Adv Exp Med Biol. Author manuscript; available in PMC 2014 Feb 19.
Published in final edited form as:
BTB-Kelch Proteins and Ubiquitination of Kainate Receptors
Kainate
receptors (KAR) form a class of glutamate receptors that have been
implicated in epilepsy, stroke, Alzheimer’s and neuropathic pain.1
KAR subtypes are known to be segregated to specific locations within
neurons and play significant roles in synaptic transmission and
plasticity.2 Increasing evidence suggests a the role for ubiqutination in regulating the number of synaptic neurotransmitter receptors.3–5
The ubiquitin pathway consists of activation (E1), conjugation (E2) and
ligation (E3). Cullins form the largest family of E3 ligase complexes.
We have recently shown that the BTB/Kelch domain proteins, actinfilin (KLHL17 , 1p36.33, https://www.ncbi.nlm.nih.gov/gene/339451#gene-expression)
and mayven (KLHL2, 4q32.3,https://www.ncbi.nlm.nih.gov/gene/11275O , bind both Cul3 and specific KAR subtypes (GluR6 and
GluR5-2b) to target these KARs for ubiquitination and degradation.5
In this chapter we will review how these interactions occur, what they
mean for the stability of KARs and their associated proteins and how, in
turn, they may affect synaptic functions in the central nervous system.
Introduction
KARs
are found pre and postsynaptically and have been implicated in the
etiology of epilepsy, as well as stroke-induced neurodegeneration and
Huntington’s disease.6,7
Epilepsy occurs when inhibitory adaptation is unable to prevent excess
neural activity. The developing cortex is particularly vulnerable and a
number of syndromes are associated with epilepsy at an early age.8 Postsynaptic injection of kainic acid causes epileptiform discharges and the death of hippocampal CA3 pyramidal neurons.9,10
Moreover, KARs are subject to developmental and activity-dependent
regulation at thalamocortical synapses and are likely to play an
important role in the development of hippocampal synaptic circuits.11–13
KARs act as excitatory glutamate- gated ion channels: KAR-mediated
excitatory postsynaptic currents were first described at mossy fiber-CA3
pyramidal cell synapses,14,15
while presynaptically, activation of KARs on inhibitory interneurons
decreases GABA release which acts to enhance electrical activity,
suggesting that presynaptic KARs may be epileptigeneic.16 Notably, the GluR6 subtype of KARs can also increase neuronal excitability via metabotropic regulation of potassium channels.17
To treat pathological conditions it will be crucial to understand the
molecular mechanisms that determine localization of specific KARs to
specific membrane domains.
Within KARs, there is a considerable diversity of properties, including unitary channel conductance, Ca2+
permeability and rectification, which arise from differences in
receptor subunit composition and RNA editing of GluR5 and GluR6.18–20
KARs are tetramers that can be assembled from any one of five receptor
subunits encoded by two separate gene families. The first of these
includes receptor subunits GluR5, -6 and -7. Each of these subunits can
form functional homomeric ion channels or heteromeric mixtures that
appear to assemble promiscuously with any available subunit GluR5, -6 or
-7.21–23 Alternative splicing of GluR5 yields two isoforms:21
GluR5-1 and GluR5-2, which has three additional splice variants
possessing distinct C-terminal sequences. The shortest variant is
designated GluR5-2a, while additional exons located in the C-terminal
region give rise to GluR5-2b and GluR5-2c; these variants share a
C-terminal type 1 PDZ-binding domain that is absent in GluR5-2a.18
The second gene family consists of KA1 and KA2 subunits that are
functional only when expressed as heteromeric assemblies with GluR5, -6
or -7.23,24
Alternative
splicing and RNA editing of ionotropic glutamate receptors play
important roles in receptor assembly and trafficking.25–27
Regulatory steps in the assembly of KA2-containing KARs are governed by
at least two trafficking signals located in the cytoplasmic terminal
(C-tail) of the KA2 subunit. The first is an arginine(R)-rich motif which
operates as an endoplasmic reticulum (ER) retention signal preventing
the insertion of homomeric KA2 receptors into the plasma membrane.28
The second is a di-leucine motif (LL) which mediates the internalization and
subsequent relocalization of surface-expressed KA2 subunits.28 Similarly, GluR5-2b carries a positively charged amino acid motif that acts as a novel ER retention signal.29
In contrast, GluR6, which is highly expressed at the plasma membrane,
has a forward trafficking signal in its C-terminal domain critical for
ER exit.30,31
These
differences in targeting appear to convey specific roles to specific
KAR subtypes: In GluR6 knockout mice, mossy fiber long-term potentiation
(LTP) was reduced, whereas GluR5 knockout mice exhibited normal LTP.32
The activation of KARs also modulates neurotransmitter release from a
number of hippocampal synapses, including GABA release at inhibitory
terminals that synapse onto CA1 pyramidal cells.16,33–35
In hippocampal slices, kainate depresses GABA-mediated synaptic
inhibition and increases the firing rate of interneurons. These effects
are explained by two populations of KARs in CA1 interneurons: GluR6/KA2
located in the somatodendritic compartment and GluR5-GluR6 or GluR5-KA2
at presynaptic terminals.35 It is anticipated that this segregation of KARs will allow us to design drugs that specifically target each function.
Recent
evidence suggests that the ubiquitin-proteasome pathway and synaptic
activity affects the composition of postsynaptic proteins.36–38
The addition of ubiquitin to proteins leads to a variety of fates for
the tagged proteins, one of which is degradation by the 26S proteasome.39
A family of proteins called E3 ligases determines the specificity of
ubiquitin addition. E3 ligases frequently consist of a complex of
proteins that act together for specific substrate binding and ubiquitin
ligation activity. Two major families of E3 ligases have been described:
the HECT-domain family that is defined by its homology to the
C-terminus of E6-associated protein (E6AP) and the RING family that
contains either an intrinsic RING-finger domain or an associated
RING-finger protein subunit essential for ubiquitin ligase activity.40
One of the best-characterized subset of the RING E3 ligases is the
Skp1/Cul1/F-box protein complex (SCF), in which Cul1 binds an adaptor
molecule, Skp1.41,42
Skp1 associates with an F-box protein that in turn binds a
phosphorylated substrate. The Cul1 component of the SCF E3 ligase
belongs to an evolutionarily conserved family of proteins known as
cullins, of which there are six closely related members (Cul1, 2, 3, 4A,
4B and 5) and three distant relatives (Cul7, Parc and APC2).
A major class of Kelch proteins, defined as containing a 6-fold tandem “kelch” element,43
contains an N-terminal BTB/POZ domain and C-terminal kelch repeats and
targets different substrates to the Cul3-Roc1 catalytic core.44
For example, the BTB-Kelch protein Keap1 (KLHL19), a negative regulator of the
transcription factor Nrf2, binds Cul3 and Nrf2 via its BTB and kelch
domains, respectively, targeting Nrf2 for ubiquitination and subsequent
degradation by the proteasome.45
The BTB-Kelch family also includes the closely related protein mayven (KLHL2) ,
an actin-binding protein and gigaxonin (KLHL16, GAN), which is mutated in a human
autosomal recessive neurodegenerative disorder named giant axonal
neuropathy.46 Mutations in E3 ubiquitin ligases have also been associated with Parkinson’s disease and breast cancer.47
KAR Regulation by the Ubiquitin-Proteasome Pathway
To
search for proteins involved in the regulation of KARs, we performed a
yeast two-hybrid screen of an adult rat brain cDNA library using the
C-terminus of GluR6 as bait. Strong interactions were detected between
GluR6 and actinfilin (KLHL17, Actinfilin (AF) is a novel BTB/Kelch protein that
was identified as a brain-specific actin-binding protein in postsynaptic
densities (PSDs).48
Co-immunoprecipitation studies performed using HEK293 cell and rat
brain extracts show that actinfilin binds GluR6 and GluR5-2b, but not
with other glutamate receptors and ion channels.5 Because actinfilin is highly similar to another brain BTB/Kelch protein member, mayven (KLHL2) 49 (55% amino acid identity), we cloned this cDNA and, upon expression, found that it also co-immuno-precipitated with GluR6.
Actinfilin (KLHL17) was found to interact with Cul3 to promote proteasomal degradation of GluR6 in vitro and in vivo.5
Expression of GluR6 with an HA-tagged ubiquitin (Ub-HA) in HEK293 cells
showed a characteristic ladder indicating that GluR6 was ubiquitinated.
Conversely, treatment with the 26S proteasomal inhibitor, MG132,
greatly enhanced ubiquitination and stabilized GluR6 expression,
demonstrating that GluR6 protein is fairly short-lived. Furthermore,
co-immunoprecipitation studies verified that actinfilin interacts with
Cul3, but not Cul1, in brain. The interactions of cullins with their
adaptors o%en cause mutual degradation. Importantly, Cul3 appears to
specifically regulate KAR levels in vivo (Fig. 1):
In synaptosomes prepared from heterozygous Cul3 knockout mice, GluR6
levels are substantially increased, a small effect is observed on KA2
levels, but significantly, no effect on AMPA or NMDA receptors can be
detected. These data suggest that Cul3 promotes degradation of KARs.We also found that actinfilin (AF) (KLHL17) was localized synaptically in hippocampal
and cortical neurons and that it negatively regulates KAR expression (Fig. 2).
A high degree of colocalization of AF and GluR6 was observed in
dendritic spines. To establish tools to determine the role of
actinifilin in the trafficking and/or synaptic localization of GluR6, we
have developed a short hairpin inhibitory RNA (RNAi) to actinfilin that
eliminates actinifilin expression (Fig. 2D).
Specifically, we found that decreasing actinfilin levels via RNAi and
overexpressing an inactive Cul3 both induced increased surface GluR6
expression at synapses, suggesting that actinfilin-Cul3-mediated
degradation may provide an important mechanism for regulating neuronal
GluR6.
Proposed
regulation of KARs by AF and Cul3. The Cul3-based E3 ligase consists of
Cul3, which acts as a scaffold to bring several essential proteins in
close proximity. These proteins include Nedd 8 (Nd8), Rbx1 and a
ubiquitin conjugating enzyme (E2). Substrate specificity is mediated by
proteins containing a BTB domain that bind Cul3 in its amino-terminal
end. Other domains on the BTB domain-containing proteins are involved in
recognition of substrates; in the cases of actinfilin(KLHL17) and mayven(KLHL2) , these
are kelch domains.
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