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The Journal of Neuroscience, June 15, 2002, 22(12):4878-4884
Light Response of Retinal ON Bipolar Cells Requires a Specific
Splice Variant of G o
Anuradha
Dhingra1, *,
Meisheng
Jiang3, *,
Tian-Li
Wang1, *,
Arkady
Lyubarsky2,
Andrey
Savchenko2,
Tehilla
Bar-Yehuda1,
Peter
Sterling1,
Lutz
Birnbaumer3, and
Noga
Vardi1
Departments of 1 Neuroscience and
2 Ophthalmology, University of Pennsylvania, Philadelphia,
Pennsylvania 19104, and 3 Department of Anesthesiology,
University of California, Los Angeles, Los Angeles, California
90024
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ABSTRACT |
Glutamate released onto retinal ON bipolar neurons binds to a
metabotropic receptor to activate a heterotrimeric G-protein (Go) that ultimately closes a nonspecific cation
channel. Signaling requires the  subunit (Go),
but its effector is unknown. Because Go is transcribed
into two splice variants (o1 and
o2) that differ in the key GTPase domain, the
next step in elucidating this pathway was to determine which splice
variant carries the signal. Here we show by reverse transcription-PCR
and Western blots that retina expresses both splice variants.
Furthermore, in situ hybridization and immunostaining on
mouse retina deficient in one splice variant or the other show that
both o1 and o2 are expressed by ON
bipolar cells but that o1 is much more abundant. Finally, electroretinography performed on mice deficient for one splice
variant or the other shows that the positive b-wave (response of ON
bipolar cells to rod and cone input) requires o1 but not o2. Thus, the light response of the ON bipolar
cell is probably carried by its strongly expressed splice
variant, Go1.
Key words:
G-protein; Go splice variants; splice variant
knock-out mouse; mGluR6; metabotropic glutamate receptor; retina; ERG
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INTRODUCTION |
At the first visual synapse,
glutamate modulates three-quarters of the postsynaptic neurons (ON
bipolar cells) via a metabotropic glutamate receptor (mGluR6) (Nomura
et al., 1994 ; Vardi and Morigiwa, 1997; Vardi et al., 1998, 2000) that
couples to a heterotrimeric G-protein (Go) (Weng
et al., 1997; Nawy, 1999; Dhingra et al., 2000). Signaling requires the
subunit (Go), but, the next step in this
pathway, which ultimately closes a nonspecific cation channel, is
unknown (Nawy and Jahr, 1990; Shiells and Falk, 1990; de la Villa et
al., 1995; Euler et al., 1996). To identify an effector for
Go seems important even beyond the visual
system because, although effectors are known for the  / subunits
of this most abundant brain G-protein (Dolphin, 1998), no effectors have yet been identified for the subunit.
Go is transcribed into two splice variants,
o1 and o2, which
contain identical helical domains near the N terminus (NT) but
different GTPase domains near the C terminus (CT) (Goldsmith et al.,
1988; Hsu et al., 1990; Strathmann et al., 1990; Tsukamoto et al.,
1991; Horn and Latchman, 1993). Because the GTPase domain binds both
receptor and effector, it is crucial for coupling (for review, see
Neer, 1994; Clapham, 1996; Gudermann et al., 1997). Therefore, the next
step in elucidating this pathway was to determine which splice variant
carries the signal. Previous studies that colocalized
Go with mGluR6 in ON bipolar dendrites could
not distinguish between different splice variants because the antibody (Ab) used recognizes both (Vardi, 1998), nor did the
o null mouse, which lacks the ON response,
resolve this question because it is missing both splice variants (Jiang
et al., 1998; Dhingra et al., 2000). Here, using mice deficient in one
splice variant or the other, we show that both are expressed by ON
bipolar dendrites but that only Go1 matters
for the light response.
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MATERIALS AND METHODS |
Animals were deeply anesthetized by intraperitoneal injection
(for rat, 45 mg/kg pentobarbital; for mouse, a mixture of 85 µg/gm
ketamine and 13 µg/gm xylazine), and the eyes were enucleated; animals were then killed by anesthetic overdose (three times the initial doses). Animals were treated in compliance with federal regulations and University of Pennsylvania policy. The eye was incised
at the ora serata and fixed by immersion in buffered 3 or 4%
paraformaldehyde with (for immunocytochemistry) or without (for
in situ hybridization) 0.01% glutaraldehyde for 1 hr. It was then rinsed in buffer, soaked overnight in 30% buffered sucrose, and embedded in a mixture of two parts 20% sucrose in phosphate buffer
and one part of tissue freezing medium. Radial cryosections were
10-15 µm thick.
Reverse transcription-PCR. Retina was homogenized in
solution containing 4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol, and total RNA was isolated
by the acid-guanidium phenol-chloroform method (Chomczynski and
Sacchi, 1987). The reverse transcription (RT) reaction was performed at
42°C for 50 min with 1-5 µg of total RNA in 20 µl of buffer
containing 50 mM Tris-HCl, pH 7.4, 60 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 U/ml
RNase inhibitor, 0.5 mM of each dNTP, 500 pmol of
random hexamer (or 100 pmol of oligo-dT), and 200 U of
SuperScript II Moloney murine leukemia virus reverse
transcriptase (Invitrogen, Gaithersburg, MD). PCR reaction was
performed in a buffer containing 10 mM Tris, pH
8.3, 50 mM KCl, 2.5 mM
MgCl2, 0.4 mM dNTP, 0.2 µM 5' and 3' primers, 2 µl of
reverse-transcribed cDNA, and 2.5 U of AmpliTaq (PerkinElmer Life
Sciences, Branchburg, NJ). Thirty cycles (94°C for 1 min, 52°C for
1 min, and 72°C for 2 min) were performed on a programmable
thermocycler (PerkinElmer Life Sciences). The sequences of PCR primers
(synthesized by Invitrogen) were the same as those used to generate the
long probe for in situ hybridization (see below).
In situ hybridization. o1 and
o2 sequences were amplified by RT-PCR from
whole retina. The sequences of PCR primers were as follows:
o1 upstream, 5'-catcctccgaaccagggtc-3';
o1 downstream, 5'-caagccacagccccggag-3';
o2 upstream, 5'-catcctccgaaccagggtc-3'; and
o2 downstream, 5'-ggcgatgatgacgtccgt-3'. We
also designed a set of probes at the most diverse region of
o (which gave a shorter reaction product):
o1 upstream, 5'-gctcttcgactccatctgt-3'; o1 downstream, same as for the first set;
o2 upstream, 5'-gacagcatctgcaacaac-3'; and
o2 downstream, same as for the first set. PCR
products were subcloned into PCRII vector (Invitrogen) or pBluescript
(Stratagene, La Jolla, CA), and the authenticity of the products
was verified by direct sequencing.
33P-labeled riboprobe was made by in
vitro transcription. Briefly, the reaction was performed by
incubating the linearized plasmid DNA in 20 µl of solution containing
40 U of RNA polymerase, 10 mM DTT, 20 U of RNase
inhibitor, 0.5 mM ATP, GTP, and CTP, 250 µM UTP, and 25 µCi of
[-33P]UTP. After incubating at 37°C
for 2 hr, the reaction was treated with RNase-free DNase I and
precipitated by LiCl. RNA was further purified by phenol-chloroform
extraction, precipitated by ethanol, and finally dissolved in DEPC water.
To test whether the antisense probes cross-hybridized, we applied
the sense transcripts of o1 and
o2 to a nitrocellulose membrane and then tried
to hybridize them with the antisense probes. Each antisense hybridized
to its own sense but not to the other; therefore, the in
situ hybridization probably reflects genuine distribution of
o1 and o2 mRNAs.
Retina sections were hybridized overnight (~18 hr) with
33P-labeled probes in in situ
hybridization buffer containing 10% dextran sulfate, 50% formamide,
4× SSC, 0.1% SDS, and 2× Denhardt's solution at
106 cpm/50 µl. After overnight
hybridization at 58 or 65°C, slides were washed twice at 10°C above
the hybridization temperature in 2× SSC-50% formamide, once at room
temperature in 2× SSC, followed by one wash at 10°C above the
hybridization temperature in 1× SSC-0.1% SDS. Slides were rinsed in
0.1× SSC, incubated with 300 mM ammonium
acetate, dehydrated in ethanol, and finally dipped in photographic
emulsion (Kodak NTB-2; Eastman Kodak, Rochester, NY) and exposed for
1-5 weeks.
Immunocytochemistry. Staining was performed according to a
standard protocol: soak in diluent containing 10% normal goat serum, 5% sucrose, and 0.3% Triton X-100 in phosphate buffer; incubate in
primary antibodies overnight at 4°C; wash and incubate (3 hr) in
anti-rabbit F(ab)2 fragment conjugated to a
fluorescent marker; and rinse and mount in Vectashield (Vector
Laboratories, Burlingame, CA). To stain a 4%
paraformaldehyde-fixed retina for protein kinase C (PKC), we needed to
retrieve the antigenicity with 0.5% sodium borohydrate (8 min) and use
both Triton X-100 (0.75%) and Tween 20 (0.2%) as detergents. For
double staining, incubation was done as for single labeling,
with both primary antibodies and both secondary antibodies
simultaneously. Some sections were incubated in horseradish
peroxidase-conjugated secondary antibody and visualized with
3,3'-diaminobenzidine (DAB) reaction product. For electron microscopy,
DAB reaction product was intensified by the gold-substituted silver-intensified peroxidase method. The tissue was then osmicated (1.5% osmium tetroxide, 60 min), stained with 1% uranyl acetate in
70% methanol (60 min), dehydrated in 80, 90, and 100% methanol, cleared in propylene oxide, and embedded in Epon 812. Ultrathin sections were mounted on Formvar-coated slot grids and stained with
uranyl acetate.
Antibodies. The antibody against o1
(Ab 1718) was raised in rabbit against the peptide EYPGSNTYED, and the
antibody against o2 (Ab 1715) was prepared
against peptide EYTGPSAFTE (both are gifts from Dr. D. Manning,
University of Pennsylvania, Philadelphia, PA). In addition,
three antibodies against o that recognize both splice variants on Western blots were used. (1) A polyclonal antibody (Ab 9072) was raised in rabbit against the peptide
ANNLRGCGLY located at the C terminus (gift from Dr. D. Manning). This sequence is identical to that of
o1 peptide but is different in one amino acid
from the corresponding peptide of o2 (in which
the bolded asparagine is replaced by lysine). (2) A polyclonal antibody
was raised in rabbit against a peptide DGISAAKDV located at the N terminus (gift from Kwen-Jen Chang, The Burroughs Wellcome Co., Research Triangle Park, NC) (Chang et al., 1988; Codina et al., 1991). This peptide is identical in both splice variants. (3) A
monoclonal antibody (mAb 3073) raised in mouse against the purified bovine protein (Chemicon; Li et al., 1995). The antibody to PKC was
monoclonal raised in mouse (mAb 5; Amersham Biosciences, Little Chalfont, UK).
Selective disruption of Go1 and
Go2 expression. Standard molecular
biology techniques, to be reported in detail elsewhere, were used to
construct targeting vectors of two types (Rudolph et al., 1993, 1994;
Jiang et al., 2002). For disruption of Go1, a
replacement-type targeting vector was made. It contained a genomic segment of the Go gene with exons 6, 7.2, 8.2, 7.1, 8.1, and 9.1, of which exon 7.1 was disrupted by insertion of a
neomycin selection cassette, followed by an internal ribosome entry
site, and the open reading frame of -galactosidase.
Go1-targeted embryonic stem (ES) cells were
obtained by selection for G418 resistant clones. For disruption of
Go2, an insertion-type vector was made with
the same genomic segment, but, instead of disrupting exon 7.1, the
codon for Cys-255 of exon 7.2 was replaced by a stop codon. In
addition, a double neomycin-thymidine kinase selection cassette was
placed at the end of the genomic segment to allow for positive and
negative selection strategies.
Go2-targeted ES cells were generated by the
"hit-and-run" procedure in which insertion of the mutated homology
and selection cassettes is selected for in the presence of G418, and
subsequent excision of the wild-type duplicate with attending loss of
both selection cassettes was obtained by negative selection in the presence of FIAU
(2-fluoro,2-deoxy-5-iodouracyl--D-arabinofuranoside). After germline transmission, the resulting F2 mice (50:50
129Sv/C57BJ/6) either lacked Go2 but preserved
expression of Go1 or lacked Go1 but preserved expression of
Go2 and, in addition, expressing -galactosidase (M. Jiang and L. Birnbaumer, unpublished
observations) (vide infra for differential
Go-protein expression data).
Electroretinographic recordings. The experimental apparatus,
methods of light stimulation and quantification, electroretinographic (ERG) recording, and cone signal isolation have been described in
detail previously (Lyubarsky et al., 1999, 2002). Briefly, a mouse was
dark adapted for 2 hr, and then, under dim red light, it was deeply
anesthetized by intraperitoneally injecting ketamine (20 µg/gm)
plus xylazine (8 µg/gm). The animal was immobilized in a
holder, the pupils were dilated with 1% tropicamide, and the eyes were
protected with a drop of methylcellulose. A platinum recording
electrode contacted both corneas, and a tungsten reference electrode
was inserted subcutaneously on the forehead. The animal in its holder
was then placed inside a light-proof Faraday cage, and light stimuli
were delivered through several ports. Stimulus intensity and spectral
composition were controlled with neutral density and bandpass
interference filters. Light intensities were calibrated and converted
to estimated numbers of photoisomerization per photoreceptor
(R*) as described previously (Lyubarsky et al., 1999,
2000).
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RESULTS |
Both splice variants of Go (o1 and
o2) are expressed in retina
To determine which transcripts are expressed, we first performed
RT-PCR on rat whole retina. Both transcripts were amplified: the bands
were at the expected sizes (530 for o1 and 510 for o2), and the PCR products were cut at the
expected positions by the appropriate restriction enzyme
(ClaI applied to o1 and HindIII applied to o2) (Fig.
1A,B).
In several experiments in which the PCR products were sequenced, there
was good agreement with published sequences for hamster
o1 or o2
sequences.
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Figure 1.
Retina expresses both splice variants of
o. A, Primers for amplifying
o1 and o2 mRNA; restriction enzymes
cutting sites are also indicated. B, mRNA from rat
retina was reverse transcribed and amplified using the specific primers
shown in A. In lanes 2 and
4, the PCR products were cut with restriction enzymes.
C, Dot blots show that o1 antibody does
not recognize the o2 peptide
(o2 pep) and that
o2 antibody does not recognize the o1
peptide (o1 pep). Neither antibody
reacted with bovine serum albumin (BSA). D,
Western blots of whole rat retina show, for
o1, a single prominent band at ~43 kDa and,
for o2, a prominent band at ~40 kDa plus a
weak band at ~50 kDa.
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To determine whether both proteins are expressed, we used antibodies
specific for each splice variant. The specificity of the antibody was
tested by dot blots: the antibody for o1
reacted with the o1 peptide but not with the
o2 peptide, and the antibody for
o2 reacted with the
o2 peptide but not with the
o1 peptide (Fig. 1C). By SDS-PAGE
of retinal homogenates followed by Western blotting, the
o1 antibody detected a prominent band at ~43
kDa, and the antibody for o2 detected a
prominent band at ~40 kDa; both agree with their known molecular
weights of 40 and 39 kDa (Fig. 1D). The antibody for
o2 detected an additional band at ~50 kDa.
We conclude that the retina expresses RNA transcripts and proteins of
both splice variants of o.
o1 is expressed by rod bipolar cells
First, we tested the distribution of o1
mRNA by in situ hybridization. Antisense riboprobe for
o1 transcript applied to sections of fixed rat
retina showed strong labeling in the inner nuclear layer (INL) and
ganglion cell layer. Labeling was strongest in the upper tiers of the
INL, the location of rod bipolar cell somas. Labeling in the
outer nuclear layer (ONL) and in photoreceptor inner segments was weak
and was not significantly different from background obtained with sense
probes for o1 (Fig.
2A,B).
A shorter, more specific probe gave less signal but the same labeling
pattern. Also, the same probes applied to mouse retina gave weaker but similar labeling.
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Figure 2.
Bipolar cells strongly stain for the
o1 splice variant. A, B,
In situ hybridization (rat). Antisense probe gave
labeling in the INL and in the ganglion cell layer
(GCL). Strongest labeling is in the upper tier of the
INL, the location of rod bipolar somas. Sense probe gave low
background. Left panels, Differential interference
contrast shows the retinal layers of the section in A.
PR, Photoreceptors. C-E, Immunostaining
with the o1-specific antibody visualized with
3,3'-diaminobenzidine (rat). C, Staining is strong in
bipolar dendrites (Den) in OPL and weaker in bipolar
somas (BP) in the INL and in the IPL. D,
Staining with the preimmune serum was negative. E,
Staining with the antibody preabsorbed with the o1
peptide was also negative. F-H, Immunostaining with the
o1-specific antibody visualized with FITC (mouse).
F, In the OPL and INL, staining in wild-type mouse
resembles that in rat. In the IPL, note the thin bands of higher
intensity (black arrows), a thicker band of lower
intensity (brackets), and a band devoid of staining
(white arrow). G, H, No
staining above general background was observed when the same antibody
was applied, respectively, to an o1 or
o1+2 null mouse. ko, Knock-out;
wt, wild type.
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Next, we tested the distribution of o1
protein. An antibody specific for this splice variant, applied to both
rat and mouse retinas, showed staining similar to that obtained
previously for o (Vardi et al., 1993; Vardi,
1998). Stained somas were numerous in the upper tiers of the
INL, with dendrites reaching high in the outer plexiform layer
(OPL) (Fig. 2C,F). Electron microscopy showed the stain in dendrites that invaginate the rod terminal (data
not shown). Thus, rod bipolar somas and dendrites stain for
o1. The bipolar axons crossing the INL were
stained for o1, but, as they cross the inner
plexiform layer (IPL), they were unstained. The IPL was also stained
but more weakly than OPL. By confocal microscopy, the IPL showed two
intense strata at ~25 and 55% of IPL depth, a lack of stain between
~25 and 35%, and weak stain elsewhere (0% is the interface between
INL and IPL). Photoreceptors were unstained. Staining pattern for
o1 was specific because (1) retinal sections
from rat incubated with preimmune serum or with antibody preabsorbed
with the o1 peptide were negative (Fig.
2D,E), and (2) retinal sections
from an o1 null mouse and an
o null mouse were negative (Fig.
2G,H).
o1 is expressed by all types of ON cone
bipolar cell
Because rod bipolar cells and ON cone bipolar cells differ in
response kinetics, components of their transduction cascades might also
differ (Berntson and Taylor, 2000; Wu et al., 2000). Therefore, we
asked whether rod and cone bipolar cells express the same splice
variant of Go. Double immunostaining showed that all ON bipolar cells express o1. First, rod
bipolar cells identified with antibody for PKC stained for
o1, but additional somas positive for
o1 were negative for PKC; these are cone
bipolar cells (Fig. 3, left).
Similarly, rod bipolar dendritic terminals projecting high in the OPL
stained for both PKC and o1, but processes at midlevel in the OPL, in which cone bipolar dendrites terminate, stained
only for o1 (Fig. 3, left). Second,
we labeled all ON bipolar cells using a monoclonal antibody for
Go. This antibody is suitable because it stains
exactly the same set of cells as the antibody against the C terminus,
which was shown to label all ON bipolar cells (Vardi, 1998). In this
double staining, all somas stained by the monoclonal antibody also
stained for o1 (Fig. 3, right).
Interestingly, stain for o and
o1 completely colocalized, even in the inner
plexiform layer, suggesting that in retina all the
o-positive cells express at least the
o1 splice variant.
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Figure 3.
All ON cone bipolar cells stain for
o1 (rat). Left, All rod bipolar somas
(stained for PKC) also stained for o1 (double
arrowhead), but some somas stained only for o1
(long arrow). In the OPL (bracketed), rod
bipolar dendrites stained for both PKC and o1
(arrowhead), but some dendrites stained only for
o1 (short arrow). These probably belong
to ON cone bipolar cells. Right, All ON bipolar somas
were identified by staining with the monoclonal antibody for
o. All of these somas also stained for
o1. Also, in the IPL, the two staining patterns were the
same, indicating that the retina expresses primarily the
o1 splice variant.
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ON bipolar cells and certain amacrine cells express low levels
of o2
A specific antisense probe for o2 mRNA
distributed similarly to that of o1: strong
labeling in the INL, somewhat weaker in the ganglion cell layer, and
none in the outer nuclear layer and photoreceptor inner segments (Fig.
4A). A shorter, more
specific probe distributed similarly, but less intensely, whereas the
sense probe distributed randomly and rather weakly (Fig.
4B).
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Figure 4.
Bipolar cells and stratum 1 of the IPL weakly
express the o2 splice variant. A,
B, In situ hybridization (rat). Antisense
probes specific for o2 message hybridized strongly in
the INL and weakly in the ganglion cell layer (GCL).
Sense probes gave weak, diffuse background. Left,
Differential interference contrast for retinal section in
A. C-F, Immunostaining of mouse retina,
visualized with FITC. D-F were captured using the same
confocal laser intensity and gain parameters. C,
Antibody against the CT peptide of o1 applied to the
o1 null retina faintly stained the OPL, bipolar somas
(BP), and stratum 1 of the IPL
(brackets). D, Antibody against the NT
peptide applied to the o1 null retina gave similar
staining: faint in the OPL, bipolar somas, and puncta throughout the
IPL, plus more intense staining in stratum 1. E, NT
antibody applied to o knock-out (KO)
retina gave weak diffuse background staining. F, NT
antibody applied to o2 knock-out retina gave strong
labeling, resembling that obtained with this and other
anti-o antibodies in wild-type retina.
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To test the distribution pattern of the o2
protein was difficult. The antibody for o2
capriciously stained cone outer segments in rat (but not in mouse), and
it also stained Muller cells. The staining of the Muller cell was
clearly unspecific because this cell also stained with the preimmune
serum applied to wild type and with the antibody applied to
o or o2 null mice.
Thus, the antibody for o2, under our staining
conditions, was unsuitable for immunocytochemistry. Instead, we used
three antibodies shown by Western blots to recognize both
o1 and o2 and applied
them to retina of the o1 null mouse. The
monoclonal antibody gave no staining. However, the other two antibodies
that recognize both splice variants, one against the CT and one against
the NT, both gave faint staining in the OPL, bipolar somas, and stratum 1 of the IPL (Fig. 4C,D). To distinguish this
stain from background, we first imaged staining from the
o null retina. This general background served
as a reference at the confocal microscope (Fig. 4E).
Then, using the same laser intensity and gain parameters, we imaged
staining from the o1 null retina. For both CT
and NT antibodies, staining was stronger than in
o null retina, and the pattern was repeatable
(Fig. 4, compare E with C, D). Both antibodies applied to the o2 null retina gave
identical staining as in the wild type (Fig. 4F).
Thus, both by in situ hybridization and by
immunocytochemistry, bipolar cells weakly express
o2.
Next, to determine whether the stained bipolar cells represent only a
subset of bipolar cells, we stained the o1
null retina for PKC and anti-o (C terminus).
All cells that stained for PKC also stained for
o, but ~8% of the cells (13 of 75 in one
animal; 1 of 100 in the other) that stained for
o were unstained for PKC (Fig.
5). The difference between the two
animals could be attributed to weaker expression in cone bipolar cells
or to regional differences. The cells that are
o2 positive (but PKC negative) were not
identified. We can rule out the possibility that they are OFF bipolar
cells because it has been established (using the same antibody as in
this experiment) that o is absent from OFF cone bipolar cells (Vardi, 1998). Thus, o2 is
expressed in rod bipolar cells and in at least some ON cone bipolar
cells.
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Figure 5.
Rod bipolar and some cone bipolar cells stain for
Go2. Rod bipolar cells were identified by staining for
PKC; staining for o2 was achieved by staining an
o1 null mouse with anti-Go-CT. All
bipolar somas stained for PKC also stained for o2
(double arrowhead), but some somas were stained only for
o2 (arrow).
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The light response requires o1 but
not o2
To test which splice variant is crucial for the ON bipolar cell
light response, we examined the electroretinogram in mice lacking
either o1 or o2.
Three different lighting conditions were used: scotopic (dark adapted
plus dim stimulus), saturated (dark adapted plus saturating stimulus),
and photopic (light adapted plus strong stimulus). Under all
conditions, mice lacking o1 also lacked the
b-wave, which is generated by the ON bipolar cells (Robson and
Frishman, 1995) (Fig. 6, middle
column; two animals, four eyes). This lack of b-wave does not
reflect lack of photoreceptor activity because the a-wave, which
originates in suppression of the photoreceptor dark current (Hagins et
al., 1970; Hood and Birch, 1993; Breton et al., 1994; Lyubarsky and
Pugh, 1996; Pugh et al., 1998), was present. Under the "saturated"
condition, o1 null mice gave a reduced, but
still profound, a-wave (78 ± 34 µV). Under photopic conditions,
in which the negative wave is likely a combination of a-wave and OFF
bipolar response, the negative wave was as large or greater than in the
wild type. This response pattern resembled that obtained when both
splice variants were eliminated (o null)
(Dhingra et al., 2000). Light responses in mice lacking
o2 were indistinguishable from wild type (Fig.
6, compare left with right columns; three
animals, six eyes). The average peaks for Go2
null mouse versus wild type were as follows (respectively, in
microvolts): rod-generated b-wave, 249 ± 89 versus 220 ± 72; saturated a-wave, 283 ± 51 versus 319 ± 97; and cone-generated b-wave, 146 ± 49 versus 109 ± 32.
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Figure 6.
Rod- and cone-driven b-waves are present in the
electroretinogram of Go2 null mouse but absent from the
Go1 null mouse. A, Animals dark adapted
for 2 hr were stimulated with dim flashes that elicited a rod-driven,
corneal-positive b-wave in the wild-type mouse and in
Go2 null but not in the Go1 null. Flash
intensities estimated as photoisomerizations (R*) per
rod ( ), and number of responses (n) averaged
for each trace were as follows: = 9; n = 18. B, Dark-adapted animals were stimulated with an
intense flash isomerizing ~0.1% of the rhodopsin. This elicited in
wild-type and Go2 null mice a negative a-wave
(shading), followed by a positive b-wave. The
Go1 null mice showed an a-wave (although somewhat
reduced) but no b-wave. = 105;
n = 4. C, Mice were adapted to a
background (white light, 9100 R*
rod 1 sec1) that completely
suppressed the rod cGMP-activated current. Rods were then stimulated
with an intense white flash that isomerized ~0.4% of the M-cone
pigment and 0.03% of the UV-cone pigment. All of the animals showed a
cone-driven a-wave. A typical cone-driven b-wave (positive-going
response with superimposed oscillations, peaking ~70-90 msec after
the flash) was present in wild-type and o2 null mice but
was absent in the Go1 null animals. For all of the
records, n = 16. KO, Knock-out;
WT, wild type.
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DISCUSSION |
We showed by several techniques (RT-PCR, Western blotting,
in situ hybridization, and immunostaining with antibodies
directed against different domains) that both splice variants of
Go, Go1 and
Go2, are expressed in retinal ON bipolar
cells. However, Go1 is much more abundant and
is essential for the bipolar cell light response. Mice lacking the
Go1 splice variant are devoid of the scotopic
and photopic b-waves, whereas mice lacking the Go2 splice variant show a normal ERG. We
noticed that the rod a-wave in the o1 null
mouse was reduced relative to the wild type. The difference is not
clear and seems insignificant because in the complete
Go null, both a-waves were within normal
range. Nevertheless, the lower amplitude does not compromise the
conclusion that the response of the bipolar cell is absent because this
range of photoresponses should have elicited a b-wave ON response.
Thus, the ON responses in rod and all ON cone bipolar cells depend only on Go1.
Importance of identifying the crucial splice variant in the
mGluR6 cascade
The identification of o1 as the critical
splice variant permitted its use (in constitutively active form) as
bait in a yeast two-hybrid assay. One strong interactor encodes the subunit of the photoreceptor phosphodiesterase (PDE6). However,
available probes for PDE- did not localize it to ON bipolar cells,
and therefore it is unlikely to be the effector in the ON response (Nawy, 1999). Another interactor encodes Ret-RGS1 (regulator of G-protein signaling), and this protein is expressed in dendritic tips
of at least one type of ON cone bipolar cell (Dhingra et al., 2001).
Ret-RGS is unlikely to be the actual effector for Go1, although it may assist. Nevertheless,
these preliminary observations suggest the value of having narrowed
down the search for effectors for the specific splice variant of
o.
Possible function of Go2 in ON bipolar cells
If only Go1 is crucial for the ON response,
what is the function of Go2? We propose several
possibilities. (1) Go2 may contribute to
fine-tuning of the mGluR6 cascade in certain ON bipolar cells. The ON
bipolar cells are classified into rod bipolar cells and approximately
five different types of cone bipolar cells that differ by their
morphology (Cohen and Sterling, 1990; Boycott and Wässle, 1991;
Euler and Wässle, 1995), and they are thought to divide the range
of temporal frequencies by responding with different kinetics
(Sterling, 1998; Freed, 2000a,b; Masland, 2001; Roska and Werblin,
2001). All ON bipolar cells express the same receptor (mGluR6) (Masu et
al., 1995; Vardi and Morigiwa, 1997; Vardi et al., 2000), and the same
G-protein subunit (o1) and subunit
(G13) (Huang et al., 2000). Because
Go2 is expressed only in a subtype of the ON
bipolar cells, it may differentially contribute to shaping response
kinetics. (2) Go2 could couple different
receptors to different biochemical cascades.
Go1 and Go2 differ in
the C-terminal domain that is generally involved in specific
interaction with the receptor and the effector (Conklin et al., 1996).
An example for effector specificity is given by Helisoma neurons, in
which o2, but not o1,
inhibits Ca2+ channels (Man-Son-Hing et
al., 1992). An example for receptor specificity is given by rat
pituitary GH3 cells, in which Go1 inhibits the
Ca2+ channel by coupling muscarinic
receptor, whereas Go2 inhibits this channel by
coupling somatostatin receptors (Kleuss et al., 1991; Chen and Clarke,
1996; Degtiar et al., 1997). In rat retina, rod bipolar cells and a
subset of cone bipolar cells express, in addition to mGluR6, the
metabotropic glutamate receptors mGluR1 and mGluR5 (Koulen et al.,
1997). Therefore, it is possible that Go2 couples
these receptors to a second-messenger cascade. Because these receptors
typically regulate intracellular [Ca2+]
(via PLC pathway) (Abe et al., 1992) and because intracellular [Ca2+] is critical for adaptation in rod
bipolar cells (Shiells and Falk, 1999; Nawy, 2000; Berntson and Taylor,
2000), Go2 may contribute to adaptation. (3)
Go2 may be involved in some aspects of
development. Expression patterns of Go1 and
Go2 during development suggest that it mediates
several processes in development (Strittmatter et al., 1990; Rouot et
al., 1992; Duc-Goiran et al., 1999). Thus, similar function may apply
also in retina. It is important, however, to note that whatever the
function in development of Go is, it is not
crucial for gross morphology because all of the
o knock-outs (Go /, Go1/,
and Go2/) have normal brain and retinal
morphology (Valenzuela et al., 1997; Jiang et al., 1998; Dhingra et
al., 2000; this study).
| |
FOOTNOTES |
Received Jan. 18, 2002; revised March 27, 2002; accepted April 3, 2002.
*
A.D., M.J., and T.-L.W. contributed equally to this work.
This work was supported by National Eye Institute Grants EY11105
(N.V.), EY00828 (P.S.), and EY02660 (E.N.P.), the Research to Prevent
Blindness Foundation (E.N.P.), and National Institutes of Health
Grant DK19318 (L.B.). We thank Yi-Jun Shi and Sally Shrom for
excellent technical assistance. We also thank David Manning for
donating the antibodies for Go (C terminus),
Go1, and Go2 and Kwen-Jen Chang
for donating the antibody for Go (N terminus).
Correspondence should be addressed to Dr. Anuradha Dhingra, 123, Anat-Chem Building, Department of Neuroscience, University of
Pennsylvania, Philadelphia, PA 19104. E-mail:
annu{at}retina.anatomy.upenn.edu.
T.-L. Wang's present address: Howard Hughes Medical Institute and
Johns Hopkins Oncology Center, The Johns Hopkins University, Baltimore,
MD 21231.
| |
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A. Dhingra, E. Faurobert, N. Dascal, P. Sterling, and N. Vardi
A Retinal-Specific Regulator of G-Protein Signaling Interacts with G{alpha}o and Accelerates an Expressed Metabotropic Glutamate Receptor 6 Cascade
J. Neurosci.,
June 23, 2004;
24(25):
5684 - 5693.
[Abstract]
[Full Text]
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ABSTRACT
INTRODUCTION
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