Abstract
The metabotropic glutamate receptor 1 (mGlu[1]) plays a pivotal role in
synaptic transmission and neuronal plasticity. Despite the fact that
several interacting proteins involved in the mGlu[1] subcellular
trafficking and intracellular transduction mechanisms have been
identified, the protein network associated with this receptor in
specific brain areas remains largely unknown. To identify novel
mGlu[1]-associated protein complexes in the mouse cerebellum, we used
an unbiased tissue-specific proteomic approach, namely
co-immunoprecipitation followed by liquid chromatography/tandem mass
spectrometry analysis. Many well-known protein complexes as well as
novel interactors were identified, including G-proteins, Homer, δ2
glutamate receptor, 14-3-3 proteins, and Na/K-ATPases. A novel putative
interactor, KCTD12, was further investigated. Reverse
co-immunoprecipitation with anti-KCTD12 antibodies revealed mGlu[1] in
wild-type but not in KCTD12-knock-out homogenates. Freeze-fracture
replica immunogold labeling co-localization experiments showed that
KCTD12 and mGlu[1] are present in the same nanodomain in Purkinje cell
spines, although at a distance that suggests that this interaction is
mediated through interposed proteins. Consistently, mGlu[1] could not
be co-immunoprecipitated with KCTD12 from a recombinant mammalian cell
line co-expressing the two proteins. The possibility that this
interaction was mediated via GABA[B] receptors was excluded by showing
that mGlu[1] and KCTD12 still co-immunoprecipitated from GABA[B]
receptor knock-out tissue. In conclusion, this study identifies
tissue-specific mGlu[1]-associated protein clusters including KCTD12 at
Purkinje cell synapses.
Keywords: glutamate receptors, cerebellum, KCTD12, immunoprecipitation,
proteomics
1. Introduction
Metabotropic glutamate receptors (mGlus) are members of class C
G-protein coupled receptors (GPCRs) activated by glutamate, the major
excitatory neurotransmitter in the central nervous system. These
receptors are involved in many physiological functions including
neuronal excitability, development, synaptic plasticity, and memory
[[44]1]. The eight members of this protein family are classified into
three groups. Group I consists of mGlu[1] and mGlu[5], which share
about 70% sequence homology and mainly couple to Gαq. Group I mGlus are
selectively activated by (S)-3,5-dihydroxyphenylglycine and are mainly
localized post-synaptically [[45]2,[46]3]. In the cerebellar cortex,
mGlu[1] is highly expressed in Purkinje cells and a subset of
interneurons, whereas mGlu[5] is expressed in Golgi and Lugaro cells
and in deep cerebellar nuclei [[47]4,[48]5,[49]6,[50]7]. At
glutamatergic synapses of Purkinje cells, mGlu[1] contributes to
long-term depression (LTD), which is important for cerebellar learning
mechanisms [[51]8]. Gene-targeted deletion of mGlu[1] results in
impaired LTD and severe ataxia [[52]9,[53]10]. This receptor also plays
an important role in the elimination of multiple climbing fiber
innervation to Purkinje cells during development [[54]11,[55]12]. These
functions critically depend on the coupling of mGlu[1] to Gαq proteins
[[56]12,[57]13]. In addition, mGlu[1] is involved in synaptic
plasticity at GABAergic synapses such as rebound potentiation which is
mediated by coupling of the receptors to Gαs [[58]14]. A number of
studies have shown that G-protein dependent as well as G-protein
independent functional properties of mGlu[1] depend on their
interaction with scaffolding and signaling proteins, including other
GPCRs and ion-channels [[59]15,[60]16]. Alternative splicing at the
mGlu[1] gene (Grm1) generates four variants, namely mGlu[1]α, mGlu[1]β,
mGlu[1]γ, and mGlu[1]δ, which share a large part of the N-terminal
sequence but differ primarily in their intracellular C-terminal domains
[[61]1]. The mGlu[1]α isoform has the longest C-terminal domain and can
physically interact with a variety of proteins through motifs that are
not present in the shorter isoforms [[62]17,[63]18]. CFTR-associated
ligand (aka Golgi-Associated PDZ And Coiled–Coil Motif-Containing
Protein) [[64]19], Homer proteins [[65]20,[66]21], Norbin
(neurochondrin) [[67]22,[68]23], protein phosphatase 1C [[69]24],
Siah-1A [[70]25], and Tamalin [[71]26] are some of the signaling and
scaffolding proteins that were reported to directly bind to mGlu[1]α
[[72]15]. This isoform was also found to form functional complexes with
the Gluδ2 receptor and the short Transient Receptor Potential Cation
channel C3 (TRPC3) [[73]27,[74]28,[75]29,[76]30,[77]31].
Most of our current knowledge about mGlu[1] interaction partners is
obtained from affinity purifications or yeast two-hybrid screenings. In
recent years, proteomic studies have emerged as a valuable tool for
studying the co-assembly of proteins in native tissue. Proteomic
approaches have the advantage of identifying stable and transient
protein–protein interactions, defining native protein complexes, and
finding novel interaction partners [[78]32,[79]33].
In the current study, we used a proteomic approach to identify protein
complexes that are associated with mGlu[1]α in the cerebellum. We
immunoprecipitated mGlu[1]α from mice cerebellar lysates and analyzed
co-purified proteins by mass spectrometry. Using this approach, we
identified multiple well-known as well as novel interactors and
generated a mGlu[1] protein interaction network. We investigated a
novel mGlu[1]α interaction partner, namely the Potassium Channel
Tetramerization Domain-containing protein 12 (KCTD12), a GABA[B]
receptor auxiliary subunit, using in vivo and in vitro methods. Our
findings showed that mGlu[1] and KCTD12 co-exist in the same nanodomain
in Purkinje cell spines, though their interaction does not depend on
direct physical binding but most likely through interposed proteins.
2. Materials and Methods
2.1. Experimental Animals
For immunoprecipitations of mGlu[1]α from the cerebellum, adult male
and female C57BL/6N wild-type (WT) (n = 23), Grm1-knock-out (KO) (n =
7), BALB/c WT (n = 4), GABA[B1] (n = 4), and GABA[B2] (n = 4) KO mice
were used. C57BL/6N WT (n = 3) and KCTD12-KO (n = 3) adult male and
female mice were used to immunoprecipitate KCTD12 from the cerebellum.
The following mice were used: C57BL/6N (Charles River, Sulzfeld,
Germany), Grm1-KO (gift from GlaxoSmithKline), GABA[B1] KO [[80]34],
GABA[B2] KO [[81]35], Balb/c littermate, and KCTD12-KO mice [[82]36].
Experimental procedures on animals were approved by the Austrian Animal
Experimentation Ethics Board (GZ66.011/28-BrGT/2009) and by the
Veterinary Office of Basel-Stadt and were in compliance with the
European Convention for the Protection of Vertebrate Animals used for
experimental and other scientific purposes, the Animal Experiments Act
2012 (TVG 2012), and the EU Directive 2010/63/EU. The authors further
attest that all efforts were made to minimize the number of animals
used.
2.2. Antibodies and Vectors
Antibodies against mGlu[1]α (Af811-1 and Af660-1) were purchased from
Frontier Institute Co. Ltd. (Hokkaido, Japan) and KCTD12 antibodies
were generated as previously reported [[83]37].
The plasmid containing the open reading frame for the mouse mGlu[1]α
DNA and lentiviral particles packaged for mouse KCTD12 DNA was obtained
from Genecopoeia (Rockville, MD, USA). The expression of KCTD12 was
under the control of the cytomegalovirus (CMV) promoter and both the
plasmid and lentiviral vectors contained the neomycin resistance gene.
The plasmid containing the open reading frame for mouse GABA[B2]-YFP
DNA under the control of the CMV promoter was previously reported
[[84]38].
2.3. Immunoprecipitation of Proteins from Mouse Cerebellum (P2 Fraction)
To immunoprecipitate mGlu[1]α and KCTD12 and mouse cerebella were
dissected and pooled. Homogenization was performed in ice-cold 10 mM
Tris-HCl, pH 7.4 buffer containing 320 mM sucrose, 1 mM
phenylmethylsulphonyl fluoride, 1 mM NaF, 1 mM Na[3]VO[4], and complete
EDTA-free protease inhibitors (Roche, Vienna, Austria) using a
motorized homogenizer (Sartorius, Göttingen, Germany) at a speed of
1400 rpm and 10 strokes. Lysates were centrifuged for 10 min at 1000× g
and 4 °C to remove unbroken cells and tissue debris. This process was
repeated 2 more times. Supernatants were collected and centrifuged for
40 min at 17,000× g at 4 °C. The pellet (P2 fraction) was resuspended
in ice-cold 25 mM Tris-HCl, pH 7.4 buffer containing 1 mM NaF, 1 mM
Na[3]VO[4], and complete EDTA-free protease inhibitors. The protein
concentration was determined by the Bradford protein assay using bovine
serum albumin as the standard protein.
P2 samples were centrifuged for 30 min at 20,000× g and 4 °C. The
pellet was suspended for 1 h in ice-cold 25 mM Tris-HCl, pH 7.4 buffer
containing 0.5% sodium deoxycholate, 1% NP-40, 0.1% sodium dodecyl
sulfate (SDS), 137 mM NaCl, 3 mM KCl, 1 mM phenylmethylsulfonyl
fluoride, 1 mM NaF, 1 mM Na[3]VO[4], and complete EDTA-free protease
inhibitors. Detergent lysates were then centrifuged for 60 min at
20,000× g and 4 °C. The supernatant was aspirated carefully and
incubated with primary antibodies (0.2 µg/100 µg proteins) for 2 h at 6
°C with constant rotation. Dynabeads Protein-A (Invitrogen, Waltham,
MA, USA) were added at the ratio of 1 µL:20 µg of protein followed by
incubation for 1 h at 6 °C. The immunoprecipitation eluates were
obtained by heating the samples in 1× Laemmli buffer plus 20 mM
dithiothreitol for 10 min at 70 °C and 5 min at 56 °C to prevent
oligomerization.
2.4. Immunoblotting
Samples were loaded on NuPAGE Bis-Tris 4–12% precast gels and proteins
were resolved at 80 V in MOPS SDS buffer. Proteins were transferred to
a polyvinylidene difluoride membrane overnight at 150 mA, 6 °C. The
membranes were stained with Ponceau-S for 10 min and incubated in 5%
dry milk blocking solution for 1 h at room temperature. Membranes were
then incubated in primary antibodies for 2–3 overnights and 6 °C
(1:3000). Immunoreactive bands were detected by incubation in
horseradish peroxidase-conjugated secondary antibodies (Invitrogen)
followed by the ECL Prime reagent. Chemiluminescence was visualized
with the Fusion SL-4 Vilber Lourmat imaging system (Peqlab, Erlangen,
Germany).
2.5. Mass Spectrometry (LC–MS/MS) Analysis of Proteins
Eluates were lyophilized in a Christ RVC 2-18 concentrator and loaded
on precast NuPAGE 4–12% Bis-Tris gels (Invitrogen). After 1.5 cm
running in a MOPS SDS buffer, the gels were stained for 1 h in
Coomassie R-250 at room temperature. Destaining was performed overnight
in 40% methanol and 10% glacial acetic acid solution.
Protein bands were excised from gels and digested with trypsin obtained
from porcine pancreas (Sigma-Aldrich, Vienna, Austria), as previously
described [[85]39]. Tryptic digests were analyzed using an UltiMate
3000 nano-HPLC system coupled to an LTQ Orbitrap XL mass spectrometer
(Thermo Scientific, Bremen, Germany). The peptides were separated on a
homemade fritless fused silica micro-capillary column (75 µm i.d. × 280
µm o.d. × 10 cm length) packed with 3 µm reversed phase C18 material
(Reprosil). The solvent for HPLC was 0.1% formic acid (solvent A) and
0.1% formic acid in 85% acetonitrile (solvent B). The gradient profile
was as follows: 0–2 min, 4% B; 2–55 min, 4–50% B; 55–60 min, 50–100% B;
and 60–65 min, 100% B. The flow rate was 250 nL/min.
The LTQ Orbitrap XL mass spectrometer was operated in the
data-dependent mode selecting the top 10 most abundant isotope patterns
with a charge of 2+ or 3+ from the survey scan with an isolation window
of 2 for the mass-to-charge ratio (m/z). Survey full scan MS spectra
were acquired from 300 to 2000 m/z at a resolution of 60,000 with a
maximum Injection Time (IT) of 20 ms and automatic gain control (AGC)
target 1 × 10^6. The selected isotope patterns were fragmented by
collisional-induced dissociation (CID) with a normalized collision
energy of 35 and a maximum injection time of 55 ms.
Data analysis was performed using Proteome Discoverer 1.3 (Thermo
Scientific) with the search engine Sequest. The raw files were searched
against the Mus musculus database (167,940 entries) extracted from the
NCBInr. The precursor and fragment mass tolerances were set to 10 ppm
and 0.02 Da, respectively, and up to two missed cleavages were allowed.
Carbamidomethylation of cysteine and oxidation of methionine were set
as variable modifications. Peptide identifications were filtered at a
1% false discovery rate.
2.6. Generation of a Stable Cell Line Expressing KCTD12
HEK293 cells were seeded for 24 h and transduced with lentiviral
particles containing KCTD12 DNA at a multiplicity of infection-10 and
selected with G418 (600 μg/mL). After 48 h, the cells were trypsinized
and transferred to a new dish. G418 was added to the medium for the
next 3 passages until dead cells were sparse. From that stage onwards,
cells were split regularly when almost 90% were confluent without
adding G418 to the growth medium. Cells were maintained in an incubator
at 37 °C, 5% CO[2], and 95% humidity. Confirmation of KCTD12 protein
translation was obtained by Western blot and immunofluorescence.
2.7. Immunofluorescence
KCTD12-expressing cells were quickly washed with ice-cold PBS and fixed
in ice-cold 4% paraformaldehyde for 45 min. Fixed cells were washed 3
times in tris-buffered saline followed by 1 h incubation in blocking
solution at room temperature. Cells were incubated overnight with
primary antibodies (rabbit anti-KCTD12 1:3000; guinea pig anti mGlu[1]α
1:1000) at 6 °C. Cells were washed three times with tris-buffered
saline and incubated with secondary antibodies [Cy3 donkey-anti rabbit
IgG 1:400 (Jackson ImmunoResearch, Ely, UK); Alexa 488-goat anti guinea
pig IgG, 1:1000 (Invitrogen)] overnight at 6 °C in the dark. Cells were
washed twice in tris-buffered saline, a coverslip was mounted using
Vectashield, and analysis was performed using a Zeiss Axioimager M1
microscope equipped with a metal halide lamp.
2.8. Transient Transfection and Immunoprecipitation
Stable cell lines were transfected with plasmids containing the
mGlu[1]α DNA and the GABA[B2]-YFP DNA. DNA and lipofectamine
(Invitrogen) were diluted in Optimem, mixed at a ratio of 1:3, and
incubated for 20 min at room temperature. The mixtures were added to
the growth medium and the cells were kept in an incubator for 72 h. The
cells were washed with warm PBS and lysed in an ice-cold buffer used
for immunoprecipitation. Lysates were centrifuged for 40 min at 17,000×
g and 4 °C. The supernatant was aspirated carefully and KCTD12 was
immunoprecipitated using a guinea pig polyclonal antibody raised
against KCTD12 following the same protocol as described above.
2.9. Freeze-Fracture Replica Immunogold Labeling (FRIL)
FRIL was performed according to previously published procedures
[[86]40,[87]41]. Two C57BL/6N WT and two KCTD12-KO mice were perfused
transcardially for 10 min with a solution containing 1%
paraformaldehyde and 15% of a saturated solution of picric acid in 0.1
M phosphate buffer at a rate of 5 mL/min. The brains were quickly
extracted from the skull and the cerebellums were cut with a vibratome
(Leica Microsystems VT1000S, Vienna, Austria) into 140 µm thick coronal
sections from where samples of the cerebellar cortex molecular layer
(Crus 2 lobule) were dissected out under a stereomicroscope. The slices
were cryoprotected in 30% glycerol in 0.1 M phosphate buffer and
high-pressure frozen by means of an HPM 010 machine (Bal-Tec, Balzers,
Liechtenstein). The frozen slices were then freeze-fractured at −115 °C
and replicated with a first layer of carbon (5 nm), shadowed by
platinum (2 nm), and followed by a second carbon layer (15 nm) in a
freeze-etching BAF 060 device (Bal-Tec). After thawing, the tissue
attached to replicas was digested with stirring at 80 °C overnight in
SDS solubilization buffer for FRIL.
For immunogold labeling of replicas, blocking was performed first in a
solution consisting of 5% bovine serum albumin and 0.1% Tween-20 in
tris-buffered saline, pH 7.4 at room temperature. Replicas were then
incubated in primary antibodies (guinea pig anti-KCTD12 1:90; rabbit
anti mGlu[1]α 1:300) for 72 h at 6 °C. Following extensive washes and 1
h blocking, replicas were incubated with gold-conjugated secondary
antibodies of 5 and 10 nm (British Biocell International, Cardiff, UK;
BBI 1175; BBI 009076) for 48 h at 6 °C. Incubation of the primary and
secondary antibodies was in a sequence with the antibodies for the
detection of mGlu[1]α always incubated first. The specificity of the
antibodies was tested on tissue obtained from KO mice as well as by
omitting the primary antibody. Replicas were mounted on
pioloform-coated mesh copper grids and examined with a Philips CM120
transmission electron microscope (Philips, Eindhoven, The Netherlands).
2.10. Network Construction
We constructed a Protein–Protein Interaction (PPI) network for
immunoprecipitated proteins identified in all 6 replicate experiments.
To obtain reliable protein interactions, we extracted only the
validated interactions for mice. The mouse network was modelled using
the 1-step neighbors of the putative interacting proteins with high
confidence. We comprehensively merged the interactions from multiple
interaction databases, including iRefIndex
([88]https://irefindex.vib.be; accessed on 12 September 2022), Mentha
([89]https://mentha.uniroma2.it; accessed on 12 September 2022),
InnateDB-all ([90]https://www.innatedb.com; accessed on 19 September
2022), EBI-GOA ([91]https://www.ebi.ac.uk/GOA/index; accessed on 19
September 2022), MiNT ([92]https://mint.bio.uniroma2.it; accessed on 12
September 2022), IMEX ([93]https://www.imexconsortium.org; accessed on
19 September 2022), and IntAct ([94]https://www.ebi.ac.uk/intact/home;
accessed on 19 September 2022). Indeed, the seven interaction databases
cover the mouse interactome. To ensure data quality, the constructed
network consists only of non-redundant and validated protein
interactions. To this aim, we extracted protein interactions from each
database and then combined them into the final network after removing
overlapping proteins. The network was visualized with the Cytoscape
software 3.9.1 ([95]https://cytoscape.org accessed on 19 September
2022).
2.11. Network Analysis
There is evidence that the functional importance of proteins might be
inferred from their topological properties, specifically their key
positions in the protein interaction network [[96]42,[97]43]. To gain
information on the network and its participating proteins, two
centrality indices were evaluated for each protein: the degree and
betweenness. The degree centrality shows how many direct neighbors a
node in the network connects to. A protein is considered as a hub in
the network if its degree centrality is high. Betweenness shows the
bridge role of a protein for other proteins in the network [[98]44]. A
protein presenting high betweenness centrality is an important
connector in the network, playing a key mediator role. The degree
centrality is measured by counting the neighborhood of a node in the
network, thus identifying network hubs. Betweenness is the centrality
measure based on shortest-path calculations.
A given network G(N,E) consists of a set of nodes (N) and a set of
edges (E) between them. An edge e[ij] connects node n[i] with node
n[j]. In this paper, the extracted network is unweighted and
undirected. In an undirected graph, e[ij] and e[ji] are considered
identical. Therefore, the neighborhood
[MATH: ℵ :MATH]
[i] for a node n[i] is defined as its direct connected neighbors, as
follows:
[MATH:
ℵ={n<
mi>j : eij ∈E} :MATH]
(1)
The degree Di of a node is defined as the number of nodes |
[MATH: ℵ :MATH]
[i]| in its neighborhood
[MATH: ℵ :MATH]
[i].
The betweenness centrality is calculated based on the shortest paths.
For each node n[k] in the network, we counted the total number of
shortest paths from node n[i] to node n[j], called p(v[i], v[j]) as
well as the number of those paths that pass through n[k], called
p(v[i], v[j], v[k]). The betweenness B(v[k]) of a node v[k] is defined
as follows:
[MATH:
B(nk<
/msub>)=∑ni
≠nk≠n
jp
(ni,
nk, nj)d(ni,nj
)
:MATH]
(2)
Pathway analysis and the identification of enriched GO terms were
performed using DAVID and GOrilla based on the 10% highest scoring
proteins in the network [[99]45,[100]46,[101]47].
3. Results
3.1. Immunoprecipitation of mGlu1α from Mouse Cerebellum
To identify protein complexes that can associate with mGlu[1]α, we
immunoprecipitated the receptor from mouse cerebellar extracts
([102]Figure 1A). Pilot experiments were performed to determine the
optimal conditions for immunoprecipitation. Two main conditions have
been optimized: (1) solubilization and (2) the choice and concentration
of antibody. Optimal detergent conditions were determined empirically
by assessing the amount of native mGlu[1]α extracted from cerebellar
lysates by Western blot. Under our experimental conditions, a
combination of 0.5% deoxycholate, 0.1% SDS, and 1% NP-40 was most
effective in extracting mGlu[1]α. Three different antibodies were
tested for immunoprecipitation: a guinea pig polyclonal antibody raised
against the amino acids 945-1127 (Af660-1) within the C-terminal region
of mGlu[1]α and two rabbit polyclonal antibodies raised against amino
acid residues 945-1127 (Af811-1) or 1179-1199 (AB1551). Only the
antibodies directed against the sequence 945-1127 were able to
specifically immunoprecipitate mGlu[1] and were used in subsequent
experiments. A concentration of 0.2 µg of antibody per 100 µg of
proteins resulted in the most efficient immunoprecipitation of the
receptor. Three immunoprecipitation replicate experiments were then
performed with antibody Af811-1 and three additional ones with antibody
Af660-1. Immunoprecipitations from Grm1-KO cerebellar lysates were
carried out as controls for both antibodies.
Figure 1.
[103]Figure 1
[104]Open in a new tab
Multiepitope affinity purification identifies native mGlu[1] in mouse
cerebellar protein extracts. (A) Schematic of the immunoprecipitation
procedure and of the mGlu[1]α structure. (B) Primary mGlu[1]α amino
acid sequence. Red indicates the protein coverage of mGlu[1]α
identified by MS analysis. (C) MS/MS spectra of the mGlu[1] unique
peptide YDYVHVGTWHEGVLNIDDYK. Monoisotopic m/z 808.38007 Da (−1.56
mmu/−1.93 ppm). MH+: 2423.12564 Da, RT: 32.51 min. Fragment match
tolerance used for search: 0.8 Da. Fragments used for search: b;
b-H[2]O; b-NH[3]; y; y-H[2]O; and y-NH[3]. Ion series: b (shown in
red), y (shown in blue).
In the LC–MS/MS analysis, mGlu[1]α was successfully identified in all
six immunoprecipitation experiments from WT, but not from Grm1-KO
animals. In all experiments, the highest SEQUEST score was indeed
observed for mGlu[1]α. The protein sequence coverage of the receptor
was between 31.6 and 49.5% ([105]Figure 1B,C; [106]Table 1). The number
of unique peptides specifically corresponding to mGlu[1]α ranged from 7
to 11. Additional 19 to 28 peptides were in common between mGlu[1]α and
mGlu[1]β ([107]Table 1; [108]Supplementary Table S1). mGlu[1]β is a
short alternatively spliced variant of the Grm1 gene in which the last
318 amino acid residues of the mGlu[1]α variant are replaced by 20
different residues [[109]1]. Available evidence suggests that mGlu[1]α
and mGlu[1]β variants heterodimerize both in vitro and in vivo
[[110]48,[111]49,[112]50] which affects receptor trafficking to the
plasma membrane of Purkinje cell [[113]46,[114]47]. In line with these
previous findings, we were able to identify specific mGlu[1]β peptides
in five out of six experiments, despite the fact that the specific
portion of the mGlu[1]β variant is quite short. Therefore, our results
provide further support to the notion that mGlu[1]α and mGlu[1]β
heterodimerize in Purkinje neurons.
Table 1.
Identification of mGlu[1]α by LC–MS/MS in six immunoprecipitation
experiments.
Coverage (%) Unique Peptides ^1 (Number) Peptides (Number)
35.11 11 33
31.61 7 29
43.20 10 34
49.54 11 39
46.79 11 37
31.61 8 27
[115]Open in a new tab
^1 Peptides not shared with mGlu[1]β.
3.2. mGlu1α Co-Immunoprecipitated Proteins
Proteomic analysis using the Mus musculus NCBInr database with a 99%
confidence threshold yielded hundreds of proteins from each experiment.
Immunoglobulins, keratins, and proteins co-immunoprecipitated from
lysates of Grm1-KO mice were considered as non-specific interacting
proteins and, therefore, removed from the list of putative mGlu[1]
interaction partners. We identified a total of 304 proteins in the data
pooled from the six experiments ([116]Supplementary Table S2).
Among the identified proteins, 25 proteins were consistently detected
in all 6 replicate experiments ([117]Table 2) and were, therefore,
considered putative interacting proteins with a high confidence.
Several of these proteins have been previously described as mGlu[1]
interactor partners, such as Homer proteins, the G-protein α/o subunit,
inositol 3-phosphate receptors, and Gluδ2, as well as protein kinase C
and calcium/calmodulin-dependent protein kinase II (CaMKII)
[[118]1,[119]20,[120]51,[121]52,[122]53]. Other previously recognized
mGlu[1] interactors were detected in this study, although they were not
included in [123]Table 2 because they were not identified in all six
replicates. Among them, the G-protein α/q subunit, the predominant
G-protein coupled to mGlu[1] [[124]1], was positively detected in four
experiments out of six ([125]Supplementary Table S3). Likewise, TRPC3
was also identified in four experiments ([126]Supplementary Table S3)
in agreement with previous reports establishing TRPC3 as a downstream
effector of mGlu[1]-dependent synaptic transmission in Purkinje cells
[[127]28]. Ca[V]2.1 calcium channels were reported to interact with
mGlu[1] in Purkinje neurons [[128]54] and were among the
co-immunoprecipitated proteins ([129]Supplementary Table S2).
Table 2.
mGlu[1] interacting proteins identified in all six immunoprecipitation
experiments.
Protein ID Protein Name Gene ID Unique Peptides (Number) NCBI ID
[130]P97772 metabotropic glutamate receptor 1 Grm1 48 [131]NP_058672.1
[132]P18872 guanine nucleotide-binding protein G(o) subunit α Gnao1 11
[133]NP_034438.1
[134]P62874 guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit
β-1 Gnb1 6 [135]NP_001153488.1
[136]Q8BW86 ρ guanine nucleotide exchange factor 33 Arhgef33 14
[137]NP_001138924.1
[138]Q6PIC6 sodium/potassium-transporting ATPase subunit α -3 Atp1a3 26
[139]NP_001361556.1
[140]Q6PIE5 sodium/potassium-transporting ATPase subunit α-2 Atp1a2 14
[141]NP_848492.1
[142]P14094 sodium/potassium-transporting ATPase subunit β-1 Atp1b1 7
[143]NP_033851.1
[144]Q91V14 solute carrier family 12 member 5 Slc12a5 29
[145]NP_001342409.1
[146]P56564 excitatory amino acid transporter 1 Slc1a3 6
[147]NP_683740.1
[148]O35544 excitatory amino acid transporter 4 Slc1a6 7
[149]NP_033226.1
[150]Q61625 glutamate receptor ionotropic, δ-2 Grid2 20
[151]NP_032193.1
[152]Q99JP6 homer protein homolog 3 Homer3 14 [153]NP_001139625.1
[154]P16330 2′,3′-cyclic-nucleotide 3′-phosphodiesterase Cnp 10
[155]NP_001139790.1
[156]Q91VR2 ATP synthase subunit γ, mitochondrial Atp5c1 6
[157]NP_065640.2
[158]Q8VEM8 phosphate carrier protein, mitochondrial Slc25a3 5
[159]NP_598429.1
[160]P48962 ADP/ATP translocase 1 Slc25a4 3 [161]NP_031476.3
[162]P61982 14-3-3 protein γ Ywhag 7 [163]NP_061359.2
[164]P68254 14-3-3 protein θ Ywhaq 8 [165]NP_035869.1
[166]P11881 inositol 1,4,5-trisphosphate receptor type 1 Itpr1 62
[167]NP_034715.3
[168]Q7TNC9 inositol polyphosphate-5-phosphatase A Inpp5a 10
[169]NP_898967.2
[170]P63318 protein kinase C γ type Prkcg 16 [171]NP_035232.1
[172]P28652 calcium/calmodulin-dependent protein kinase type II subunit
β Camk2b 4 [173]NP_001167524.1
[174]P35802 neuronal membrane glycoprotein M6-a Gpm6a 2
[175]NP_705809.1
[176]P46097 synaptotagmin-2 Syt2 4 [177]NP_001342655.1
[178]Q6WVG3 BTB/POZ domain-containing protein KCTD12 Kctd12 9
[179]NP_808383.3
[180]Open in a new tab
We further detected mGlu[5], though only in one experiment, that was
shown to form functional heterodimers with mGlu[1] [[181]55,[182]56]
despite the fact that their colocalization is restricted to Golgi and
Lugaro cells and deep nuclei in the adult rodent cerebellum
[[183]4,[184]5,[185]6,[186]7]. GABA[B] and GABA[A] receptor subunits
were also recovered ([187]Supplementary Table S2), consistent with the
identification of mGlu[1] also at GABAergic synapses [[188]57,[189]58].
Among the proteins identified with high confidence, we also detected
novel putative interactors ([190]Table 2). These included the ρ guanine
nucleotide exchange factor 33 (RhoGEF33), excitatory amino acid
transporters, 14-3-3 adapter proteins, Na^+/K^+ ATPase subunits,
synaptotagmin-2 (Syt2), the K^+/Cl^- cotransporter KCC2, and the KCTD12
protein. The KCTD family of proteins includes 21 members that share a
common tetramerization T1 domain [[191]59]. KCTD8, 12, 12b, and 16 form
homo- and hetero-oligomers and directly bind to the GABA[B2] receptor
subunit via their T1 domains [[192]38,[193]60,[194]61]. The expression
of KCTD12 and 12b strongly desensitizes the GABA[B] receptor response
[[195]62].
3.3. mGlu[1] Interactome
To gain further insight into the interactions between the identified
and other neuronal proteins, we constructed a network based on
validated interactions as available from the literature and collected
in several databases (see [196]Section 2.10). We constructed a network
from the most reliably identified proteins ([197]Table 2). The network
had 643 nodes and 964 edges; the section centered on mGlu[1] is
displayed in [198]Figure 2. The highlighted proteins belong to the
starting list of high-confidence interactors; they are endowed with
high scores in the degree and/or betweenness centralities
([199]Supplementary Table S4) to signify close interactions and
important mediating roles. Out of the 304 proteins identified by
LC–MS/MS analysis, in at least one of the experiments, 26.6% were
present among the nodes of the network. Pathway and gene ontology (GO)
enrichment analysis was carried out on network proteins
([200]Supplementary Tables S5 and S6). Pathway enrichment analysis, as
expected, identified among the terms with highest score: glutamatergic
synapse, protein–protein interaction at synapses and long term
potentiation. Noteworthy, also circadian entrainment, the cGMP-PKG
signaling pathway and neurexins and neuroligins showed high
significance. GO term enrichment highlighted the known role of mGlu[1]
in synaptic plasticity, glutamatergic signaling and locomotor behavior
[[201]63], but also regulation of Ca^2+/Na^+ antiporter activity and
mitogen-activated protein kinases [[202]64].
Figure 2.
[203]Figure 2
[204]Open in a new tab
Protein network associated with mGlu[1] in mouse cerebellar synapses.
The nodes represented by high-confidence mGlu[1] putative and
established interactors are shown in orange, mGlu[1] is shown in red,
and KCTD12 in green. A few GO-extracted functionalities are given at
the side of protein communities having a high confidence mGlu[1]
interactor as the starting protein.
3.4. Interaction between mGlu[1]α and KCTD12 in the Cerebellum
We then focused on the putative interaction between mGlu[1]α and KCTD12
because (1) KCTD12 directly interacts with the GABA[B2] subunit of
GABA[B] receptors [[205]38,[206]62], which are also members of the
class C GPCRs and share structural similarities with mGlu[1] [[207]17];
(2) KCTD12 is highly expressed in the dendritic spines and shafts of
Purkinje cells [[208]37,[209]62]; and (3) KCTD12 co-purifies G-protein
βγ subunits even in the absence of GABA[B2] [[210]65]. Interestingly,
in our proteomic approach, G-protein βγ subunits were also
co-immunoprecipitated with mGlu[1]α.
At first, we performed co-immunoprecipitation experiments followed by
Western blots using cerebellar lysates from WT, Grm1-KO, and KCTD12-KO
mice. KCTD12 was consistently co-immunoprecipitated with mGlu[1]α in WT
but not Grm1-KO eluates ([211]Figure 3). The co-assembly between the
two proteins was further examined by reverse co-immunoprecipitation
from cerebellar lysates of WT and KCTD12-KO mice. When KCTD12 was
immunoprecipitated, bands corresponding to mGlu[1]α (monomer around 145
kDa and dimer/multimer above 205 kDa) were also detected in WT but not
KCTD12-KO eluates ([212]Figure 3). However, the efficiency of mGlu[1]α
co-immunoprecipitation with KCTD12 appeared very low and might be due
to the preferential and strong binding of KCTD12 to GABA[B] receptors.
Taken together, these results support a direct or indirect interaction
of mGlu[1]α with KCTD12 in the cerebellum.
Figure 3.
[213]Figure 3
[214]Open in a new tab
mGlu[1]α and KCTD12 co-immunoprecipitants from the cerebellum. Left:
mGlu[1]α was immunoprecipitated from the cerebellum of WT and Grm1-KO
mice using the anti-mGlu[1]α antibody raised in guinea pigs and
immunoblotted using anti-mGlu[1]α (top) and anti-KCTD12 (bottom)
antibodies raised in rabbit. Lanes: WT-input; WT-eluate; KO-input;
KO-eluate. Right: KCTD12 was immunoprecipitated from the cerebellum of
WT and KCTD12-KO mice using the anti-KCTD12 antibody raised in guinea
pigs and immunoblotted using the anti-mGlu[1]α raised (top) and anti
KCTD12 (bottom) antibodies raised in rabbits. Lanes: WT-input;
WT-eluate; KO-input; KO-eluate. Non-specific bands were detected
between 90 and 70 kDa with the anti-mGlu[1]α antibody in eluates of
KCTD12-Co-IPs.
3.5. The Co-Existence of mGlu1α and KCTD12 in the Same Microdomain of
Cerebellar Purkinje Cell Dendritic Spines
Next, we studied the spatial relationship between mGlu[1]α and KCTD12
in the mouse cerebellar cortex using the freeze-fracture replica
immunogold labeling (FRIL) technique. Protoplasmic face immunogold
labelling for both mGlu[1]α and KCTD12 was observed at postsynaptic
elements of Purkinje cells. At Purkinje cell spines, immunogold
particles corresponding to KCTD12 were mainly found peri-synaptically
([215]Figure 4A,B), similar to mGlu1α [[216]58,[217]66]. At dendritic
shafts, KCTD12 was also detected at extra-synaptic sites with or
without mGlu1α in close vicinity ([218]Figure 4C). The nearest neighbor
analysis confirmed that labeling for mGlu[1]α and KCTD12 co-existed in
a nano-domain in both dendritic spines and shafts of Purkinje cells
(mean distance ± s.e.m.: 26.70 ± 2.0 nm for spines; 36.86 ± 3.44 nm for
dendrites) ([219]Figure 4D). The distance between gold particles
detecting mGlu[1]α was also analyzed (mean distance ± s.e.m.: 19.73 ±
0.85 nm for spines; 22.90 ± 0.95 nm for dendrites), thus revealing that
the distribution of their nearest neighbor distance was shorter when
compared to gold particles detecting KCTD12 (two-sample
Kolmogorov-Smirnov test, spines: p = 0.0028; dendrites: p = 0.0001) and
that this is probably due to homodimerization ([220]Figure 4D). The
specificity of immunogold labeling was tested by labeling replicas
obtained from mGlu[1]-KO and KCTD12 KO cerebellum as well as omitting
primary antibodies.
Figure 4.
[221]Figure 4
[222]Open in a new tab
FRIL shows co-distribution of mGlu[1]α and KCTD12 in the same
nano-domain at dendritic spines and shafts of cerebellar Purkinje
cells. (A,B) Electron micrographs showing co-localization of mGlu[1]α
(5 nm) and KCTD12 (10 nm) in spines. A pseudocolor (green) has been
used to simplify the identification of the postsynaptic density (PSD).
(C) A Purkinje cell dendrite shows KCTD12 labeling at extra-synaptic
sites (arrowhead). (D) Cumulative distributions of the nearest neighbor
distance (NND) of gold particles detecting mGlu[1]α and KCTD12 in
Purkinje cell spines and dendritic shafts. Abbreviations: AT, axon
terminal; PC, Purkinje cell. Scale bars: (A,B) 200 nm and (C) 500 nm.
Data were analyzed by means of the two-sample Kolmogorov-Smirnov Test.
p values < 0.05 were considered significant.
3.6. In Vitro Analysis of the mGlu1α–KCTD12 Interaction
To further examine the interaction between mGlu[1]α and KCTD12, we
adopted an in vitro approach. HEK293 cells stably over-expressing mouse
KCTD12 were transiently transfected with a mouse mGlu[1]α plasmid
([223]Supplementary Figure S1). Since KCTD12 directly binds to the
GABA[B2] receptor subunit [[224]62], cells were transfected with
GABA[B2] as a positive control. Mock-transfected cells were used as a
negative control. KCTD12 was successfully immunoprecipitated from whole
cell lysates of mock-transfected, mGlu[1]α-transfected, and
GABA[B2]-transfected cells ([225]Figure 5). However, there was no
co-immunoprecipitation of mGlu[1]α with KCTD12, whereas the GABA[B2]
receptor was detected in the co-immunoprecipitation ([226]Figure 5).
These findings suggest that the interaction between mGlu[1]α and KCTD12
is not occurring via a direct binding but is mediated indirectly
through additional proteins.
Figure 5.
[227]Figure 5
[228]Open in a new tab
mGlu[1]α and KCTD12 do not directly interact in vitro. KCTD12 was
immunoprecipitated from KCTD12 cells that were not transfected,
transfected with mGlu[1]α, or transfected with GABA[B2]-YFP plasmids.
The anti-KCTD12 antibody raised in guinea pigs was used for
immunoprecipitation and the anti-mGlu[1]α (top left), anti-GABAB2 (top
right), and anti-KCTD12 (bottom) antibodies raised in rabbit were used
for immunoblots. Lanes are from whole cell lysates of
mock-transfected-input; mock-transfected-eluate; mGlu[1]α
transfected-input; mGlu[1]α transfected-eluate; GABA[B2]-YFP
transfected-input; and GABA[B2]-YFP transfected-eluate. Non-specific
bands were detected between 90 and 70 kDa with the anti-mGlu[1]α
antibody in eluates.
3.7. The mGlu1α Receptor–KCTD12 Interaction Is Not Mediated by GABAB
Receptors
Since Purkinje cells abundantly express GABA[B] receptors [[229]67], we
investigated whether the in vivo interaction between mGlu[1]α and
KCTD12 occurs through binding of KCTD12 to GABA[B] receptors. We
immunoprecipitated mGlu[1]α from cerebellar lysates of WT, GABA[B1]-KO,
and GABA[B2]-KO mice. If the interaction between mGlu[1]α and KCTD12
were due to GABA[B] receptors, then KCTD12 should not be detected in
the immunoprecipitants of mGlu[1]α from GABA[B1]-KO and GABA[B2]-KO
lysates. However, a band corresponding to the molecular weight of
KCTD12 was detected in mGlu[1]α immunoprecipitants from both
GABA[B1]-KO and GABA[B2]-KO lysates ([230]Figure 6), suggesting that
the interaction of mGlu[1]α with KCTD12 is not mediated by GABA[B]
receptors.
Figure 6.
[231]Figure 6
[232]Open in a new tab
The interaction between the mGlu[1]α and KCTD12 is not mediated by
GABA[B] receptors. mGlu[1]α was immunoprecipitated from WT,
GABA[B1]-KO, and GABA[B2]-KO cerebellar lysates. The anti-mGlu[1]α
antibody raised in guinea pigs was used for immunoprecipitation. The
anti-mGlu[1]α (top) and anti-KCTD12 (bottom) antibodies raised in
rabbits were used for immunoblots. Lanes: WT-input; WT-eluate;
GABA[B1]-KO-input; GABA[B1]-KO-eluate; GABA[B2]-KO-input;
GABA[B2]-KO-eluate.
4. Discussion
In this study, we determined that the mGlu[1] interactome at mouse
cerebellar synapses comprises 304 proteins forming a dense network
involved in a number of neuronal functions including receptor
trafficking, intracellular signaling, and synaptic plasticity. Using a
highly conservative approach to select a small number of high
confidence interacting partners, i.e., detected in all 6 experimental
replicates, we could identify 25 proteins that interact directly or
indirectly with mGlu[1], some of which were already known. Among the
novel interactors, we focused on KCTD12 because of its direct binding
to GABA[B2] [[233]62] which has strong structural similarities to mGlus
[[234]17]. Taking a high-resolution imaging approach, we showed, by
means of immuno-electron microscopy, that KCTD12 and mGlu[1]α were
present in the same nanodomain in Purkinje cell spines. However, using
a recombinant mammalian cell line co-expressing both proteins, we were
unable to co-immunoprecipitate mGlu[1]α with KCTD12. We excluded the
possibility that the GABA[B] receptor mediates the interaction between
KCTD12 and mGlu[1]α. Taken together, our findings suggest that KCTD12
interacts with mGlu[1]α indirectly, possibly via G-protein subunits
[[235]65] or other proteins that interact with mGlu[1]α.
We adopted a proteomic approach based on co-immunoprecipitation and
LC–MS/MS. The isolation of protein complexes from brain tissues using
immunoprecipitation coupled to MS provides a significant advantage over
other methods to detect protein–protein interactions as it allows for
the analysis of complexes within native physiological cellular domains.
However, proteomic results can be confounded by false positive
interactions due to antibody cross-reactivity. To overcome this
problem, we have used tissue obtained from Grm1-KO mice as control
samples. This strategy helped us to ensure the selection of truly
interacting proteins expressed in the mouse cerebellum.
Using only interacting partners with high confidence, we built a
protein interaction network that had over 600 nodes. However, we are
aware that many bona fide interacting proteins might have been excluded
from such a conservative approach. Our findings show that approximately
one-fourth of the proteins identified by our proteomic approach
overlapped with the network proteins, on one hand supporting the
validity of the network for cerebellar synapses also. On the other
hand, our data indicate a molecular complexity of the cerebellar
mGlu[1]interactome greater than the models generated based on the
currently available databases and the extant literature. Future studies
can avail the additional identified proteins to expand our knowledge on
the mGlu[1] interactome both in terms of its fundamental as well as
synapse-specific molecular components.
Our proteomic approach identified several proteins already described as
mGlu[1] interactor partners, such as Homer proteins that directly
interact with the C-terminal domain of group I mGlus [[236]20,[237]68].
Likewise, a number of effectors involved in the classical signaling
pathway of group I mGlus, namely the hydrolysis of phosphoinositides
and release of Ca^2+ from intracellular stores, were detected by our
LC–MS/MS analysis of co-immunoprecipitated proteins. These effectors
included the inositol 3-phosphate receptor type 1, phospholipase Cβ4,
calmodulin, protein kinase Cγ, and TRPC3 besides the G-protein αq and
αi subunits. Amid the mGlu[1] high confidence interacting proteins, we
also detected subunits of CaMKII, a highly abundant serine/threonine
kinase in the post-synaptic density of Purkinje cells. This finding is
consistent with studies showing the presence of multiple CaMKII
consensus motifs in the mGlu[1] C-terminal domain
[[238]69,[239]70,[240]71] and their phosphorylation by CaMKII through a
direct interaction [[241]70]. As for the interactors, we identified
members of the 14-3-3 protein family, small-size proteins (27–32 KDa)
highly enriched in the brain and involved in multiple cellular
signaling events by functioning as adaptors and scaffolds. The 14-3-3
proteins have been reported as important modulators of glutamatergic
synapses, potentially integrating multiple signaling pathways
[[242]72]. They interact with several G-protein coupled receptors,
including GABA[B] and α2-adrenergic receptors [[243]73] but
interactions with mGlu[1] have never been reported thus far.
Nevertheless, a 14-3-3 isoform was shown to mediate the group I
mGlu-agonist-induced down-regulation of KCNK3 potassium channel
activity through protein kinase C [[244]74], thus supporting the
hypothesis of a role in mGlu[1] signaling. Although with low efficiency
(in only one experiment, but the one yielding the highest number of
interactors), we detected mGlu[5] among the interaction partners of
mGlu[1]. In the adult rodent cerebellum, only Golgi and Lugaro cells
and neurons in the deep nuclei were shown to co-express mGlu[1] and
mGlu[5] [[245]4,[246]5,[247]6,[248]7]. Group I mGlus are known to form
heterodimers in recombinant systems and cultured neurons
[[249]55,[250]56] and to co-purify from brain membranes
[[251]55,[252]75]. While the existence of functional mGlu[1]/mGlu[5]
heterodimers in native tissue remains to be unambiguously demonstrated,
our findings provide further support to this notion. The low efficiency
may be compatible with the restricted co-expression of these two
receptors in just a few cerebellar cell types.
In our experiments, we consistently detected a high number of unique
peptides for the neuron-specific α3 catalytic subunit as well as other
subunits of the Na^+/K^+-ATPase, which contributes to maintaining the
polarity of mammalian cells by actively exporting Na^+ and importing
K^+ [[253]76]. Despite the fact that the Na^+/K^+-ATPase was previously
shown to interact with glutamate transporters and ionotropic receptors
[[254]76], possible non-specific co-immunoprecipitation cannot be ruled
out because of its very high expression on the neuronal plasma
membranes. Among the newly discovered interactors, RhoGEF33, which
belongs to a membrane-bound protein family of upstream regulators of
Rho GTPases, is involved in different cellular processes including GPCR
downstream signaling. The role of RhoGEF33 in relation to mGlu[1] may
depend on its Rho GTPase function [[255]77] involved in the regulation
of synaptic structure and efficacy [[256]78].
Among the novel putative interactors identified in our study, we
focused on KCTD12 because of its direct binding to the GABA[B2]
receptor subunit [[257]62] which has structural similarities to mGlus
[[258]17]. We reasoned that KCTD12 could similarly interact with
mGlu[1]and regulate its signaling. Indeed, we were able to
co-immunoprecipitate KCTD12 using antibodies against mGlu[1]α. Reverse
co-immunoprecipitation experiments similarly allowed the detection of
mGlu[1]α, although with lower efficiency. In addition, we showed that
mGlu[1]α and KCTD12 colocalize in the same nanodomain in Purkinje cell
spines, strengthening the idea of a functional interaction in neurons
in vivo. However, our nearest neighbor analysis suggested that the
distance between KCTD12 and mGlu[1]α was incompatible with a direct
physical interaction. Consistent with this hypothesis, we were unable
to co-immunoprecipitate mGlu[1]α with KCTD12 from HEK293 cell extracts
expressing these two proteins. Previous studies reported a functional
interaction between mGlu[1]α and GABA[B] receptors at Purkinje cell
synapses [[259]79,[260]80] and showed that the activation of GABA[B]
receptors facilitates the mGlu[1]-mediated LTD induction
[[261]51,[262]80,[263]81]. Furthermore, the two receptors were shown to
co-immunoprecipitate from cerebellar extracts [[264]51,[265]79].
However, their interaction was shown to be indirect and mediated via
Gβγ subunits released upon GABA[B] receptor activation [[266]82]. In
line with these findings, we observed that KCTD12 was
co-immunoprecipitated with the mGlu[1]α even in the absence of the
GABA[B1] and GABA[B2] receptor subunits, suggesting that the
interaction between KCTD12 and mGlu[1]α is independent of GABA[B]
receptors. A likely candidate to mediate the interaction between
mGlu[1]α and KCTD12 is the Gβ subunit of the G protein. KCTD12 also
binds to Gβ [[267]38] independently of GABA[B] receptors [[268]65]. It
is, therefore, conceivable that mGlu[1] binds G proteins bound to
KCTD12. This would also explain the weak co-immunoprecipitation of
mGlu[1] with anti-KCTD12 antibodies. Future studies should explore
whether Gβ subunits do indeed mediate this interaction.
Irrespective of the interaction mechanism between mGlu[1]α and KCTD12,
a still open question is the potential functional role of this
interaction. KCTD12 mediates fast GABA[B] receptor desensitization
through its binding to the activated Gβγ subunits [[269]65], thereby
uncoupling them from the inwardly rectifying K^+ (GIRK or Kir3) and
voltage-gated Ca^2+ channels (VGCCs) modulated by GABA[B] receptor
activation [[270]62]. It can be surmised that KCTD12 similarly
modulates mGlu[1] complex postsynaptic responses. In Purkinje cells,
both VGCCs, namely Ca[V]2.1 and Ca[V]3.1 [[271]54,[272]83], and
potassium channels [[273]84,[274]85] were shown to functionally
interact with mGlu[1]. KCTD12 may thus regulate mGlu[1]α-mediated
responses through these channels. In this respect, it is interesting to
note that the related KCTD8 and KCTD12b proteins were shown to directly
bind to and co-localize with Cav2.3 channels at the presynaptic zone in
projections from the medial habenula where they modulate
neurotransmitter release [[275]86]. Future studies will be necessary to
clarify the functional significance of the mGlu[1]α–KCTD12 interaction.
5. Conclusions
The mGlu[1] interactome presented here serves as a molecular framework
for further exploring the signaling, trafficking, and involvement in
the cerebellar function of this receptor. Some of the proteins
identified by our proteomic approach are novel putative interactors
whereas others were already known to interact directly or indirectly
with mGlu[1]. Among the novel interactors, we focused on KCTD12 and
showed that it coexists with mGlu[1]α in the same nanodomain in
Purkinje cell spines. However, biochemical analyses suggested an
indirect interaction via additional proteins that warrants follow-up
functional investigations.
Acknowledgments