ER, Golgi, and plasma membrane compartments. The collected iodixanol gradient fractions were probed with anti-mGluR1α, S8 ATPase, and Homer-3 antibodies as well as anti-calnexin [(endoplasmic reticulum marker (ER)], anti-GM-130 (Golgi marker) and anti-pan-Cadherin (PM marker) antibodies. Homer-3 and the S8 ATPase cosedimented with some of the mGluR1α receptors in enriched ER compartment (fractions 11–14, Fig. 2a). The S8 ATPases showed an extra peak in fractions 3–8 which can represent another subpopulation of proteasome complexes (Drews et al. 2007).

Fig. 2.

Fig. 2

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A complex of Homer-3/the S8 ATPase and mGluR1α is enriched in the endoplasmic reticulum of rat hippocampal HC2S2 cells. (a) Lysates prepared from differentiated rat hippocampal HC2S2 cells were subjected to a high-speed iodixanol density gradient centrifugation. Western blot analyses of collected fractions revealed that Homer-3 exclusively co-sediments with the S8 ATPase and mGluR1α in an enriched-ER compartment probed by the anti-calnexin antibody (fractions 11–14, boxed with broken lines). Anti-pan-cadherin (PM marker, fractions 3 and 4) and anti-GM-130 (Golgi marker, fractions 5–7) antibodies showed location of those mGluR1α receptors already transported from the ER. (b) Agarose-conjugated monoclonal antibodies against the α2 subunit immunoprecipitate the 26S proteasome from rat hippocampal HC2S2 cell lysates. Prior to performing specific protein detection, proteins were subjected to 4–20% gradient SDS–PAGE followed by Sypro ruby protein gel stain. Sypro staining revealed a ladder of proteasomal subunits typically seen with proteasome pull down (Kriegenburg et al. 2008; lane I) in comparison to the total cell lysate (lane II). (c) The precipitated proteins used in panel (b) were also transferred to nitrocellulose membranes and probed with anti-S8 antibodies (upper panel) and anti-pan α (α1, α2, α3, α5, α6 and α7, lower panel) to confirm that anti-α2 antibodies can pull down the entire 26S complex. (d, e) Ultimately, the immunoprecipitation products were probed with anti-Homer-3 (d) and anti-mGluR1α (e) antibodies. Results indicate that mGluR1α and Homer-3A can be pulled down with the 26S proteasome. The asterisks indicate location of IgG-heavy chains.

Because the S8 ATPase is a subunit of the 26S proteasome, we next investigated whether Homer-3 and mGluR1α can bind to the 26S proteasome in rat hippocampal HC2S2 cells. To answer this question, we first confirmed that an immobilized monoclonal antibody (MCP21) to the proteasomal 20S core, α2 can pull down the hippocampal rat 26S proteasome complex using Sypro ruby staining (Fig. 2b) plus specific antibodies against the 20S core complex and the S8 ATPase of 19S regulatory complex (Fig. 2c). Fig. 2d and e shows that both Homer-3 (Fig. 2d) and mGluR1α (Fig. 2e) bind to the 26S proteasome in HC2S2 cells. Similar set of experiments conducted in HEK293 cells expressing Flag-tagged mGluR1α verify the above results (data not shown). Collectively, data obtained in HEK293 cells and HC2S2 cells indicate the physiological existence of a complex of mGluR1α/long Homer and S8 ATPase-containing 26S proteasomes located in an enriched ER compartment.

Homer-3 co-fractionates with proteasomes and mGluR1α in vivo

We next examined the subcellular localization of Homer-3 and the S8 ATPase in the prefrontal cortex (PFC) of C57BL/6J male mice. The microarray data provided by the Allen Institute for Brain Science indicates Homer-3 (experiment no.356087) express moderately in the neocortex, besides the hippocampus or cerebellum. However, studies in rats have found that mGluR1α is expressed in the cortex (Petralia et al. 1997). Because of the above fact, we tested the protein levels of Homer-3 and mGluR1α as well as the S8 ATPase in PFC. We were able to observe detectable levels of these three proteins in PFC. Three PFCs were dissected, homogenized and mixed, followed by iodixanol gradient fractionation. We used a linear iodixanol gradient with nine high-resolution layers optimized for ER and Golgi separation (Chen et al. 2003). Western blotting of collected fractions (Fig. 3a) revealed that some endogenous Homer-3 co-sediments with fractions containing the S8 ATPase. In addition, some mGluR1α receptors are present in fractions containing Homer-3/S8 ATPase (fractions 8–13, Fig. 3a). Finally, Fig. 3a suggests that Homer-3/S8 ATPase and mGluR1α coexist in a single complex within the ER-Golgi compartment as they co-sediment with calreticulin-enriched vesicles. In this experiment, we used calreticulin, a soluble luminal analog of calnexin (Danilczyk et al. 2000), as an ER marker because calnexin showed only a partial enrichment in fractions 8–13. It should be noted, however, that Homer-3 proteins were segregated into at least two populations located in 8–13 and 17–18 fractions. Homer-3 proteins in fraction 17 and 18 may represent other complexes containing Homer-3 proteins.

Fig. 3.

Fig. 3

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Homer-3 and mGluR1α receptors are associated with the S8 ATPase of the 26S proteasome in vivo. (a) To analyse the in vivo compartmentalization of mGluR1α, Homer-3, and S8 ATPase, extracts of three PFCs (prefrontal cortex) isolated from male C57BL/6J mice (~25 g) were loaded on to a column of iodixanol discontinuous gradient (adapted from Chen et al. 2003). Eighteen fractions of ~700 μL were collected and equal volume of fractions were analysed by Western blotting. Homer-3, mGluR1α and the S8 ATPase cosediment in fractions 8–13 which are enriched in calreticulin, a marker protein for the ER-Golgi. (b) An antibody to the proteasomal S8 ATPase subunit co-immunoprecipitates Homer-3 (lane II, upper panel) and mGluR1α (lane II, lower panel) after incubation of mouse pre-frontal cortex tissue lysates with anti-S8 antibodies immobilized on protein A/G-agarose beads. Lane III shows 10% input of the PFCs. Lane I in panel (b) shows precipitation with non-specific immunoglobulins.

PFC tissue lysates were subjected to immunoprecipitation of the S8 ATPase with a monoclonal anti-S8 ATPase antibody. Homogenized PFCs were incubated with beads coupled to mouse IgG as control. The interaction of Homer-3 with the S8 ATPase was then detected by immunoblotting with anti-Homer-3 rabbit polyclonal antibodies (Fig. 3b, upper panel lane II). The same immunoprecipitation of the S8 ATPase was probed with anti-mGluR1α antibodies to confirm the presence of mGluR1α with Homer-3 and the S8 ATPase (Fig. 3b, lower panel lane II). Figure 3b lane I and III (upper and lower panels) represents an immobilized IgG on the beads and 10% of input, respectively. In the presence of β-mercaptoethanol, the anti-mGluR1α antibodies detect the monomeric form of the receptors dominantly as previously described (Ray and Hauschild 2000). These in vivo experiments showed that Homer-3 is associated with the S8 ATPase and mGluR1α in an ER enriched compartment, thus suggesting a physiological interaction between Homer-3 and the S8 ATPase in vivo.

Homer-3 co-localizes with the S8 ATPase in rat primary neurons

Primary hippocampal neurons isolated from day 18 embryonic Sprague–Dawley rat plated in a 24-well culture plate at density of 5 × 105 per well were observed by confocal fluorescence microscopy. Cells were incubated with a rabbit polyclonal anti-Homer-3 antibody and a mouse monoclonal anti-S8 ATPase antibody followed by visualisation with the corresponding fluorescent secondary antibodies. Homer-3 shows abundant punctate structures located around the nucleus (Fig. 4a) where the ER apparatus is typically found in large vesicular structures (Mauceri et al. 2004). The S8 ATPase shows mostly diffuse small punctate structures scattered throughout the cytoplasm and nucleus, reflecting the S8 ATPase in the 26S proteasome (Fig. 4b). While we did not observe frequent close colocalization of the two proteins, the z-stack image of the perforated square in Fig. 4d confirmed colocalization of Homer-3 and S8 ATPase (Fig. 4e) indicating that some of these proteins are concentrated in the same structures. The Homer-3 and S8 ATPase signals overlapped in perinuclear structures and adjacent compartments, possibly reflecting the large pools of Homer-3 and proteasomes localized to the ER-Golgi compartment as described previously (Roche et al. 1999). Similar results were achieved when HEK293 cells were transiently transfected with HA-tagged Homer-3 and GFP-S8 ATPase (data not shown). In addition to the cell body of hippocampal neurons, colocalization of Homer-3 and the S8 ATPase appears to occur in the neuronal processes (Fig. 4f–h) indicated by white arrows in magnified image in Fig. 4i.

Fig. 4.

Fig. 4

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Co-localization of the S8 ATPase and Homer-3 in rat hippocampal neurons. (a–e) Rat hippocampal neurons were cultured on coverslips and processed for immunocytochemistry. Staining of the permeabilized cells for the S8 ATPase (red) and Homer-3 (green) demonstrated partial overlap of S8 and Homer-3 (arrows in panel e), particularly in the perinuclear region of the cells. Images are a projection of a z-stack. (f–i) These panels demonstrate neuronal process in hippocampal neurons stained for Homer-3 (green) and the S8 ATPase (red). Arrows in panel i point to punctate clusters along the length of the neuronal branch containing both S8 and Homer-3, which appeared yellow. The region indicated by the white dashed box in the merge panels (d and h) are magnified in panels (e) and (i).

Homer-3 increases the ubiquitination and degradation of mGluR1α

As Homer-3 itself is not targeted for proteasomal degradation, unlike Homer-1A (Ageta et al. 2001), we asked if Homer-3 can be a negative regulator of mGluR1α receptors. To answer this question, we verified that mGluR1α receptors can be ubiquitinated in the cells (Figure S1a). Degradation of the receptor was inhibited by MG132 (Figure S1b) and PS-341 (Figure S1c) proteasomal inhibitors. The receptor is degraded in HEK293 cells with a half-life of approximately 12 h (Figure S1d and e). We tested if transient over-expression of increasing amounts of (His)6-TYG tagged Homer-3 can influence endogenous mGluR1α protein abundance. Figure 5a shows that over-expression of Homer-3 induces significant down-regulation of mGluR1α in a dose-dependent manner while no significant changes were observed in the S8 ATPase and 20S proteasome complexes (probed with anti-pan α antibody). To determine whether Homer-3A is necessary for degradation of mGluR1α, we also studied the degradation of endogenous mGluR1α in dPC12 cells stably expressing either WT Homer-3A11, the N-terminus of Homer-3A11, or the empty vector. We expressed the N-terminus of Homer-3A11 (residues 1–142) because this region of Homer proteins interacts with the C-terminal tails of metabotropic glutamate receptors but not the proteasomal ATPase. We demonstrated that both WT and the N-terminus of Homer-3A11 are expressed by western analysis of cell extracts (Figure S2a). We then investigated whether the presence of Homer-3A11 can decrease the stability of mGluR1α receptors. Cell lysates of dPC12 cells stably expressing WT Homer-3A11, the N-terminus of Homer-3A11 or the empty vector were probed with anti-mGluR1α antibodies. Over-expression of WT-Homer-3A11 significantly reduces the levels of mGluR1α (Fig. 5b and c, 64 ± 3%, p < 0.001). Expressing the N-terminus of Homer-3A11 had no effect on the degradation of mGluR1α over a 24-h period (Fig. 5b and c). The N-terminus of Homer-3A11 lacks the C-terminus necessary for the interaction with the S8 ATPase and therefore cannot facilitate degradation of mGluR1α by the 26S proteasome. As expected, given the specificity of the interaction of the Homer-3A11 with mGluR1α, there was no effect of the Homer 3A11 on the degradation of the α7 nAChRs endogenously expressed in dPC12 cells (Figure S2b). Next, cell lysates were subjected to p62 pull down. The agarose-immobilized p62-derived Ubiquitin-associated domain (UBA) domain pulls down ubiquitinated proteins (Rezvani et al. 2009). Figure 5d and e shows that over-expression of WT Homer-3A11, but not empty vector or N-terminus of Homer-3A11, significantly increased the levels of ubiquitinated mGluR1α receptors (43 ± 6%, p < 0.005).

Fig. 5.

Fig. 5

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Homer-3A11 increases the degradation of mGluR1α receptors in differentiated PC12 cells. (a) Differentiated PC12 (dPC12) cells were transiently transfected with increasing amounts of Homer-3A11. WB analyses of cell lysates showed Homer-3A11, probed with an anti-TYG antibody, induces a dose-dependent decrease in endogenous mGluR1α receptor levels after 24 h while no changes were observed for the level of the S8 ATPase and 20S proteasomes. An anti-GAPDH antibody was used for loading control. Experiments were performed two times with similar results. (b, c) The total cell lysates from dPC12 cells stably transfected with the corresponding empty vector, NT-Homer-3A11, or WT-Homer-3A11 in the presence of emetine (protein synthesis blocker) for 24 h were subjected to SDS–PAGE, followed by Western blot analyses using anti-mGluR1α antibodies (b). The results indicated that WT-Homer-3A11 significantly increases the degradation of endogenous mGluR1α (panel c; ***p < 0.001) while the truncated form (NT-Homer-3A11) and empty vector have no effect (n = 3, panel c). (d) P62 pull down was performed on the cell lysates used in panel (b). Western blot analysis with anti-mGluR1α antibodies detected a ladder of ubiquitinated mGluR1α in each sample. Bands around and under 95-kDa may represent truncated receptors retained on beads. (e) Quantification of the p62 pull-down blots showed that WT-Homer-3A11, but not NT-Homer-3A11, and empty vector produced a modest but significant increase in the levels of ubiquitinated mGluR1α (panel e; **p < 0.005, n = 3).

Homer-3 silencing substantially increases cellular and cell surface mGluR1α levels

We further tested the hypothesis that the Homer-3/S8 ATPase interaction can mediate degradation of mGluR1α receptors by the 26S proteasome by transfecting dPC12 cells with siRNA against Homer-3. To functionally silence Homer-3, we obtained Homer-3 siRNA constructs (clone 3–1) from Qiagen. Clone 3–1 showed the maximum silencing effect (Parisiadou et al. 2008) and Figure S2c. A scrambled siRNA was used as a control. Fourty-eight hours after transfection, cell lysates were prepared and subjected to Western blot analyses. These experiments showed that silencing of Homer-3 leads to an increase in the levels of total mGluR1α (Fig. 6a). There were no significant changes in the level of proteasome components (Fig. 6a) with the decreased level of Homer-3 (65 ± 10%). Silencing Homer-3 did not alter the level of α7 nAChRs subunit (Fig. 6a). Quantitative analysis confirmed a significant increase in the total levels of mGluR1α (Fig. 6b, 200 ± 21%; p < 0.001) but not α7.

Fig. 6.

Fig. 6

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Long Homer-3s regulate the stability and plasma membrane level of mGluR1α receptors in differentiated PC12 cells. (a) Cells were transfected with siRNA against Homer-3 in 6-well plates. Fourty-eight hours after transfection, cell lysates were prepared. As expected, western blot analysis indicates a considerable decrease in Homer 3 (~60% reduction). Western blot analysis revealed that the amount of total mGluR1α was significantly increased in siRNA-transfected cells. Probes for other proteins showed no change in the amounts of proteasome components (20S core and the S8 ATPase) and nicotinic acetylcholine receptor α7. (b) Quantification of mGluR1α and α7 showed that siRNA against Homer-3 causes an approximate doubling in mGluR1α receptors (b, 200 ± 21%; ***p < 0.001). The graph in panel (b) shows cumulative data from three experiments. Anti-GAPDH (a) antibody was used as a loading control. (c) The reduction in Homer-3 levels led to a significant increase in mGluR1α levels in PM fractions in dPC12 cells. Lane I: scrambled siRNA. Lane II: siRNA against Homer-3. (d) A surface biotinylation technique was employed to biochemically measure the relative levels of surface mGluR1α receptors in the presence of Homer-3 siRNA in dPC12 cells. While no changes were observed for the amount of the S8 ATPase, biotinylated receptors show an increase at the surface in Homer-3 knockdown cells compared with scrambled siRNA. Anti-Pan-cadherin (c) antibody was used as a loading control as well as a PM marker. Experiments in panel c and d were performed twice with similar results.

We next asked whether the absence of Homer-3 leads to an increase in mGluR1α receptors trafficking to the plasma membrane where receptors exert physiological effects by binding to their ligands. To answer this question, we employed two methods to purify plasma membrane receptors. We used a differential centrifugation technique to purify plasma membrane proteins from the dPC12 cell lysates shown in Fig. 6a. Figure S3a demonstrates the method utilized for membrane and cytosol fractionation involving a series of ultracentrifugation steps. Figure S3b confirmed that the isolated membrane fractions (crude membrane fraction or P2 and washed membrane fraction or P’2) are free of contaminating cytosol by immunoblotting for the Golgi and ER markers (GM-130 and KDEL) while they are enriched in the plasma membrane marker, pan-cadherin. Therefore, the final plasma membrane fraction obtained by the above method is a pure and intact purified plasma membrane fraction (Fig. 6c). In Fig. 6d, a surface biotinylation assay was used in dPC12 cells transfected with scrambled siRNA or siRNA against Homer-3. Using a cell surface protein isolation kit (see Supporting information), the outer membrane proteins on dPC12 cells were labelled with biotin followed by purification with the NeutrAvidin agarose resin. Isolated cell surface proteins and intracellular extracts (flow-through) were subjected to Western blot analysis. Figure S3c shows that there is no trace of HSP90 (a cytoplasmic marker) in the purified cell surface proteins, whereas HSP90 was detected in the total cell lysates as well as intracellular extracts. However, the pan-cadherin (plasma membrane marker) was only detected in the plasma membrane fractions. This indicates that there was minimal contamination with intracellular proteins of the isolated plasma membranes containing surface mGluR1α proteins. Results obtained in panels c and d established that reducing levels of Homer-3 enhanced the trafficking of mGluR1α towards the plasma membrane.

Silencing of Homer-3 increases mGluR1α in rat cortical neurons

Because rat cortical neurons express mGluR1α (Petralia et al. 1997; Gupta et al. 2005), we studied the effect of Homer-3 silencing in the cortex by confocal microscopy. Embryonic day 18 rat cortical neurons were transfected with clone 3–1 siRNA. After 48 h, cells were fixed and processed for immunofluorescence with primary anti-mGluR1α and anti-Homer-3 antibodies followed by incubation with fluorescent secondary antibodies. Control cells were stained with primary anti-GABAA and Homer-3 antibodies. The percentage of cortical neurons transfected with Homer-3 siRNA was 5–10%. Figure S4a and c shows that Homer-3 siRNA decreased the levels of Homer-3 protein by ~60% in transfected cells (red signal in panel S4a), which in turn resulted in increased mGluR1α by 46%±14 (green signal in panel S4c). Images quantified with ImageJ software revealed a significant increase of mGluR1α signals in siRNA-positive cells (n = 12) compared with untransfected cells (n = 12, Figure S4e, p < 0.05). No significant changes were observed when GABAA signals (red signal in Figure S4d) were measured in the presence and the absence of Homer-3 siRNA (green signal in Figure S4b) when they were quantified (n = 14, Figure S4e).

Discussion

The data presented here shine new light upon the molecular mechanisms involved in the degradation of group I metabotropic glutamate receptors, which have a key role in memory acquisition and storage in the CNS (Anwyl 2009). A large number of proteins interact with the 26S proteasome (Guerrero et al. 2008), particularly with the six ATPases in the base of the 19S particle (Ferrell et al. 2000). For example, gankyrin, an ankyrin-repeat liver oncoprotein, interacts with the S6 (rtp3) ATPase of the 26S proteasome and the ubiquitin protein ligase MDM2 to control p53 degradation (Dawson et al. 2006). The S8 ATPase interacts with proteins with diverse functions such as transcription factor Sp1 (Su et al. 2000), vitamin D receptor (Masuyama and MacDonald 1998), Prp19 (Sihn et al. 2007), MYO18B (Inoue et al. 2006), tumor suppressor p53 (Zhu et al. 2007), and ataxin-3 (Wang et al. 2007). These proteins are either targets for proteasomal degradation or they deliver other proteins for degradation via the S8 ATPase.

In this study, a Y2H screen with the cDNA for the human S8 ATPase against a HeLa cDNA library detected a specific interaction with Homer-3A11. The genetic and biochemical approaches were extended to show the S8 ATPase interacts with Homer-3A11, -A01 and -B11 according to our initial Y2H experiments. Further experiments are required to determine whether all eight isoforms in the long Homer-3 family (Homer-3A00, -3A01, -3A10, -3A11, -3B00, -3B01, -3B10 and -3B11) interact with the S8 ATPase. Even though exons 2–5 of three homer genes show 80% homology, exons 6–10 of Homers show only 20–30% homology (Xiao et al. 1998; Soloviev et al. 2000b). The low amino acid sequence similarity between Homer genes at the C-terminus and addition of two three-amino-acid insertions in the coiled-coil domain of long Homer-3s enables Homer-3A and B to be the only interaction partners of the S8 ATPase. A similar scenario has been reported when a Ubiquitin-regulatory X (UBX)/coiled-coil domain-containing protein, 2B28, specifically interacts with Homer-2 proteins but not with Homer-1 proteins using coiled-coil and UBX domains (Ishibashi et al. 2005). We believe that all eight isoforms in the long Homer-3 family should be able to mediate proteasomal degradation of mGluR1α receptors through the S8 ATPase. As other examples in the literature show (Koehnke et al. 2010), these alternative splicing forms (3A and 3B) may facilitate the degradation of mGluR1α with different speed or affinity, providing a mechanism to help specify synaptic connections. The temporal and spatial expression of these isoforms as well as their function as an mGluR1α regulator through the S8 ATPase will be elucidated in future publications.

Ultracentrifuge gradient fractionation of proteins extracted from rat hippocampal HSC2S2 cells and mouse brain tissue lysates show some long Homer-3, S8 ATPase, and mGluR1α co-sediment supporting the formation of a complex. The occurrence of mGluR1α in the same fractions along with Homer-3s is expected. The co-sedimentation with the S8 ATPase supports the notion that Homer-3s may facilitate the interaction between mGluR1α and 26S proteasomes. Homer-3/S8 ATPase and mGluR1α selectively co-sediment in enriched calreticulin (a classical ER marker) fractions in the mouse PFC. In addition to being an ER resident protein, calreticulin plays a role in ER-Golgi vesicle trafficking (Dupuis et al. 1993; Day et al. 2001) which suggests that a complex of the 26S proteasome, together with Homer-3 and mGluR1α, is located in a sub-compartment of the ER/Golgi interspace. Proteasome pull-down experiments further demonstrated that long Homer-3s and mGluR1α associate with the 26S proteasome, suggesting that Homer-3 delivers mGluR1α to the proteasome for degradation.

Our results also show that inhibition of proteasomal catalytic activity alters the ubiquitination levels and degradation of mGluR1α receptors. The accumulation of polyubiquitinated mGluR1α receptors is a characteristic of ER proteasomal degradation rather than plasma membrane events which may involve monoubiquitination as a signal for receptor endocytosis (Torres et al. 2009). Indeed, a large amount of mGluR1α is found in the ER which is enriched in neuronal dendritic spines in the brain (Petralia et al. 1997). More importantly, we demonstrated that over-expression of Homer-3A11 increases the ubiquitinated levels of mGluR1α receptors which is accompanied by a large increase in mGluR1α receptor degradation. It has been shown that Siah1A is a RING-finger-containing E3 ubiquitin ligase for mGluR1α receptors (Moriyoshi et al. 2004). Like some other E3 ligases (Sato et al. 2009), Siah1 needs an adaptor protein for ubiquitination of target proteins (Li et al. 2002). Thus, the long Homer-3 protein may recruit mGluR1α to the Siah1A ligase complex for polyubiquitination, functioning as an adaptor protein in the complex. A similar function was described for Homer-2 which binds 2B28 protein, a UBX-Ubiquitin-associated domain (UBA) containing protein, to enhance the proteasomal degradation of the IκB protein (Ishibashi et al. 2005). This type of conditional degradation of mGluR1α receptors by ubiquitin-mediated proteolysis can play a critical role in signal transduction pathways that control neural plasticity in neuronal cells. Ubiquitination and degradation of mGluR1α is not significantly enhanced by the truncated N-terminus of Homer-3A11 which has only an mGluR1α receptor-binding domain and no S8 ATPase binding domain. Thus, deletion of the C-terminus of Homer-3A11 abolished its ability to recruit an mGluR1α receptor for degradation by the 26S proteasome complex. This notion is corroborated by the fact that Homer-3 siRNA substantially decreases Homer-3 levels and causes two-fold increase in mGluR1α in dPC12 cells as well as ~50% increase in mGluR1α in rat cortical neurons. The binding of mGluR1α via Homer-3s to the 26S proteasome suggests that long Homer-3s may bind to the cytosolic tails of mGluR1α leading to receptor extraction from the ER and degradation by the proteasome (Figure S5). We assume that the expression of the C-terminus alone will lead to similar results observed with the N-terminus alone, because the C-terminus alone can only bind to the S8 ATPase and, due to the lack of EVH1 domain, it is not able to transport the mGluR1α for proteasomal degradation. Examples of ubiquitin-like proteins which mediate proteasomal degradation of substrates are ubiquilin and erasin which coordinate the degradation of selected substrates in the ER-associated protein degradation pathway (Lim et al. 2009).

While our data strongly indicate that Homer-3A11 can regulate mGluR1α turnover through its interaction with the S8 ATPase, we cannot rule out that the Homer-3A11 can directly be targeted by the S8 ATPase for proteasomal degradation due to the presence of a potential PEST domain [a sequence enriched in proline (P), glutamic acid (E), serine (S) and threonine (T)] between positions 156 and 168 (RSQSAD-TPGPTER). Another similar example concerns the human papillomavirus protein E7, which facilitates the degradation of the retinoblastoma tumour suppressor protein through its interaction with the S4 subunit of the proteasome while E7 itself is also known to be targeted for the proteasomal degradation by binding to the proteasome regulatory cap (Berezutskaya and Bagchi 1997; Gonzalez et al. 2001). Certainly, there are many other details which are beyond the scope of this study, and we have ongoing experiments to answer these questions in future publications.

There is evidence that Homer-1C can protect the mGluR1 receptor by masking the PEST domain of receptor presumably through the coiled-coil domain (Ciruela et al. 2000). We believe that the Homer family with 17 isoforms can be involved in opposing activities to regulate the turnover and the trafficking of mGluR1α. Opposing functions of isoforms have been described for other proteins such as Plic family isoforms where Plic-2 negatively regulates G protein-coupled receptor endocytosis and its closely related isoform, PLIC-1, does not affect receptor endocytosis (N’Diaye et al. 2008). More interestingly, Plic-1 shows an opposite degradative effect on binding to GABAA receptors (Bedford et al. 2001) compared with nicotinic receptors (Ficklin et al. 2005), probably due to the arrangement of protein partners. The members of the Homer family may also locate in multiple sorting stations, serving to filter and direct group one of mGluRs to their final destination positively or negatively. Therefore, while the CC-domain and not the N-terminal domain of Homers can be used for masking the PEST domain of mGluR1α, the exclusive interaction of Homer-3A11 to the S8 ATPase can switch the Homer-3A11 to a negative regulator of mGluR1α in response to synaptic activity. In this later scenario, the axis of Homer-3A/S8 ATPase can mediate mGluR1α degradation. Because mGluR1α is a transmembrane protein which is located initially in the ER compartment, it is likely that the axis of Homer-3A/S8 ATPase only exists at the ER level as part of ER-associated protein degradation pathway, while the free Homer-3 can still play a protective/messenger role for the mGluR1α in other cellular compartments, including at the plasma membrane.

Our data are not in contradiction with previously published data suggesting that Homer-3 along other long Homer family proteins facilitates mGluR1α signal transduction at the synapse (Ango et al. 2001). There is a growing body of evidence that suggests adaptor proteins in neuronal cells may have dual functions based on their cytoplasmic locations, interaction partners, or neural activity. An example is ubiqulin-1 with dual functions in the proteasomal degradation and the transport to the aggrosome based on its interaction partners (Heir et al. 2006).

Our results reveal a novel function of long Homer-3 members in controlling the level of mGluR1α receptors at the ER level. Other data has shown that over-expression of WT-Homer-3 (and not mutant forms) causes a reduction in glutamate-induced Ca2+ release by mGluR1α (Mizutani et al. 2008) when compared with cells expressing mGluR1α alone. This would be expected from our data because the over-expression of long Homer-3 proteins would increase the degradation of the receptor and consequently reduces the surface expression of mGluR1α.

A possible, but untested, alternative physiological function is the involvement of long Homer-3s in the translocation of proteasomes into neuronal sub compartments. While proteasomes are ubiquitous in the cytoplasm and in the nucleus, they can be enriched in other organelles such as the outer surface of the endoplasmic reticulum or synaptic spines (Palmer et al. 1996; Brooks et al. 2000; Wojcik and DeMartino 2003; Bingol and Schuman 2006). The proteasomal S8 (rpt6) ATPase appears to have an even more important role in controlling synaptic functions because this ATPase is phosphorylated by calcium calmodulin dependent kinase II to control the recruitment of proteasomes into dendritic spines (Bingol et al. 2010). Co-localization of S8 ATPase with Homer-3 in neuronal processes in a primary rat hippocampal neuron suggests that Homer-3 may direct the 26S proteasome to particular sub-compartments. In a similar context, interaction of Homer-3 with the transcription factor PAX6 links the synaptic activation to the neuronal transcriptional activity (Cooper and Hanson 2005). Such a translocation mechanism is attractive because there is evidence for selective secretory trafficking originated from the soma toward the proximal segments of dendrites (Horton and Ehlers 2003; Ehlers 2007). Interaction between Homer-3 and dynein light chain 2, a component of myosin-V motor complexes (Rual et al. 2005), may be the molecular mechanism by which long Homer-3s traffic 26S proteasomes from the soma to the neuronal process.

A growing body of observations shows that the stability of several receptors and, therefore, receptor number at the cell surface is controlled by the UPS through adaptor proteins (Bedford et al. 2001; Ficklin et al. 2005; Ratnam and Teichberg 2005; Saliba et al. 2008; Rezvani et al. 2009) which, therefore, controls synaptic plasticity (Hegde 2010). In conclusion, we identified the long Homer-3 proteins as selective regulators of the levels of mGluR1α receptors by facilitating their degradation.

Supplementary Material

supplement

Appendix S1. Supplementary Materials and methods.

Figure S1. The mGluR1α receptor is regulated by the ubiquitin-proteasome pathway.

Figure S2. Homer-3A has no effect on α7-containing nicotinic acetylcholine receptors.

Figure S3. Plasma membrane purification using ultra-centrifugation or cell surface biotinylation methods.

Figure S4. Homer-3 siRNA selectively increase mGluR1α receptor signals in rat cortical neurons.

Figure S5. Long Homer-3s shuttle the mGluR1α receptor to the 26S proteasome via interaction with the S8 ATPase.

Table S1. List of antibodies, suppliers and concentrations used in this study.

Table S2. The yeast two-hybrid assay showed that the N-terminal region of the S8 ATPase, which includes the putative coiled-coil motif, is indispensable for the interaction with Homer-3A11.

Table S3. The yeast two-hybrid assay indicated that neither the short Homer-3 (Homer-3D) nor the Homer-1 or -2 subfamilies of Homer-proteins interact with the S8 ATPase.

Table S4. Homer-3A11 is a selective partner for the S8 ATPase of the 19S regulatory complex.

Acknowledgements

The study was supported in part by a Core Support Grant from the Alzheimer Research Trust, the Neurosciences Support Group at the Queen’s Medical Centre (NSG@QMC) to RJM, NIHR21 (No. 1180103304) to MDB and the support provided by the Divioson of Basic Biomemdcal Sciences of the University of South Dakota to KR. We thank Nupur J. Kikani, Sanam Sane, Rebecca K. Autenried, and Justine M. Ferguson for excellent technical support. We thank Dr. Kimberley R. Tolias and Feng Liu for providing the rat cortical cultured cells. The authors have declared that no conflict of interest exists.

Abbreviations used:

ER

endoplasmic reticulum marker

EVH1

(Ena/VASP) homology 1

PFC

prefrontal cortex

UPS

ubiquitin-proteasome system

Y2H

yeast two-hybrid

Footnotes

Supporting information

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplement

Appendix S1. Supplementary Materials and methods.

Figure S1. The mGluR1α receptor is regulated by the ubiquitin-proteasome pathway.

Figure S2. Homer-3A has no effect on α7-containing nicotinic acetylcholine receptors.

Figure S3. Plasma membrane purification using ultra-centrifugation or cell surface biotinylation methods.

Figure S4. Homer-3 siRNA selectively increase mGluR1α receptor signals in rat cortical neurons.

Figure S5. Long Homer-3s shuttle the mGluR1α receptor to the 26S proteasome via interaction with the S8 ATPase.

Table S1. List of antibodies, suppliers and concentrations used in this study.

Table S2. The yeast two-hybrid assay showed that the N-terminal region of the S8 ATPase, which includes the putative coiled-coil motif, is indispensable for the interaction with Homer-3A11.

Table S3. The yeast two-hybrid assay indicated that neither the short Homer-3 (Homer-3D) nor the Homer-1 or -2 subfamilies of Homer-proteins interact with the S8 ATPase.

Table S4. Homer-3A11 is a selective partner for the S8 ATPase of the 19S regulatory complex.

NIHMS1758471-supplement-supplement.pdf (1.3MB, pdf)

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