From the
The phototransduction process in cones has been proposed to
involve a G protein that couples the signal from light-activated visual
pigment to the effector cyclic GMP phosphodiesterase. Previously, we
have identified and purified a G
The family of structurally homologous G proteins plays an
essential role in transducing extracellular signals from cell-surface
receptors to intracellular effectors (Stryer and Bourne, 1986; Gilman,
1987; Birnbaumer, 1990). Members of this group of proteins are
heterotrimers composed of G
The
molecular mechanism by which the G
The bovine retinal cDNA library in Uni-Zap
XR was a gift from Dr. Wolfgang Baehr (Baylor College of Medicine), and
Ha-Ras cDNA in the pTrcA vector (Invitrogen) was obtained from Dr.
Vincent Jung (Cold Spring Harbor). Rac2 cDNA was amplified from the
bovine retinal cDNA library using PCR.
Peptide CMAQELSEKELLKME, corresponding
to residues 1-14 of the deduced amino acid sequence of
G
Frozen sections of the tissues were cut at
7-9 µm on a Reichert-Jung 2800 Frigcut cryostat, transferred
to slides coated with aminopropyltriethoxysilane (Aldrich), and stained
using the avidin-biotinylated enzyme complex technique according to
instructions provided by the supplier (Pierce). All staining was
performed at 21 °C in a humidified chamber, and preimmune controls
were treated alongside the experimental sections. The experimental and
control sections were incubated for 45 min in purified IgG at a
concentration of A
A comparison of the deduced
amino acid sequence of G
The antiserum also recognizes recombinant
G
The phototransduction process in the vertebrate retina takes
place in the outer segments of rod and cone photoreceptor cells. Rods
are remarkably sensitive and can detect a single photon, but their
response is easily saturated at higher light intensity (Pugh and Cobbs,
1986). Compared with rods, cones are 100-fold less sensitive to light,
but display a faster and shorter response (Pugh and Cobbs, 1986;
Nakatani and Yau, 1986). They are also less easily saturable than rods.
The mechanisms underlying the differences in sensitivity and
transduction kinetics between these two types of photoreceptors have
been proposed to reside in the biochemistry of the transduction process
(Yau, 1994).
In rods, a retina-specific G protein (transducin)
couples the photoexcitation of rhodopsin to the activation of retinal
cGMP phosphodiesterase (Fung, 1986; Lolley and Lee, 1990; Stryer,
1991). The resulting change in the intracellular cGMP level leads to
the closure of cGMP-sensitive cation channels and the hyperpolarization
of the rod (Pugh and Cobbs, 1986). Although the phototransduction
mechanism of cones is less well characterized, electrophysiological
studies have detected similar types of cGMP-regulated cation channels
in the plasma membranes of the cone outer segments (Haynes and Yau,
1985; Cobbs et al., 1985). Moreover, GTP is required in order
for light to close the cGMP-regulated channels, suggesting a direct
involvement of a G protein in the cone phototransduction pathway
(Nakatani and Yau, 1986). At the molecular level, the cone-specific
G
In an effort to identify the G
To
address the question of whether G
Peng et al. (1992) have previously reported that
antipeptide antibodies directed against peptides corresponding to
residues 2-14 and 36-46 of G
Similar to the G
In addition to membrane
anchorage, the isoprenyl group of the G
With the cloning of the
G
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank/EMBL Data Bank with accession number(s)
U20085.
We thank Bernice Lieberman and Bruce Brown for
excellent technical assistance and Marcia Lloyd for performing the
immunostaining. We also thank Dr. Janmeet Anant and Michael Bova for
helpful discussion and critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
complex composed of a
G
isoform and an immunochemically distinct G
subunit (G
) from bovine retinal cones (Fung, B. K.-K.,
Lieberman, B. S., and Lee, R. H. (1992) J. Biol. Chem. 267,
24782-24788; Lee, R. H., Lieberman, B. S., Yamane, H. K., Bok,
D., and Fung, B. K.-K. (1992a) J. Biol. Chem. 267,
24776-24781). Based on the partial amino acid sequence of this
cone G
, we screened a bovine retinal cDNA library and
isolated a cDNA clone encoding G
. The cDNA insert of
this clone includes an open reading frame of 207 bases encoding a
69-amino acid protein. The predicted protein sequence of G
shares a high degree of sequence identity (68%) with the G
(G
) subunit of rod transducin. Similar to rod
G
, it terminates in a CIIS motif that is the site for
post-translational modification by farnesylation. Messenger RNA for
G
is present at a high level in the retina and at a
very low level in the lung, but is undetectable in other tissues.
Immunostaining of bovine retinal sections with an antipeptide antibody
against the N-terminal region of G
further shows a
differential localization of G
to cones with a pattern
indistinguishable from that of G
. This finding
suggests that G
is a component of
cone transducin involved in cone phototransduction and color vision.
, G
, and G
subunits. In most
biological systems, the G
subunits are generally recognized as the
signal carrier that dictates the specificity of signaling pathways. The
G
and G
subunits, which form a tightly associated G
complex, usually play a more passive role by promoting interactions
between the G
subunit and the receptor (Fung, 1983; Florio and
Sternweis, 1985). However, there is now a growing body of evidence
demonstrating that G
also participates in a wide range of
other G protein functions in some systems. These include the promotion
of cholera toxin- and pertussis toxin-catalyzed ADP-ribosylation of the
G
subunit (Yamane and Fung, 1993), interaction with phosducin (Lee
et al., 1987, 1992b) and receptor kinase (Haga and Haga, 1992;
Inglese et al., 1992; Pitcher et al., 1992), and
regulation of the activities of effectors such as adenylate cyclase
types II and IV, phospholipase A
, phospholipase C, and
cardiac K
channels (Clapham and Neer, 1993).
complex regulates a
diversity of signaling processes is still not fully understood. To
date, five forms of G
(Watson et al., 1994) and multiple
forms of G
subunits have been identified by biochemical,
immunological, and molecular cloning studies (Simon et al.,
1991). At the amino acid level, the G
subunits are highly
conserved. In contrast, the G
subunits are more divergent. Because
of the diversity of the G
sequences, it is generally believed that
the G
subunit determines the functional specificity of the
G
complex. However, it is unclear what combinations of G
and G
subunits occur physiologically to account for the
differences in their functions. The G
complexes isolated from
most tissue preparations are heterogeneous mixtures containing
different forms of G
and G
subunits (Gautam et al.,
1990). An exception to this is the cone G
complex, which is
composed of G
and a novel G
subunit (Fung et
al., 1992; Lee et al., 1992a). In this study, we report
the isolation and expression of the cDNA of this G
subunit. We
further show that this G
subunit is highly homologous to the
G
subunit of rod transducin (Hurley et al.,
1984; Yatsunami et al., 1985) and is localized in cone
photoreceptors of the retina. Following the convention of naming the G
protein subunits numerically, we will refer to this newly identified
cone G
subunit as G
in this paper. We speculate
that G
may serve an important function in the
regulation of phototransduction in cones.
Materials
Retinal G protein (transducin)
G complex was isolated from bovine retinas purchased from J.
A. Lawson (Lincoln, NE) (Fung, 1983). The cone-specific
G
complex was separated from
rod-specific G
by subtractive
chromatography using an affinity column of immobilized monoclonal
antibodies against rod G
as reported previously (Fung
et al., 1992).
(
)
Recombinant Ha-Ras and Rac2 fusion proteins containing a
histidine-rich N-terminal sequence were expressed in Escherichia
coli, and the proteins were purified according to the procedure as
described for the expression of G
and stored in 40%
glycerol at -20 °C.
plus an N-terminal cysteine for coupling purpose,
was synthesized by Immuno-Dynamics, Inc. (La Jolla, CA). A polyclonal
antiserum was generated by immunization of rabbits with the synthetic
peptide coupled to keyhole limpet hemocyanin. The IgG fraction of the
antiserum was purified by chromatography on a DEAE-Affi-Gel blue column
(Bio-Rad).
Amino Acid Sequence Determination
The purified
Gcom-plex was separated on high
resolution Tricine-polyacrylamide gels (Schägger and von Jagow,
1987), transblotted onto Immobilon-P membranes (Millipore Corp.), and
visualized by staining with Coomassie Blue (Matsudaira, 1987). The
region of the membranes containing the G
subunit was
excised, cleaved with cyanogen bromide, and subjected to gas-phase
protein sequence analysis at the Protein Sequencing Facility,
University of California, Los Angeles.
Isolation of G
Degenerate PCR oligonucleotide primers corresponding to
residues KKEVKN (5`-primer) and KGIPED (3`-primer) were designed from
the partial amino acid sequence of GcDNA
Clones
. PCR mixtures
were prepared in 50 µl containing 1
PCR buffer (10 m
M Tris, pH 8.3, 50 m
M KCl, 1.5 m
M MgCl
, 0.001% gelatin), 1 µ
M degenerate
primers, 50 µ
M dNTPs, and 0.5 µl (2.5
10
plaque-forming units) of bovine retinal cDNA library in Uni-Zap
XR. Reactions were performed for five cycles with melting, annealing,
and extension at temperatures of 94, 37, and 72 °C, respectively,
followed by 25 cycles with an annealing temperature of 52 °C. A PCR
product of
150 base pairs was obtained, and the amplified cDNA
fragment was excised from an agarose gel, purified, and ligated to the
TA cloning vector (Invitrogen). The isolated cDNA clone was sequenced
to confirm that it corresponds to the nucleotide sequence predicted
from the partial amino acid sequence of G
. A
[
P]CTP-labeled mRNA probe was then synthesized
from the vector and used to screen 3
10
plaques at
50 °C. The Bluescript plasmids containing the desired insert were
rescued from the positive phagemid preparation according to the
manufacturer's instructions (Stratagene).
Expression of Recombinant G
A
fragment of GcDNA from base pairs 2 to 429 shown in
Fig. 1
was amplified by PCR and ligated into the pT7 blue vector
(Novagen). The cDNA insert was excised at the NdeI and
BamHI sites, subcloned into the pET-16b expression vector
(Novagen), and expressed as a fusion protein containing a
histidine-rich N-terminal sequence and a Factor Xa cleavage site.
Isoprenyl-
-
D-thiogalactopyranoside induction and
extraction of the G
fusion protein were carried out
according to the procedure provided by the manufacturer. The bacterial
lysate was centrifuged at 39,000
g for 20 min and
applied to a 1-ml column of His-Bind resin (Novagen). Bound
G
fusion protein was eluted from the column with 20
m
M Tris, pH 7.9, 0.5
M NaCl, 100 m
M EDTA.
The protein eluents were pooled; dialyzed for 4 h against 50 m
M Tris, pH 8.0, 100 m
M NaCl, 1 m
M CaCl
; and digested with Factor Xa for 4 h at room
temperature at a protease/protein ratio of 1:60 (w/w). Immediately
following proteolysis, G
was further purified on a
Superose-12 column. The resulting G
is structurally
similar to the native protein, except for the replacement of N-terminal
MA residues with HMDLA residues.
Figure 1:
Nucleotide sequence and predicted amino
acid sequence of bovine G subunit. The nucleotide
sequence of G
cDNA including the coding region and a
portion of the 5`- and 3`-noncoding regions is shown. The nucleotide
residues are numbered starting with the ATG initiation codon.
Nucleotides 5` to the ATG codon are indicated by negative numbers. The deduced protein sequence is listed below the
corresponding nucleotide triplets. The region of the polypeptide
determined by partial amino acid sequencing is
underlined.
Protein Isoprenylation
In vitro isoprenylation of recombinant G, Rac2, or Ha-Ras
protein was carried out at 37 °C in a final volume of 25 µl of
buffer (50 m
M Tris, pH 7.5, 20 m
M KCl, 5 m
M MgCl
, 0.5 m
M dithiothreitol). The reaction
mixture for farnesylation contained 1.5 µ
M purified
protein, 2 µ
M [
H]farnesyl
pyrophosphate (15 Ci/mmol; American Radiolabeled Chemicals), 20
µ
M geranylgeranyl pyrophosphate, and 4 µl of
nuclease-treated rabbit reticulolysate (Promega). Geranylgeranylation
was carried out under the same conditions, except that the
[
H]farnesyl pyrophosphate and geranylgeranyl
pyrophosphate were replaced with
[
H]geranylgeranyl pyrophosphate (40 Ci/mmol;
American Radiolabeled Chemicals) and farnesyl pyrophosphate,
respectively. After 1 h of incubation, the reaction was terminated by
the addition of an equal volume of 2
SDS-PAGE sample buffer,
and the proteins were separated on high resolution
Tricine-polyacrylamide gels. To quantify the amount of radioactivity,
regions of the gel containing G
, Rac2, or Ha-Ras were
excised; digested in 20% H
O
at 65 °C
overnight; and analyzed by liquid scintillation counting.
RNA Blot Analysis
Total RNA from bovine retina was
isolated by acid guanidinium thiocyanate/phenol/chloroform extraction
(Chomczynski and Sacchi, 1987). RNA from other frozen bovine tissues
was prepared by guanidinium thiocyanate extraction and LiCl
precipitation (Cathala et al., 1983), and poly(A)RNA was further purified by binding to oligo(dT)-cellulose
columns. The RNA samples were electrophoresed on 0.9% agarose gels
containing 2.2
M formaldehyde and then transferred to
nitrocellulose membranes. Hybridization with nick-translated bovine
G
cDNA (10
cpm/ml) and human G
cDNA (10
cpm/ml) probes was carried out for 24 h at
38 °C in buffer containing 50% formaldehyde, 5
SSC, 50
m
M NaH
PO
, pH 7.0, 2
Denhardt's solution, 0.1% SDS, and 50 µg/ml denatured salmon
sperm DNA. The final washing of the membranes was performed in a
solution containing 0.1
SSC and 0.1% SDS at 42 °C for 30
min. Autoradiography was carried out by exposure to Kodak X-Omat AR
film for 42 h at -80 °C using an intensifying screen.
Immunostaining of Bovine Retinas
Fresh bovine eyes
were obtained from a local slaughterhouse and the cornea, iris, lens,
and vitreous body were removed. The remaining eyecup was fixed at 4
°C for 4 days by immersion in 4% formaldehyde freshly prepared from
paraformaldehyde. Strips of retina attached to the underlying choroidal
layer were dissected from the scleral layer and infiltrated for 12 h in
30% sucrose. The infiltrated strips were then immersed in Tissue-Tec
O.C.T. compound (Miles Inc.), frozen in liquid nitrogen, and stored at
-80 °C.
= 0.01 A units, for 30 min in goat anti-rabbit secondary antibody, and for
30 min in avidin-biotin complex. To visualize the cell bodies, the
sections were counterstained for 1 min in Mayer's hematoxylin.
Sections were viewed in a Zeiss PM III photomicroscope and photographed
with Kodak Ektachrome 50T film.
Analytical Assays
Protein concentrations were
determined by Coomassie Blue binding (Bradford, 1976) using
-globulin as a standard. SDS-PAGE of proteins was performed by the
method of Schägger and von Jagow (1987) (16.5% separation gel in
Tricine buffer). Western blotting of proteins on Immobilon-P membranes
was carried out according to a modified procedure of Towbin et al. (1979). After sequential incubation of the membranes with 5%
bovine serum albumin, primary antibodies, and peroxidase-conjugated
goat anti-rabbit IgG (Boehringer Mannheim), the immunoreactive bands
were detected by chemiluminescence with the ECL Western blotting
reagent (Amersham Corp.).
cDNA Sequence Encoding the Cone G
We have previously reported the purification of a
cone GSubunit
complex composed of G
and an
immunochemically distinct G
subunit (Fung et al., 1992;
Lee et al., 1992a). Furthermore, analysis of this cone G
peptide obtained by cyanogen bromide cleavage revealed an internal
amino acid sequence that differs from all other known G
subunits.
To obtain the complete sequence of cone G
, we first amplified from
a bovine retinal cDNA library a fragment of this cDNA by PCR using a
set of degenerate oligonucleotide primers derived from amino acid
sequence information. We then used this PCR fragment as a probe to
screen the cDNA library and isolated one clone containing the entire
coding region of this cone G
. Sequence analysis of the cDNA insert
of the clone revealed a 207-base pair open reading frame encoding a
69-amino acid protein with a calculated molecular mass of 7700 Da
(Fig. 1). The deduced amino acid sequence matches perfectly with
the sequence of the cyanogen bromide-generated peptides, indicating
that the cDNA encodes cone G
. This newly identified G
was
subsequently named G
.
and other known sequences of
G
subunits is shown in Fig. 2. As expected for a cone G
protein subunit, G
shares the strongest sequence
identity (68%) with G
of retinal rod transducin. The
differences between these two forms are concentrated at the N-terminal
region and in the region between residues 22 and 33 of
G
. In contrast, G
exhibits a lesser
degree of sequence identity to other forms of G
subunits, ranging
from 30% (for G
) to 39% (for G
and
G
). Like other members of the family, the G
subunit terminates with a C XXX motif (where C =
cysteine and X = any amino acid), which has been shown
in G
(Fukada et al., 1990; Lai et
al., 1990; Fung et al., 1994) and brain G
(Mumby
et al., 1990; Yamane et al., 1990) to be the signal
sequence for multiple post-translational modifications, including
isoprenylation, proteolysis, and carboxyl methylation.
Figure 2:
Comparison of amino acid sequences of
various G subunit isoforms. The protein sequence predicted from
the G
cDNA is aligned with the amino acid sequences of
G
(Hurley et al., 1984; Yatsunami et
al., 1985), G
(Gautam et al., 1989;
Robishaw et al., 1989), G
(Gautam et
al., 1990), G
(Fisher and Aronson, 1992), and
G
(Cali et al., 1992). Identical amino acid
residues are indicated by white letters on a
black background.
Tissue-specific Expression of G
To examine the tissue distribution of the
GmRNA
mRNA, we carried out Northern blot analysis of total
RNA from bovine retina and poly(A)
-enriched RNA from
several other bovine tissues. As shown in Fig. 3 A, the
G
probe recognized a 2.3-kilobase mRNA transcript,
which is expressed in the retina, and a 2.9-kilobase mRNA transcript,
which is present at a very low level in the lung. All other tissues
tested, however, do not show a detectable level of G
message. When the same blot was hybridized to the probe derived
from human G
cDNA as a control (Jiang et
al., 1991), a 3.8-kilobase transcript of G
mRNA
was readily detected in all preparations of tissue poly(A)
RNA (Fig. 3 B). This result indicates that mRNA of
G
is predominantly expressed in retinas.
Figure 3:
Tissue distribution of G
mRNA. Total RNA (7 µg) from bovine retina and poly(A)
RNA (4 µg) from various other bovine tissues were hybridized
to a
P-labeled probe derived from bovine G
cDNA ( A) or from human G
cDNA
( B) according to the procedure described under
``Experimental Procedures.'' The size of the G
transcript is shown relative to the 18 S and 28 S rRNA
standards.
Isoprenylation of Recombinant
G
The presence of a CIIS motif at the C
terminus of Gsuggests that it may be
post-translationally modified by isoprenylation. To explore this
possibility, we determined the ability of recombinant G
to incorporate radiolabeled isoprenoid groups. Recombinant
G
containing a histidine-rich N-terminal sequence was
expressed as a fusion protein and purified by affinity chromatography.
The purity of the fusion protein obtained was >90% as estimated by
Coomassie Blue staining of the purified proteins separated by SDS-PAGE
(Fig. 4 B, lane 3). This purified
fusion protein was digested with Factor Xa to remove the N-terminal
histidine-tagged sequence, and recombinant G
generated
from the proteolysis was separated from Factor Xa by gel filtration on
a Superose-12 column (Fig. 4 B, lane 4). In vitro isoprenylation of recombinant G
was carried out
in rabbit reticulolysate in the presence of either
[
H]farnesyl pyrophosphate or
[
H]geranylgeranyl pyrophosphate. Recombinant
Ha-Ras and Rac2 proteins, previously shown to be farnesylated and
geranylgeranylated, respectively (Casey et al., 1989; Kinsella
et al., 1991a), were used as controls to test the labeling
reaction. Compared with [
H]geranylgeranyl
pyrophosphate, incubation in the presence of
[
H]farnesyl pyrophosphate results in the
incorporation of 8.3- and 23-fold higher levels of radioactivity into
G
and Ha-Ras, respectively (). The lower
efficiency in labeling of G
relative to Ha-Ras may be
due to dimerization of the G
subunit in the absence of
the G
subunit (see Fig. 4and below). In
contrast, the labeling of Rac2 protein with a CSLL motif is 63-fold
greater in the presence of [
H]geranylgeranyl
pyrophosphate compared with [
H]farnesyl
pyrophosphate. Taken together, these results strongly suggest that
G
is modified by farnesylation. It is noteworthy that
G
, which is highly homologous to G
,
is also farnesylated (Fukada et al., 1990; Lai et
al., 1990; Fung et al., 1994). On the other hand,
geranylgeranylation has been shown to occur in G
, a
less homologous form found in the brain (Mumby et al., 1990;
Yamane et al., 1990). Evidence based on previous studies
indicates that the specificity of the isoprenylation is dictated by the
carboxyl residue of the C XXX sequence (Kinsella et
al., 1991b; Reiss et al., 1991). In agreement with these
findings, both G
and G
, which are
farnesylated, terminate in a serine residue at the carboxyl terminus,
whereas a leucine residue is present at the carboxyl terminus of all
other forms of geranylgeranylated G
subunits.
Figure 4:
Specificity of peptide antisera against
G. Purified bovine cone and rod G
subunits
( A) and the recombinant G
subunit
( B) were resolved by SDS-PAGE and analyzed by Western blotting
using the peptide antiserum against the N-terminal region of
G
. Proteins were purified as described under
``Experimental Procedures.'' Lane 1,
G
subunit (3 µg for SDS-PAGE, 1.5 µg for
immunoblotting); lane 2, G
subunit (3
µg for SDS-PAGE, 1.5 µg for immunoblotting); lane 3,
purified recombinant G
with additional N-terminal
polyhistidine residues (4 µg for SDS-PAGE, 0.1 µg for
immunoblotting); lane 4, purified recombinant G
following cleavage of the N-terminal polyhistidine residues by
Factor Xa (4 µg for SDS-PAGE, 0.1 µg for immunoblotting).
(His)G
indicates the
polyhistidine-tagged G
fusion
protein.
Specificity of the Antipeptide Antiserum
Specific
antiserum was generated against the synthetic peptide corresponding to
residues 1-14 of the deduced amino acid sequence of
G. To test the specificity of the antiserum, we
purified bovine retinal rod-specific G
and
cone-specific G
and carried out immunoblot
analysis of these proteins with the antipeptide antiserum. As shown in
the autoradiogram in Fig. 4 A, the antiserum specifically
recognizes the G
subunit from the G
preparation ( lane 1), but not the G
subunit from
the purified G
complex ( lane 2).
expressed in E. coli, which was used in the
isoprenylation experiments. As shown in the immunoblot in
Fig. 4B, the G
fusion product
containing the N-terminal histidine-rich residues ( lane 3) as well as G
generated following
Factor Xa cleavage ( lane 4) are detectable with the
antipeptide antiserum. The antiserum is also immunoreactive to a
polypeptide with an apparent molecular mass of 16 kDa present in the
Factor Xa-treated preparation. Since this immunoreactive peptide
appears only after cleavage with Factor Xa protease and the antiserum
did not cross-react with Factor Xa (data not shown), the 16-kDa
polypeptide is most likely a product of the irreversible dimerization
of recombinant G
after the histidine-tagged residues
were removed.
Immunocytochemical Localization of
G
To investigate whether Gis
specifically localized to cone photoreceptors, bovine retinal sections
were stained with antiserum raised against the N-terminal peptide of
G
. As shown in Fig. 5, the immunostaining was
exclusively observed in cones. The bovine retinal sections incubated
with the antiserum show staining in the cone outer segments, the myoid
region of inner segments, cone cell bodies, axons, and synaptic
terminals. The most intense staining was found in the outer segments.
In the retinal sections treated with the preimmune serum, however, no
staining was observed in these regions. These findings confirmed that
G
is specifically localized in cone photoreceptors
within the retina and raise the possibility that it is the G
subunit of cone transducin involved in phototransduction and color
vision.
Figure 5:
Immunocytochemical localization of
G in bovine retinas. Formaldehyde-fixed frozen bovine
retinal sections were incubated with the peptide antisera against
G
as described under ``Experimental
Procedures.'' The sections were counterstained for 1 min in
Mayer's hematoxylin to visualize the cell bodies. A,
cone inner and outer segments ( long arrows), somata
( short arrows), axons, and synaptic terminals
( arrowheads) stained positive, whereas the same cellular
components of rods were negative. B, shown are the negative
control sections for A incubated in preimmune
IgG.
subunit (Lerea et al., 1986), cGMP phosphodiesterase
(Gillespie and Beavo, 1988; Charbonneau et al., 1990; Li
et al., 1990), and the cGMP-regulated channel (Bonigk et
al., 1993) have been identified, and a G
complex
composed of G
and an immunochemically distinct G
subunit has also been purified from bovine retinal cones (Fung et
al., 1992; Lee et al., 1992a). The deduced amino acid
sequences of these cone-specific proteins are all highly homologous to
those of their corresponding rod-specific counterparts, strongly
suggesting that they are the key components of the cone
phototransduction machinery.
subunit that associates with G
in retinal cones, we
screened a bovine retinal cDNA library and isolated a cDNA clone
encoding G
. The predicted protein sequence for
G
suggests that it represents a new isoform of the
G
subunit family. Sequence comparison further reveals that
G
shares a relatively higher degree of homology with
G
(68% sequence identity) than with other known
isoforms of G
subunits (ranging from 30 to 39% identity). The
similarity in structure between G
and G
strongly implies that, like G
in rods,
G
may play a role in cone phototransduction.
is differentially
localized to one type of photoreceptor in the retina, we generated a
rabbit antipeptide antibody against the N-terminal region of
G
and performed immunocytochemistry on the sections of
bovine retina. The result shows that G
, like its
molecular partner G
, is present only in cones. Our
immunostaining data also show a distinct distribution of G
in the outer segments, inner segments, cell bodies, axons, and
synaptic terminals, with the highest staining intensity in the outer
segments. This immunostaining pattern of G
in cones is
indistinguishable from the staining pattern obtained by using a
G
-specific antipeptide antibody (Lee et al.,
1992a), suggesting that G
and G
form
a physiologically functional complex in all of these regions in cones.
also stained
exclusively the cone outer segment, suggesting that G
is also localized in cones. A comparison of the deduced amino
acid sequences of G
and G
eliminates
the possibility that these antibodies simply cross-reacted with
G
. Since G
and G
are
both absent in the cone outer segment (Lee et al., 1992a; Peng
et al., 1992) and G
and G
do
not form a complex in transfected cells (Pronin and Gautam, 1992), it
seems possible that G
may be associated with other
forms of G
subunits (von Weizsacker et al., 1992; Watson
et al., 1994).
subunit of
rod transducin, G
is found to be post-translationally
modified by farnesylation. This is in contrast to the G
subunits
of the brain G proteins, which are modified by geranylgeranylation
(Mumby et al., 1990; Yamane et al., 1990). Since the
farnesyl groups are less hydrophobic than geranylgeranyl groups,
farnesylated G
is likely to associate only loosely
with the disc membranes. Consistent with this finding, the
G
complex can be readily eluted from cone
membranes at low ionic strength (Lee et al., 1992a), and the
majority of the protein was found in the supernatant during isolation.
In contrast, brain G
protein containing a more hydrophobic
geranylgeranyl group can be extracted only in the presence of
detergents (Eide et al., 1987).
subunit may have other
biological functions. In the rod phototransduction pathway, the
carboxyl-terminal peptides of G
containing a
chemically attached farnesyl group appear to stabilize metarhodospin II
and to uncouple rhodopsin-transducin interaction (Kisselev et
al., 1994). Methylation of the farnesyl cysteine in G
also facilitates the interaction of transducin with rhodopsin
(Fukada et al., 1994). Since all other G
subunits except
those in photoreceptors are shown or predicted to be modified by
geranylgeranylation, we speculate that the farnesylation of
G
may indicate a specific functional requirement for
the phototransduction pathway in cones.
subunit reported here, we believe that all the key
protein components of the cone phototransduction machinery are now in
hand. It may soon be possible to produce all the protein components of
the cGMP cascade in a suitable eukaryotic expression system, to
reconstitute the cone phototransduction pathway in vitro, and
to study the chemical properties of each component in detail. The
future challenge is to describe quantitatively the entire
phototransduction process and to provide an explanation for the kinetic
and sensitivity differences between rods and cones.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.