©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Characterization of the G Protein Subunit of Cone Photoreceptors (*)

Olivia C. Ong (1)(§), Harvey K. Yamane (1), Kim B. Phan (1), Henry K. W. Fong (4), Dean Bok (1) (2), Rehwa H. Lee (2) (3), Bernard K.-K. Fung (1)(¶)

From the (1) Jules Stein Eye Institute and the (2) Department of Neurobiology, UCLA School of Medicine, Los Angeles, California 90024, the (3) Molecular Neurology Laboratory, Veterans Administration Medical Center, Sepulveda, California 91343, and the (4) Doheny Eye Institute and Departments of Ophthalmology and Microbiology, University of Southern California School of Medicine, Los Angeles, California 90033

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 complex composed of a Gisoform 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 Gshares 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 Gis 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 Gfurther shows a differential localization of Gto cones with a pattern indistinguishable from that of G. This finding suggests that Gis a component of cone transducin involved in cone phototransduction and color vision.


INTRODUCTION

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, 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 Kchannels (Clapham and Neer, 1993).

The molecular mechanism by which the G 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 Gand 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 Gsubunit 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 Gin this paper. We speculate that Gmay serve an important function in the regulation of phototransduction in cones.


EXPERIMENTAL PROCEDURES

Materials

Retinal G protein (transducin) G complex was isolated from bovine retinas purchased from J. A. Lawson (Lincoln, NE) (Fung, 1983). The cone-specific Gcomplex was separated from rod-specific Gby subtractive chromatography using an affinity column of immobilized monoclonal antibodies against rod Gas reported previously (Fung et al., 1992).

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.() 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 Gand stored in 40% glycerol at -20 °C.

Peptide CMAQELSEKELLKME, corresponding to residues 1-14 of the deduced amino acid sequence of Gplus 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 Gsubunit 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 GcDNA Clones

Degenerate PCR oligonucleotide primers corresponding to residues KKEVKN (5`-primer) and KGIPED (3`-primer) were designed from the partial amino acid sequence of G. 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 10plaque-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 10plaques 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 Gfusion 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 Gfusion 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, Gwas further purified on a Superose-12 column. The resulting Gis 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 GcDNA 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% HOat 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 GcDNA (10cpm/ml) and human GcDNA (10cpm/ml) probes was carried out for 24 h at 38 °C in buffer containing 50% formaldehyde, 5 SSC, 50 m M NaHPO, 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.

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= 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.).


RESULTS

cDNA Sequence Encoding the Cone GSubunit

We have previously reported the purification of a cone G complex composed of Gand 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.

A comparison of the deduced amino acid sequence of Gand other known sequences of G subunits is shown in Fig. 2. As expected for a cone G protein subunit, Gshares the strongest sequence identity (68%) with Gof 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, Gexhibits a lesser degree of sequence identity to other forms of G subunits, ranging from 30% (for G) to 39% (for Gand G). Like other members of the family, the Gsubunit 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 GcDNA 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 GmRNA

To examine the tissue distribution of the GmRNA, 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 Gprobe 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 Gmessage. When the same blot was hybridized to the probe derived from human GcDNA as a control (Jiang et al., 1991), a 3.8-kilobase transcript of GmRNA was readily detected in all preparations of tissue poly(A)RNA (Fig. 3 B). This result indicates that mRNA of Gis 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 GcDNA ( A) or from human GcDNA ( B) according to the procedure described under ``Experimental Procedures.'' The size of the Gtranscript 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 Gto incorporate radiolabeled isoprenoid groups. Recombinant Gcontaining 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 Ggenerated 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 Gwas 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 Gand Ha-Ras, respectively (). The lower efficiency in labeling of Grelative to Ha-Ras may be due to dimerization of the Gsubunit in the absence of the Gsubunit (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 Gis 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 Gand 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 Gsubunit ( 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 Gwith additional N-terminal polyhistidine residues (4 µg for SDS-PAGE, 0.1 µg for immunoblotting); lane 4, purified recombinant Gfollowing cleavage of the N-terminal polyhistidine residues by Factor Xa (4 µg for SDS-PAGE, 0.1 µg for immunoblotting). (His)Gindicates the polyhistidine-tagged Gfusion 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).

The antiserum also recognizes recombinant Gexpressed in E. coli, which was used in the isoprenylation experiments. As shown in the immunoblot in Fig. 4B, the Gfusion product containing the N-terminal histidine-rich residues ( lane 3) as well as Ggenerated 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 Gafter 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 Gis 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 Gas 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.




DISCUSSION

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 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 Gand 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.

In an effort to identify the G subunit that associates with Gin retinal cones, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding G. The predicted protein sequence for Gsuggests that it represents a new isoform of the G subunit family. Sequence comparison further reveals that Gshares 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 Gand Gstrongly implies that, like Gin rods, Gmay play a role in cone phototransduction.

To address the question of whether Gis differentially localized to one type of photoreceptor in the retina, we generated a rabbit antipeptide antibody against the N-terminal region of Gand 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 Gin the outer segments, inner segments, cell bodies, axons, and synaptic terminals, with the highest staining intensity in the outer segments. This immunostaining pattern of Gin cones is indistinguishable from the staining pattern obtained by using a G-specific antipeptide antibody (Lee et al., 1992a), suggesting that Gand Gform a physiologically functional complex in all of these regions in cones.

Peng et al. (1992) have previously reported that antipeptide antibodies directed against peptides corresponding to residues 2-14 and 36-46 of Galso stained exclusively the cone outer segment, suggesting that Gis also localized in cones. A comparison of the deduced amino acid sequences of Gand Geliminates the possibility that these antibodies simply cross-reacted with G. Since Gand Gare both absent in the cone outer segment (Lee et al., 1992a; Peng et al., 1992) and Gand Gdo not form a complex in transfected cells (Pronin and Gautam, 1992), it seems possible that Gmay be associated with other forms of G subunits (von Weizsacker et al., 1992; Watson et al., 1994).

Similar to the Gsubunit of rod transducin, Gis 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 Gis 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).

In addition to membrane anchorage, the isoprenyl group of the G subunit may have other biological functions. In the rod phototransduction pathway, the carboxyl-terminal peptides of Gcontaining 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 Galso 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 Gmay indicate a specific functional requirement for the phototransduction pathway in cones.

With the cloning of the Gsubunit 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.

  
Table: Isoprenylation of G



FOOTNOTES

*
This work was supported in part by Grants EY05895 (to B. K.-K. F.), EY 00444 (to D. B.), and EY09936 (to R. H. L.) from the National Institutes of Health and by grants from the National Retinitis Pigmentosa Foundation (to R. H. L.) and the Wong Fund (to B. K.-K. F). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U20085.

§
Recipient of Predoctoral Training Grant EY07026 and Medical Scientist Training Grant GM08042 from the National Institutes of Health.

To whom correspondence should be addressed. Tel.: 310-825-9541; Fax: 310-794-2144.

The abbreviations used are: PCR, polymerase chain reaction; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

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.


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