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The Journal of Neuroscience, October 1, 2000, 20(19):7238-7245
Homer Proteins Regulate Coupling of Group I Metabotropic
Glutamate Receptors to N-Type Calcium and M-Type Potassium
Channels
Paul J.
Kammermeier1,
Bo
Xiao2,
Jian Cheng
Tu2,
Paul F.
Worley2, and
Stephen R.
Ikeda1
1 Laboratory of Molecular Physiology, Guthrie Research
Institute, Sayre, Pennsylvania 18840, and 2 Department of
Neuroscience, Johns Hopkins University, School of Medicine, Baltimore,
Maryland 21205
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ABSTRACT |
Group I metabotropic glutamate receptors (mGluR1 and 5)
couple to intracellular calcium pools by a family of proteins, termed Homer, that cross-link the receptor to inositol trisphosphate receptors. mGluRs also couple to membrane ion channels via G-proteins. The role of Homer proteins in channel modulation was investigated by
expressing mGluRs and various forms of Homer in rat superior cervical
ganglion (SCG) sympathetic neurons by intranuclear cDNA injection.
Expression of cross-linking-capable forms of Homer (Homer 1b, 1c, 2, and 3, termed long forms) occluded group I mGluR-mediated N-type
calcium and M-type potassium current modulation. This effect was
specific for group I mGluRs. mGluR2 (group II)-mediated inhibition of
N-channels was unaltered. Long forms of Homer decreased modulation of
N- and M-type currents but did not selectively block distinct G-protein
pathways. Short forms of Homer, which cannot self-multimerize (Homer 1a
and a Homer 2 C-terminal deletion), did not alter mGluR-ion channel
coupling. When coexpressed with long forms of Homer, short forms
restored the mGluR1a-mediated calcium current modulation in an apparent
dose-dependent manner. Homer 2b induced cell surface clusters of mGluR5
in SCG neurons. Conversely, a uniform distribution was observed when
mGluR5 was expressed alone or with Homer short forms. These studies
indicate that long and short forms of Homer compete for binding to
mGluRs and regulate their coupling to ion channels. In
vivo, the immediate early Homer 1a is anticipated to enhance
ion channel modulation and to disrupt coupling to releasable intracellular calcium pools. Thus, Homer may regulate the magnitude and
predominate signaling output of group I mGluRs.
Key words:
mGluR; Homer; calcium current; M-current; ion channel
modulation; neuron; G-protein
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INTRODUCTION |
Group I metabotropic glutamate
receptors (mGluRs) modulate ionic currents in central (Sahara and
Westbrook, 1993 ; Chavis et al., 1994; Glaum and Miller, 1995; Choi and
Lovinger, 1996) and peripheral neurons (Hay and Kunze, 1994) by
activating pertussis toxin-sensitive and -insensitive G-proteins (Pin
and Duvoisin, 1995). Modulation of calcium currents through
heterologously expressed group I mGluRs in superior cervical ganglion
(SCG) neurons proceeds through a voltage-dependent, G  -mediated
(Herlitze et al., 1996; Ikeda, 1996) pathway as well as a
voltage-independent pathway (McCool et al., 1998; Kammermeier and
Ikeda, 1999). Activation of group I mGluRs also produces inhibition of
M-type potassium currents by a G-protein-dependent mechanism (Charpak
et al., 1990; Womble and Moises, 1994; Ikeda et al., 1995).
Recently, a new family of proteins, termed Homer, has been described
that bind to the intracellular C terminus of group I mGluRs (Brakeman
et al., 1997) and may regulate function of the receptor (Tu et al.,
1998; Xiao et al., 1998). Multiple forms of Homer arising from three
different genes have been identified (Kato et al., 1998; Sun et al.,
1998; Xiao et al., 1998). The first identified Homer protein, now
termed Homer 1a, was identified on the basis of its rapid induction by
synaptic activity in models of neural plasticity (Brakeman et al.,
1997). Homer 1a encodes a single Ena/VASP homology 1 (EVH1)
domain that mediates binding to proline-rich sequences in mGluR and
other proteins (Brakeman et al., 1997). All subsequently
identified Homer proteins additionally encode a C-terminal coiled coil
domain, which mediates self-multimerization between Homer proteins
(Xiao et al., 1998), but additional short form cDNAs have been
reported (Soloviev et al., 2000). The coiled coil encoding forms of
Homer (Homer 1b, 1c, 2a, 2b, and 3; collectively termed long
forms) are constitutively expressed in many brain regions and are
enriched at the postsynaptic density (Tu et al., 1998; Xiao et al.,
1998). Long forms of Homer appear to couple group I mGluRs with
intracellular inositol trisphosphate receptors (IP3Rs) (Tu et al., 1998). Homer 1a binds both
mGluRs and IP3Rs but lacks the coiled coil domain
required to cross-link the receptors. Thus, Homer 1a may function as a
naturally occurring dominant negative protein to regulate the degree of
mGluR-IP3R coupling. Homer proteins may also
regulate the coupling between NMDA receptors and intracellular calcium
pools through their interaction with Shank proteins (Tu et al.,
1999).
In addition to releasing intracellular calcium, group I mGluRs regulate
membrane ion channels and activate MAP kinase pathways (Peavy and Conn,
1998). In this report, the functional effects of Homer were
investigated from the perspective of the G-protein pathways that
mediate N-type calcium and M-type potassium channel modulation via
group I mGluRs. Consistent with previous studies, long and short forms
of Homer appeared to compete to regulate the coupling to
G-protein-dependent ion channels in a manner contrasting with their
regulation of IP3R coupling. These studies
suggest a mechanism by which Homer proteins can modulate both the
magnitude and the predominate output of its effector pathways.
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MATERIALS AND METHODS |
Cell isolation and DNA injection. Detailed
descriptions of the isolation and injection procedures have been
described previously (Ikeda, 1997). Briefly, the superior cervical
ganglia were dissected from adult rats and incubated in Earle's
balanced salt solution (Life Technologies, Rochelle, MD) containing 0.4 mg/ml trypsin (Worthington, Freehold, NJ), 0.6 mg/ml collagenase
D (Boehringer Mannheim, Indianapolis, IN), and 0.05 mg/ml DNase I
(Sigma, St. Louis, MO) for 1 hr at 35°C. Cells were then centrifuged
twice, transferred to minimum essential medium (Fisher Scientific,
Pittsburgh, PA), plated, and placed in an incubator at 37°C to await
DNA injection. Injection of cDNA was performed with an Eppendorf
(Madison, WI) 5246 microinjector and 5171 micromanipulator 4-6 hr
after cell isolation. Plasmids were stored at  20°C as a 1 µg/µl
stock solution in 10 mM Tris and 1 mM EDTA, pH
8. Group I mGluR inserts (from J.-P. Pin, Institut National de
la Santé et de la Recherche Médicale de Pharmacologie,
France) were cloned in the pRK5 vector (Genentech, South San Francisco,
CA). mGluR5-myc (myc tag TREQKLISEEDLAR, inserted between amino acids
22 and 23 of rat mGluR5a; construct from J.-P. Pin) was injected at 0.1 µg/µl. mGluR1a was injected at 0.03 µg/ml, mGluR5 at 0.1 µg/µl, and mGluR2 at 0.04 µg/µl. The Homer Zb c-terminal
deletion construct H2N, construct contains the coding sequences
for Homer 2b amino acids 2-141. All Homer cDNAs were injected at 0.1 µg/µl, unless otherwise indicated. Neurons were coinjected with
"enhanced" green fluorescent protein cDNA (0.005 µg/µl;
pEGFP-N1; Clontech, Palo Alto, CA) to facilitate later identification
of successfully injected cells. After injection, cells were incubated
overnight at 37°C, and patch-clamp experiments were performed the
following day.
Electrophysiology and data analysis. Patch-clamp recordings
were made using 7052 glass (Garner Glass, Claremont, CA). Pipette resistances were 1-3 M , yielding uncompensated series resistances of 2-7 M. Series resistance compensation of 80% was used in all recordings. Data were recorded using an Axopatch 200 or 200A from Axon
Instruments (Foster City, CA). Voltage protocol generation and data
acquisition were performed using custom data acquisition software on an
Apple (Cupertino, CA) Macintosh Quadra series computer with a MacAdios
II data acquisition board (G. W. Instruments, Somerville, MA).
Currents were sampled at 0.5-5 kHz, low-pass-filtered at 5 kHz
(calcium currents) or 1 kHz (M-currents) using the four-pole Bessel
filter in the patch-clamp amplifier, digitized, and stored on the
computer for later analysis. Experiments were performed at 21-24°C
(room temperature). Data analysis was performed using Igor software
(WaveMetrics, Lake Oswego, OR). All statistical analyses were performed
using StatView software (SAS Institute, Cary, NC). ANOVA, followed by a
post hoc test (as appropriate), was used to determine
statistical significance on all data sets. p = 0.05 was
used to determine significance.
Solutions. For calcium current recordings, the external
(bath) solution contained (in mM): 145 tetraethylammonium
(TEA) methanesulfonate (MS), 10 HEPES, 15 glucose, 10 CaCl2, and 300 nM tetrodotoxin, pH
7.4, osmolality 320 mOsm/kg. The internal (pipette) solution contained
(in mM): 120 N-methyl-D-glucamine MS, 20 TEA, 11 EGTA, 10 HEPES, 10 sucrose, 1 CaCl2, 4 MgATP, 0.3 Na2GTP, and 14 Tris-creatine phosphate, pH 7.2, osmolality 300 mOsm/kg. For M-current recordings, the external solution
contained (in mM): 150 NaCl, 2.5 KCl, 10 HEPES, 1 MgCl2, 2 CaCl2, 15 glucose,
and 300 nM tetrodotoxin, pH 7.4, osmolality 320 mOsm/kg. The internal solution contained (in mM):
150 KCl, 0.1 K4-BAPTA, 10 HEPES, 4 MgATP, and 0.1 Na2GTP, pH 7.2, osmolality 300 mOsm/kg. The
glutamate concentration in all experiments was 100 µM.
Western blot and microscopic analysis. A detailed
description of the Western blot procedure is reported elsewhere (Xiao
et al., 1998). Briefly, six SCGs, weighing ~1 mg each, from three rats were pooled in a microfuge tube and sonicated in 200 µl of 2×
SDS sample buffer. The SCG homogenates were centrifuged at 14,000 rpm
for 10 min. Various amounts of supernatants were resolved by SDS-PAGE
and transferred to nitrocellulose membranes. The blots were probed with
polyclonal Homer antibody (Xiao et al., 1998) and visualized with ECL
(New Life Sciences, Inc., Boston, MA). For the surface expression
experiments, neurons were prepared and injected as for
electrophysiological experiments but were plated on
poly-L-lysine-treated glass coverslips. The
following day, cells were exposed to the mouse anti-myc antibody
(Jackson ImmunoResearch, West Grove, PA), fixed in 4% paraformaldehyde (Fisher Scientific, Pittsburgh, PA), and subsequently exposed to the
fluorescently tagged secondary antibody (anti-mouse Cy3; Jackson
ImmunoResearch). Cells were not permeabilized to restrict labeling to
surface epitopes. Finally, the cells were mounted on glass slides with
Permafluor (Fisher Scientific) and examined using a Zeiss (Thornwood,
NY) Axiophot microscope, and the images were captured using Metamorph
software (Universal Imaging Corp., West Chester, PA).
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RESULTS |
Endogenous expression of Homer proteins in SCG neurons
As a prelude to studies of Homer effects on mGluR signaling in SCG
neurons, we determined whether Homer proteins are expressed endogenously in SCG neurons. Western blot analysis was performed on rat
SCG tissue using antibodies directed against Homer 1 (recognizes 1b and
1c), Homer 2 (recognizes 2a and 2b), and Homer 3. The presence of
endogenous Homer 2 and Homer 3 was detected by Western blotting, but
Homer 1 proteins were not detected (Fig.
1). It is likely that intranuclear cDNA
injection resulted in protein levels much higher than those of the
endogenously expressed Homer proteins, as indicated by the degree of
ion channel modulation and the surface expression pattern observed in
cells heterologously expressing only the receptor (see below).
Therefore, natively expressed Homer 2b and Homer 3 were unlikely to
confound the following experiments.
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Figure 1.
SCG neurons natively express Homer 2 and Homer 3 but not Homer 1. SCG extracts were examined for expression of Homer
isoforms. Antibodies were directed against Homer proteins derived from
each gene. The anti-Homer1 antibody (left) recognized
Homer 1b and Homer 1c. Anti-Homer2 (center) recognized
Homer 2b, and anti-Homer3 (right) recognized Homer 3. Five, 10, and 15 µl of SCG extract were loaded into lanes 1, 2, and 3 of each group, respectively. The
immunoblots were probed with Homer 1-, 2-, and 3-specific
antibody.
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Long-form Homer results in surface clusters of mGluR
SCG neurons do not natively express functional mGluRs (Ikeda et
al., 1995). This "null background" provides a system whereby specific mGluR subtypes can be heterologously expressed and studied in
isolation. To examine the surface expression of a heterologously expressed group I mGluR, mGluR5a containing an extracellular myc tag
(mGluR5-myc) was expressed in SCG neurons. Live mGluR5-myc-expressing cells were then exposed to primary anti-myc antibody, fixed in 4%
paraformaldehyde, and exposed to a fluorescently tagged secondary antibody (anti-mouse Cy3) without permeabilization. SCG neurons heterologously expressing mGluR5-myc showed a diffuse receptor distribution (Fig. 2, right).
Coexpression of Homer 1a did not appear to alter mGluR5-myc surface
expression (data not shown). However, coexpression of Homer 2b with
mGluR5-myc appeared to produce a punctate receptor distribution (Fig.
2, left), but surface expression remained high. These data
indicate that the long forms of Homer can aggregate group I mGluRs into
clusters on the membrane. This clustering is likely to be a function of
the coiled coil C-terminal tail present in the long forms of Homer
(Xiao et al., 1998), because Homer 1a did not produce the pattern.
These changes in group I mGluR distribution suggest that the function
of these receptors may also be altered by expression of Homer proteins. Therefore, the effect of Homer expression on group I mGluR-mediated modulation of N-type calcium and M-type potassium currents was examined.
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Figure 2.
Homer 2b expression induces clustering of
mGluR5-myc. Left, Surface expression pattern of three
cells (A-C represent different focusing plains)
expressing mGluR5-myc and Homer 2b. Right, Surface
expression pattern of three cells (A-C)
expressing mGluR5-myc alone.
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Long forms of Homer proteins reduce group I mGluR-mediated
N-type calcium current inhibition
When mGluR1a was expressed in SCG neurons, application of 100 µM glutamate produced a 55 ± 4% (average
inhibition ± SEM; n = 15) inhibition of the
calcium current (Fig. 3A).
Currents were elicited with a triple-pulse protocol (Elmslie et al.,
1990) consisting of a test pulse to +10 mV (the "prepulse";
indicated in Fig. 3A, filled circles), followed by a strong
depolarizing step to +80 mV, and another test pulse to +10 mV (the
"postpulse"; Fig. 3A, open circles). Glutamate produced
a strong inhibition of the current in the prepulse as well as a slight
slowing of activation. The depolarizing step to +80 mV produced partial
relief of inhibition and restored "normal" activation kinetics to
the postpulse current. These features of inhibition are hallmarks of
modulation produced by activated G-protein subunits (Hille,
1994), presumably binding directly to the channel (DeWaard et al.,
1997). Inhibition by glutamate was typically fully reversible and quite
rapid (Fig. 3A).
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Figure 3.
The long but not the short forms of Homer reduce
calcium current inhibition through group I mGluRs. A,
Sample currents (top) and time course
(bottom) illustrating inhibition by 100 µM
glutamate in a cell expressing mGluR1a. Cells were held at 80 mV and
stepped to a +10 mV test pulse for 25 msec and then to a 50 msec +80 mV
conditioning pulse, followed by a brief step back to the holding
potential and another test pulse to +10 mV. In the time course,
filled circles represent current measurements 10 msec
into the first test pulse, the prepulse; open circles
represent measurements from the postpulse. Calibration: 0.2 nA, 20 msec. B, Sample currents and time course for a cell
expressing mGluR1a and Homer 1a. Calibration: 0.2 nA, 20 msec.
C, Currents and time course for a cell expressing
mGluR1a and Homer 2b. Calibration: 0.5 nA, 20 msec. D,
Summary of data from mGluR1a (open bars) and mGluR5a
(hatched bars) cells. Error bars represent mean + SEM.
The number of cells is indicated in parentheses to the
right of each bar. *Statistically significant
differences versus controls (ANOVA, p = 0.05).
glu, Glutamate; con,
control.
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The effect of expressing different Homer forms on calcium current
inhibition mediated by two group I mGluRs, mGluR1a and mGluR5a (Fig.
3B-D), was tested. Coexpression of Homer 1a with mGluR1a had no effect on calcium current modulation by glutamate (Fig. 3B,D). In cells expressing both mGluR1a and Homer 1a,
glutamate inhibited calcium current by 53 ± 6%
(n = 11), similar to cells expressing mGluR1a alone. In
addition, an N-terminal construct of Homer 2b was also without
significant effect on calcium current inhibition by glutamate. This
construct (H2N) possesses the mGluR binding site common to all Homers
but lacks the coiled coil C-terminal tail, making it functionally
equivalent to Homer 1a (Xiao et al., 1998). In cells expressing mGluR1a
and H2N, calcium current was inhibited 62 ± 6%
(n = 5) by 100 µM glutamate
(Fig. 3D). In contrast, Homer homologs containing the
C-terminal coiled coil significantly reduced the modulation of calcium
current by glutamate (ANOVA, p = 0.05; Fig.
3C,D). Figure 2C illustrates current traces from a cell expressing mGluR1a and Homer 2b. Note that application of
glutamate produced virtually no inhibition in this example. On average,
calcium current was inhibited 13 ± 2% (n = 17)
in cells expressing mGluR1a and Homer 2b. Similarly, when Homer 3 was
coexpressed, glutamate produced only a 13 ± 5%
(n = 6) inhibition. Expression of Homer 1b and Homer 1c
yielded intermediate effects, significantly reducing the calcium
current inhibition to 38 ± 4% (n = 14) and
21 ± 6% (n = 8), respectively. Experiments with mGluR5a produced similar results. Cells expressing mGluR5a, mGluR5a plus Homer 1a, and mGluR5a plus Homer 2b were inhibited 43 ± 5% (n = 6), 42 ± 4% (n = 5), and
11 ± 2% (n = 6) by 100 µM glutamate, respectively (Fig. 3D,
hatched bars).
The presence of natively expressed long forms of Homer and the ability
of these proteins to occlude group I mGluR-mediated calcium channel
modulation raised the question of whether group I mGluRs are
endogenously expressed at low levels but with their effects masked by
the presence of endogenous Homer 2 and Homer 3. To test for this
possibility, Homer 1a and H2N were expressed alone in cells, without
injection of mGluR cDNAs. In six cells expressing Homer 1a alone and in
six cells expressing H2N alone, no detectable calcium current
inhibition was observed after application of 100 µM
glutamate (data not shown). These data indicate that although SCG
neurons express Homer 2 and Homer 3 proteins, they do not appear to
natively express functional mGluRs.
Expression of Homer protein does not alter signaling
via mGluR2
Homer proteins are known to bind specifically to group I mGluRs
(Brakeman et al., 1997). As a test for specificity, the effect of Homer
expression on calcium current inhibition mediated by mGluR2, a group II
mGluR, was tested. Expression of mGluR2 in SCG neurons produced a
potent, strongly voltage-dependent inhibition of the calcium current
(Fig. 4A). On average,
mGluR2-mediated calcium current inhibition was 64 ± 2%
(n = 7; Fig. 4D). Cells expressing
mGluR2 plus Homer 1a (Fig. 4B), Homer 2b, or Homer 3 (Fig. 4C) exhibited calcium current inhibitions of 54 ± 5% (n = 9), 57 ± 4% (n = 6),
and 71 ± 1% (n = 5), respectively (Fig. 4D). Neither the kinetics nor the voltage dependence
of mGluR2-mediated calcium current inhibition appeared to be altered by
Homer expression. These data indicate that the effects of various forms
of Homer on mGluR1a and mGluR5a calcium current modulation result from selective association with group I mGluRs.
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Figure 4.
Homer does not alter modulation through mGluR2.
A-C, Sample currents for inhibition by 100 µM glutamate in cells expressing mGluR2
(A), mGluR2 plus Homer 1a
(B), and mGluR2 plus Homer 3 (C). Calibration: A, 1 nA;
B, C, 0.7 nA; A-C, 20 msec.
D, Summary of all mGluR2 data. No group produced a level
of inhibition that differed from control (p = 0.05, ANOVA). glu, Glutamate; con,
control.
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Effects on mGluR-mediated modulation by Homer 2b are reversed by
coexpression of short-form Homer proteins
The data above demonstrate that the long and short forms of Homer
have contrasting effects. The long forms reduce calcium current
modulation, whereas the short forms (Homer 1a and the H2N construct)
are permissive. Because the mGluR binding site on each form of Homer is
well conserved (Brakeman et al., 1997; Xiao et al., 1998), the opposing
Homer isoforms may be competing for a shared mGluR binding site.
Therefore, the ability of the short forms of Homer to reverse the
occlusion by Homer 2b was examined. In cells expressing mGluR1a and
Homer 2b, coexpression of Homer 1a (Fig.
5B) or H2N (Fig.
5C) was able to significantly increase calcium current
inhibition compared with cells expressing mGluR1a and Homer 2b alone
(ANOVA, p = 0.05). Inhibition was increased from
13 ± 2% (n = 17), in mGluR1a plus Homer
2b-expressing cells to 21 ± 2% (n = 13) and
33 ± 3% (n = 7) in cells coexpressing Homer 1a
or H2N, respectively (Fig. 5D). When Homer 1a cDNA was injected at a twofold higher concentration (0.2 µg/µl), presumably inducing higher expression levels, calcium current inhibition was
increased further to 35 ± 2% (n = 8;
p < 0.05). This apparent concentration dependence
argues for competition for the mGluR1a binding site. These data suggest
that Homer 1a displays a dominant negative phenotype.
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Figure 5.
The effect of Homer 2b can be partially reversed
by coexpression of other forms of Homer. A, Sample
currents illustrating inhibition by 100 µM glutamate in a
cell expressing mGluR1a and Homer 2b (same cell as in Fig.
1C). Calibration: 0.5 nA, 20 msec. B,
Sample currents for a cell expressing mGluR1a, Homer 2b, and Homer 1a.
Calibration: 0.5 nA, 20 msec. C, Sample currents for a
cell expressing mGluR1a, Homer 2b, and H2N. Calibration: 0.7 nA, 20 msec. D, Data summary for control cells (mGluR1a only,
from Fig. 3D), Homer 2b cells (cells expressing mGluR1a
plus Homer 2b), and those coexpressing Homer 1a injected at 0.1 µg/µl [+2b, 1a (low)], 0.2 µg/µl [+2b,
1a (hi)], Homer 1b (+2b, 1b), and H2N
(+2b, H2N). Data are mean + SEM. *Significant
divergence from Homer 2b only cells (ANOVA, p = 0.05). In addition, inhibition in the +2b 1a (hi)
group was significantly larger than in the +2b 1a (low)
group. glu, Glutamate; con,
control.
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Interestingly, cells expressing mGluR1a, Homer 2b, and Homer 1b also
displayed significantly more calcium current inhibition by glutamate
than the mGluR1a plus Homer 2b-expressing cells (inhibition was
increased to 33 ± 5%; n = 9; Fig.
5D). This result was unexpected, because expression of Homer
1b alone reduced glutamate inhibition of calcium current via mGluR1a
(see Fig. 3D). One interpretation of this result is that
Homer 1b can compete with Homer 2b for the mGluR binding site,
but that receptors bound to Homer 1b are occluded less efficiently than
those bound to Homer 2b.
Homer proteins reduce mGluR1a modulation of M-current
M-type potassium current is a noninactivating, delayed
rectifier-type potassium current present in many neuronal cell types (Brown and Adams, 1980; Wang et al., 1998). Modulation of the M-current
is believed to play an important role in the regulation of cell
excitability (Marrion, 1997). The M-current modulatory pathway by
glutamate in SCG neurons (Kammermeier and Ikeda, 1999), as well as by
different neurotransmitters (Haley et al., 1998), appears to be
initiated by activation of G q/11. To examine
the effect of Homer expression on this pathway, M-current modulation was observed in the absence and presence of several forms of Homer. Cells were held at 30 mV and stepped negatively to 60 mV for 500 msec. M-current was measured as the difference in current at the
beginning and end of the step to 60 mV. Application of 100 µM glutamate to SCG neurons expressing mGluR1a produces a 66 ± 4% (n = 17) inhibition of M-type potassium
current (Fig. 6A). Note
that with application of glutamate, M-current was quickly inhibited and
partially reversed during washout of the drug (see time course; Fig.
6A, bottom). As with calcium current
inhibition, coexpression of Homer 1a or H2N had no effect on M-current
modulation by glutamate in mGluR1a-expressing cells [inhibition was
71 ± 4% (n = 6) and 63 ± 9%
(n = 6) for Homer 1a and H2N, respectively; Fig.
6B,D]. In contrast, coexpression of Homer 2b or
Homer 3 was able to reduce M-current modulation (Fig. 6C,D).
However, these long forms of Homer were less effective at blocking the
modulation of M-current than of calcium current. When Homer 2b cDNA was
injected at 0.1 µg/µl, M-current inhibition was reduced to only
51 ± 6% (n = 6; not a significant reduction,
p = 0.067), whereas similar injections of Homer 2b
strongly reduced calcium current modulation (mGluR1a cDNA concentration
was 0.03 µg/µl in all experiments; see Fig. 3C,D).
Increasing injected Homer 2b cDNA levels threefold (to 0.3 µg/µl)
significantly decreased M-current modulation via mGluR1a to 23 ± 5% (n = 8; Fig. 6C,D). Coinjection of 0.1 and 0.3 µg/µl Homer 3 cDNA reduced M-current inhibition by
glutamate to 41 ± 10% (n = 3) and 36 ± 9%
(n = 5), respectively. These data indicate that the
long forms of Homer can reduce signaling of mGluR1a to the M-channel.
However, this effect requires higher levels of Homer plasmid injection
than the analogous effect on calcium current inhibition.
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Figure 6.
Long forms of Homer can occlude M-current
modulation. A-C, Sample current traces and time courses
from M-current inhibition by glutamate in cells expressing mGluR1a
(A), mGluR1a plus Homer 1a
(B), and mGluR1a plus Homer 2b
(C). Cells were held at 30 mV and stepped to
60 mV for 500 msec every 10 sec. M-current in each time course was
taken as the difference in current at the start and end of
the step to 60 mV as M-type channels deactivated. Calibration:
A, 0.2 nA; B, C, 0.1 nA;
A-C, 20 msec. D, Summary of M-current
modulation data. *Statistical significance
(p = 0.05) compared with control (mGluR1a
alone). glu, Glutamate; con,
control.
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Effects of Homer proteins on the Gi/o and
Gq/11 pathways
Because the modulation of M-current via group I mGluRs proceeds
through a Gq/11-mediated pathway (Kammermeier and
Ikeda, 1999), and moderate levels of Homer seem to discriminate between
M-type potassium and N-type calcium current modulation, one can
speculate that the long forms of Homer preferentially occlude
activation of Gi/o over
Gq/11 by mGluRs. As discussed above (see
introductory remarks), group I mGluRs activate both the
Gi/o and Gq/11 classes of
G-proteins. Activation of Gi/o G-proteins by
group I mGluRs initiates a voltage-dependent inhibition mediated by
G-protein subunits (Herlitze et al., 1996; Ikeda, 1996). This
form of channel modulation is identifiable by the slowing of activation kinetics and partial reversal of inhibition after strong
depolarizations (Bean, 1989; Elmslie et al., 1990). In addition, group
I mGluRs in SCG neurons inhibit calcium currents through activation of Gq/11 (Kammermeier and Ikeda, 1999). This form of
modulation is readily distinguished from the
Gi/o, G-mediated inhibition, because it
does not exhibit voltage dependence or slowing of activation kinetics.
Therefore, relative changes in activation of the
Gi/o and Gq/11 pathways can
be monitored by examining the voltage dependence of group I
mGluR-mediated calcium current inhibition. To test the possibility that
Homer expression selectively influences G-protein activation by
mGluR1a, the percent inhibition of postpulse currents (in which
voltage-dependent inhibition would be partially relieved) was plotted
against that of currents in the prepulse, and the voltage dependence of
inhibition was determined from the slope of the resulting curve. For
this type of analysis, voltage-dependent inhibition would produce a
flatter slope, whereas voltage-independent inhibition would have a
slope of ~1 (Kammermeier and Ikeda, 1999). Inhibition data from cells
expressing mGluR1a alone and those expressing mGluR1a plus Homer 2b
appear to have similar slopes, indicating that the voltage dependence
is not altered (Fig. 7A). Therefore, signaling of both G-protein pathways activated by group I
mGluRs is similarly inhibited by Homer 2b.
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|
Figure 7.
Homer does not alter the voltage dependence of
calcium current inhibition by glutamate. A, Postpulse
versus prepulse inhibition plot for glutamate inhibition in cells
expressing mGluR1a (filled circles) and mGluR1a
plus Homer 2b (open circles). In this plot, slope is
inversely proportional to voltage dependence. Slopes for mGluR1a and
mGluR1a plus Homer 2b were 0.52 ± 0.09 and 0.60 ± 0.13 (slope ± 95% confidence interval). These values were not
significantly different. B, Similar plot as in
A, but for mGluR1a cells and cells expressing mGluR1a,
Homer 2b, and Homer 1a. The slopes were 0.52 ± 0.09 and 0.53 ± 0.09, respectively. Solid lines represent linear
regressions to each data set.
|
|
A related question concerning mGluR signaling is whether "naked"
mGluRs behave similarly to those bound to Homer 1a (or H2N). The data
indicate that the magnitude of maximal inhibition is not altered by the
short forms of Homer. But is there a change in G-protein coupling? This
question can be addressed by examining the voltage dependence of
glutamate inhibition in cells injected with mGluR1a alone versus cells
injected with mGluR1a, Homer 2b, and Homer 1a (or H2N). Cells injected
with mGluR1a alone would be expected to represent naked
receptors (unbound to Homer), because the receptors are overexpressed.
Those injected with mGluR1a and Homer 2b plus Homer 1a (or H2N)
represent a nearly pure population of mGluR1a-Homer 1a (or H2N)
receptors, because under these conditions Homer 1a is required to
"protect" receptors from the effects of Homer 2b. When the voltage
dependence of glutamate inhibition in these two groups was compared, it
was found to be identical (Fig. 7B). This indicates that
receptors bound to the short form of Homer appear to act similarly to
naked receptors.
| |
DISCUSSION |
This study provides evidence that the long forms of Homer act
functionally to occlude signaling from group I mGluRs to G-proteins that regulate N-type calcium and M-type potassium channels. The natural
immediate early gene form of Homer (short form), as well as a
structural homolog, act competitively to reverse the effects of long
forms of Homer. Accordingly, the immediate early gene functions as a
natural antagonist of long forms of Homer. Because Homer 1a is
dynamically expressed in many models of developmental and
activity-dependent plasticity, its physiological antagonism of
constitutively expressed long Homers provides a mechanism to regulate
group I mGluR signaling to ion channels.
Although the mechanism of Homer action on group I mGluRs remains
undefined, images of mGluR surface expression can begin to shed light
on this process. The principle effect of coexpressing long forms of
Homer with group I mGluRs in SCG neurons appears to be the formation of
cell surface receptor clusters. Levels of membrane surface-expressed
mGluR5 appeared comparable when the receptor was expressed alone or
coexpressed with Homer 2b. This is consistent with observations that
Homer long forms induce cell surface mGluR clusters in cultured CNS
neurons (Ciruela et al., 1999; Tadokoro et al., 1999). This clustering
activity contrasts with observations from HeLa and HEK-293 cells in
which long forms of Homer act to retain mGluR in endoplasmic reticular
pools and reduce cell surface expression of mGluRs (Roche et al.,
1999). We anticipate that these different effects of Homer may be
attributable to cell-specific expression of accessory proteins that
interact with Homer or mGluRs. In contrast to long forms, Homer short
forms do not induce cell surface clustering in SCG neurons, indicating that cluster formation is dependent on the C-terminal domain. This
suggests a model in which mGluR clusters form on the basis of the
physical association of the coiled coil multimerization domain of long
Homer proteins.
How might the binding of Homer influence coupling between mGluR and
G-proteins? The C-terminal cytosolic tail of group I mGluRs has a
demonstrated role in the signaling properties to G-proteins (Prezeau et
al., 1996; Mary et al., 1997). One possibility is that the physical
presence of the Homer EVH1 domain interferes with the physical
association between mGluR and G-proteins. This does not appear to be
the case, because coupling of group I mGluR is not affected by
expression of short forms of Homer, which do directly bind mGluR.
Moreover, the blocking activity of long Homers is reversed by
coexpression of short-form Homer. The effects of Homer expression
are specific for group I mGluRs, because they do not influence coupling
of mGluR2 to the same G-protein-regulated ion channels. This is
consistent with the demonstrated binding of the Homer EVH1 domain to
group I mGluRs and the absence of binding to other mGluR subtypes.
Moreover, Homer is not known to interact with G-proteins or the
effector ion channels in this study. Additionally, long forms of Homer
did not selectively occlude one G-protein signaling pathway over
another (see Fig. 7). Specifically, both Gi/o and
Gq/11 effects on voltage-dependent and
-independent inhibition of N-type channel function were similarly
blocked by long forms of Homer. Long forms of Homer have been
demonstrated to limit the mobility of group I mGluRs within the
membrane surface and may thereby limit functional access to
membrane-bound, voltage-gated ion channels. This model predicts that
"free" group I mGluRs (i.e., those not clustered with long forms of
Homer) can couple more efficiently to voltage-gated ion channels on the
cell membrane (Fig. 8).
View larger version (20K):
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|
Figure 8.
Schematic representation of the proposed mechanism
of Homer function. A, When Homer 1a associates with
group I mGluRs (or without any Homer proteins bound), receptors are
diffusely distributed in the membrane. In this state, voltage-gated ion
channels on the cell membrane are efficiently modulated, but the
calcium signal via mGluRs and the IP3 receptor is weak.
B, In the presence of long forms of Homer, group I
mGluRs cluster together, presumably because of the interaction of the
coiled coil, C-terminal tails of Homer. This interaction may also bring
the complex in association with the IP3 receptor (schematic
not shown). Under this condition, the intracellular calcium signal is
strong, but modulation of voltage-gated ion channels is weak.
|
|
Homer 1b and 1c were unexpectedly less effective than Homer 2b and 3 in
occluding actions of mGluRs (Fig. 3). For example, expression of Homer
1b was able to partially reverse the effect of Homer 2b. This result
seems to indicate that the partial effect of Homer 1b is not simply the
result of low expression, because if both were equally active,
coexpression of Homer 1b and Homer 2b would be expected to produce
results at least as great as that of Homer 2b alone. Thus, Homer 1 long
forms appear less occlusive toward group I mGluR signaling to
voltage-gated ion channels than Homer 2 or 3. This difference of
activity may provide cells with the ability to "fine tune" mGluR
activity. On the basis of the high degree of identity among the EVH1
domain products of the three Homer genes (Xiao et al., 1998),
particularly in regions that are consequential for ligand binding
(Beneken et al., 2000), and the substantial divergence between their
coiled coil domains, it is likely that the difference in efficacy will
be attributed to properties of their C-terminal domains. One
possibility that is consistent with the mechanisms noted above is that
the ability to form stable Homer multimers may be less for Homer 1 long
forms than for Homer 2 and 3. Further studies are required to define these mechanisms of Homer action and the extent to which it modifies mGluR I signaling in the in vivo context.
An interesting finding of this study is the relative potency with which
Homer 2b and 3 were able to occlude calcium current and M-current
modulation. Defined injections of the Homer 2b expression construct
were able to block calcium current modulation almost completely but
were inactive against M-current (Figs. 3 and 6). At higher levels of
cDNA injection, effects of mGluR on both currents were occluded. This
differential selectivity may result from a greater amplification in the
pathway leading to M-current modulation, compared with that of calcium
current. In a previous study (Ikeda et al., 1995) in which group I
mGluRs were expressed in SCG neurons via cytoplasmic mRNA injection,
activation of mGluRs appeared to inhibit M-current, whereas calcium
currents were unaltered. In retrospect, this result could be explained
by a lower level of mGluR expression (compared with that after nuclear
cDNA injection, as in the present study) competing with natively
expressed Homer 2 and 3. The resulting stoichiometry may have achieved
a balance that allowed modulation of M-currents but not calcium
currents. The differential sensitivity of the signaling pathways may
have a broader importance, because it implies that Homer can
selectively regulate the modulation of G-protein signals to different
effector pathways.
Previously, we demonstrated that expression of Homer 1a reduced the
amplitude and prolonged the time to peak of the intracellular calcium
transient induced by activation of mGluR in cultured Purkinje neurons.
By contrast, expression of Homer 1b did not alter this response. This
differential effect of the short and long forms of Homer was attributed
to the ability of Homer short forms to disrupt the physical linkage
produced by long forms between mGluR and the IP3
receptor. Indeed, this linkage can be demonstrated to be disrupted
using biochemical techniques in mice expressing the Homer short-form
protein. These data provide an interesting contrast to the present
study, in which Homer short form coexpression had no effect on mGluR
signaling, whereas long forms had dramatic consequences. Among the many
differences between the experimental preparations, we note that
Purkinje neurons express high levels of Homer 1 and 3 long forms, and
responses were evoked by native group I mGluRs. Nevertheless, the
results share the general principle of a physiological antagonism
between short and long forms of Homer. This comparison also
demonstrates that Homer proteins can have reciprocal effects on the
group I signal outputs, leading to release of intracellular calcium
versus inhibition of membrane ion channels. Long forms of Homer induce
receptor clustering that may allow more efficient association with
calcium release machinery but functionally isolate the receptors from
membrane-associated channels. By contrast, Homer 1a would uncouple
mGluRs from IP3R and enhance coupling to ion
channels on the membrane (see Fig. 8). Differences in the sensitivity
of effectors downstream to G-proteins, as demonstrated here between the
N-type calcium channel and M-current potassium channel, are expected to
further contribute to the consequence of Homer 1a action.
In summary, data from this study demonstrate that Homer 2b but not
Homer 1a induces cell surface clustering of a group I mGluR. In
addition, the long forms of Homer (Homer 1b, 1c, 2b, and 3) can
selectively inhibit signaling from group I mGluRs to N-type calcium and
M-type potassium channels. This occlusion can be partially reversed by
coexpression of Homer 1a or the deletion construct H2N. Higher levels
of Homer 2b and Homer 3 appear to be necessary to occlude M-current
inhibition than calcium current inhibition, perhaps because of the
amplification of the M-current modulatory pathway. Expression of the
long forms of Homer did not selectively occlude specific G-protein
pathways activated by group I mGluRs. Perhaps most interesting is the
demonstration that Homer proteins evoke reciprocal effects on group I
mGluR signals leading to regulation of membrane ion channels versus
release of intracellular calcium, suggesting a mechanism by which
synaptic activity and the consequent induction of Homer 1a can regulate
both the magnitude and predominate output of group I metabotropic
glutamate receptors.
| |
FOOTNOTES |
Received April 24, 2000; revised June 30, 2000; accepted July 6, 2000.
This work was supported by National Institutes of Health Grants GM56180
and NS37615 (S.R.I.), NS10943 (P.J.K.), and DA11742, DA10309, and
MH01152 (P.F.W.). We thank Marina King for valuable technical
assistance and J.-P. Pin, S. Nakanishi, and B. A. McCool for
supplying clones.
Correspondence should be addressed to Stephen R. Ikeda, Guthrie
Research Institute, Guthrie Foundation for Education and Research, One
Guthrie Square, Sayre, PA 18840. E-mail: sikeda{at}inet.guthrie.org.
| |
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S. Kaja, S.-H. Yang, J. Wei, K. Fujitani, R. Liu, A.-M. Brun-Zinkernagel, J. W. Simpkins, K. Inokuchi, and P. Koulen
Estrogen Protects the Inner Retina from Apoptosis and Ischemia-Induced Loss of Vesl-1L/Homer 1c Immunoreactive Synaptic Connections
Invest. Ophthalmol. Vis. Sci.,
July 1, 2003;
44(7):
3155 - 3162.
[Abstract]
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[PDF]
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B. Dziedzic, V. Prevot, A. Lomniczi, H. Jung, A. Cornea, and S. R. Ojeda
Neuron-to-Glia Signaling Mediated by Excitatory Amino Acid Receptors Regulates ErbB Receptor Function in Astroglial Cells of the Neuroendocrine Brain
J. Neurosci.,
February 1, 2003;
23(3):
915 - 926.
[Abstract]
[Full Text]
[PDF]
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P. J. Kammermeier, M. I. Davis, and S. R. Ikeda
Specificity of Metabotropic Glutamate Receptor 2 Coupling to G Proteins
Mol. Pharmacol.,
January 1, 2003;
63(1):
183 - 191.
[Abstract]
[Full Text]
[PDF]
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R. A. Hall and R. J. Lefkowitz
Regulation of G Protein-Coupled Receptor Signaling by Scaffold Proteins
Circ. Res.,
October 18, 2002;
91(8):
672 - 680.
[Abstract]
[Full Text]
[PDF]
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L. Fagni, P. F. Worley, and F. Ango
Homer as Both a Scaffold and Transduction Molecule
Sci. Signal.,
June 18, 2002;
2002(137):
re8 - re8.
[Abstract]
[Full Text]
[PDF]
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D. Bottai, J. F. Guzowski, M. K. Schwarz, S. H. Kang, B. Xiao, A. Lanahan, P. F. Worley, and P. H. Seeburg
Synaptic Activity-Induced Conversion of Intronic to Exonic Sequence in Homer 1 Immediate Early Gene Expression
J. Neurosci.,
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[Abstract]
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U. Padmanabhan, S. Dasgupta, B. B. Biswas, and D. Dasgupta
High Affinity Association of myo-Inositol Trisphosphates with Phytase and Its Effect upon the Catalytic Potential of the Enzyme
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November 16, 2001;
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[Abstract]
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[PDF]
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C. J. Swanson, D. A. Baker, D. Carson, P. F. Worley, and P. W. Kalivas
Repeated Cocaine Administration Attenuates Group I Metabotropic Glutamate Receptor-Mediated Glutamate Release and Behavioral Activation: A Potential Role for Homer
J. Neurosci.,
November 15, 2001;
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9043 - 9052.
[Abstract]
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L. Gama, S. G. Wilt, and G. E. Breitwieser
Heterodimerization of Calcium Sensing Receptors with Metabotropic Glutamate Receptors in Neurons
J. Biol. Chem.,
October 12, 2001;
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[Abstract]
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M. Sato, K. Suzuki, and S. Nakanishi
NMDA Receptor Stimulation and Brain-Derived Neurotrophic Factor Upregulate Homer 1a mRNA via the Mitogen-Activated Protein Kinase Cascade in Cultured Cerebellar Granule Cells
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION
