FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2003248941> ?p ?o ?g. }

• W2003248941 endingPage "8500" @default.
• W2003248941 startingPage "8495" @default.
• W2003248941 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 Gβγ complex composed of a Gβ3isoform and an immunochemically distinct Gγ subunit (Gγ8) 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 Gγ8, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding Gγ8. 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 Gγ8shares a high degree of sequence identity (68%) with the Gγ (Gγ1) subunit of rod transducin. Similar to rod Gγ1, it terminates in a CIIS motif that is the site for post-translational modification by farnesylation. Messenger RNA for Gγ8is 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 Gγ8further shows a differential localization of Gγ8to cones with a pattern indistinguishable from that of Gβ3. This finding suggests that Gβ3γ8is a component of cone transducin involved in cone phototransduction and color vision. 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 Gβγ complex composed of a Gβ3isoform and an immunochemically distinct Gγ subunit (Gγ8) 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 Gγ8, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding Gγ8. 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 Gγ8shares a high degree of sequence identity (68%) with the Gγ (Gγ1) subunit of rod transducin. Similar to rod Gγ1, it terminates in a CIIS motif that is the site for post-translational modification by farnesylation. Messenger RNA for Gγ8is 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 Gγ8further shows a differential localization of Gγ8to cones with a pattern indistinguishable from that of Gβ3. This finding suggests that Gβ3γ8is a component of cone transducin involved in cone phototransduction and color vision. 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, 1986Stryer L. Bourne H.R. Annu. Rev. Cell Biol. 1986; 2: 391-419Crossref PubMed Scopus (627) Google Scholar; Gilman, 1987Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4728) Google Scholar; Birnbaumer, 1990Birnbaumer L. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 675-705Crossref PubMed Google Scholar). Members of this group of proteins are heterotrimers composed of Gα, Gβ, and Gγ subunits. In most biological systems, the Gα subunits are generally recognized as the signal carrier that dictates the specificity of signaling pathways. The Gβ and Gγ subunits, which form a tightly associated Gβγ complex, usually play a more passive role by promoting interactions between the Gα subunit and the receptor (Fung, 1983Fung B.K.-K. J. Biol. Chem. 1983; 258: 10495-10502Abstract Full Text PDF PubMed Google Scholar; Florio and Sternweis, 1985Florio V.A. Sternweis P.C. J. Biol. Chem. 1985; 260: 3477-3483Abstract Full Text PDF PubMed Google Scholar). However, there is now a growing body of evidence demonstrating that Gβγ 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 Gα subunit (Yamane and Fung, 1993Yamane H.K. Fung B.K.-K. Annu. Rev. Pharmacol. Toxicol. 1993; 32: 201-241Crossref Scopus (56) Google Scholar), interaction with phosducin (Lee et al., 1987Lee R.H. Lieberman B.S. Lolley R.N. Biochemistry. 1987; 26: 3983-3990Crossref PubMed Scopus (154) Google Scholar, Lee et al., 1992bLee R.H. Ting T.D. Lieberman B.S. Tobias B.E. Lolley R.N. Ho Y.K. J. Biol. Chem. 1992; 267: 25104-25112Abstract Full Text PDF PubMed Google Scholar) and receptor kinase (Haga and Haga, 1992Haga K. Haga T. J. Biol. Chem. 1992; 267: 2222-2227Abstract Full Text PDF PubMed Google Scholar; Inglese et al., 1992Inglese J. Koch W.J. Caron M.G. Lefkowitz R.J. Nature. 1992; 359: 147-150Crossref PubMed Scopus (234) Google Scholar; Pitcher et al., 1992Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (573) Google Scholar), and regulation of the activities of effectors such as adenylate cyclase types II and IV, phospholipase A2, phospholipase C, and cardiac K+channels (Clapham and Neer, 1993Clapham D.E. Neer E.J. Nature. 1993; 365: 403-406Crossref PubMed Scopus (590) Google Scholar). The molecular mechanism by which the Gβγ complex regulates a diversity of signaling processes is still not fully understood. To date, five forms of Gβ (Watson et al., 1994Watson A.J. Katz A. Simon M.I. J. Biol. Chem. 1994; 269: 22150-22156Abstract Full Text PDF PubMed Google Scholar) and multiple forms of Gγ subunits have been identified by biochemical, immunological, and molecular cloning studies (Simon et al., 1991Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1591) Google Scholar). At the amino acid level, the Gβ subunits are highly conserved. In contrast, the Gγ subunits are more divergent. Because of the diversity of the Gγ sequences, it is generally believed that the Gγ subunit determines the functional specificity of the Gβγ complex. However, it is unclear what combinations of Gβ and Gγ subunits occur physiologically to account for the differences in their functions. The Gβγ complexes isolated from most tissue preparations are heterogeneous mixtures containing different forms of Gβ and Gγ subunits (Gautam et al., 1990Gautam N. Northup J. Tamir H. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7973-7977Crossref PubMed Scopus (97) Google Scholar). An exception to this is the cone Gβγ complex, which is composed of Gβ3and a novel Gγ subunit (Fung et al., 1992Fung B.K.-K. Lieberman B.S. Lee R.H. J. Biol. Chem. 1992; 267: 24782-24788Abstract Full Text PDF PubMed Google Scholar; Lee et al., 1992aLee R.H. Lieberman B.S. Yamane H.K. Bok D. Fung B.K.-K. J. Biol. Chem. 1992; 267: 24776-24781Abstract Full Text PDF PubMed Google Scholar). In this study, we report the isolation and expression of the cDNA of this Gγ subunit. We further show that this Gγ subunit is highly homologous to the Gγ1subunit of rod transducin (Hurley et al., 1984Hurley J.B. Fong H.K. Teplow D.B. Dreyer W.J. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6948-6952Crossref PubMed Scopus (118) Google Scholar; Yatsunami et al., 1985Yatsunami K. Pandya B.V. Oprian D.D. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1936-1940Crossref PubMed Scopus (54) Google Scholar) 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 Gγ subunit as Gγ8in this paper. We speculate that Gγ8may serve an important function in the regulation of phototransduction in cones. Retinal G protein (transducin) Gβγ complex was isolated from bovine retinas purchased from J. A. Lawson (Lincoln, NE) (Fung, 1983Fung B.K.-K. J. Biol. Chem. 1983; 258: 10495-10502Abstract Full Text PDF PubMed Google Scholar). The cone-specific Gβ3γ8complex was separated from rod-specific Gβ1γ1by subtractive chromatography using an affinity column of immobilized monoclonal antibodies against rod Gγ1as reported previously (Fung et al., 1992Fung B.K.-K. Lieberman B.S. Lee R.H. J. Biol. Chem. 1992; 267: 24782-24788Abstract Full Text PDF PubMed Google Scholar). 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. 1The abbreviations used are:PCRpolymerase chain reactionTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinePAGEpolyacrylamide gel electrophoresis. 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 Gγ8and stored in 40% glycerol at −20°C. polymerase chain reaction N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine polyacrylamide gel electrophoresis. Peptide CMAQELSEKELLKME, corresponding to residues 1-14 of the deduced amino acid sequence of Gγ8plus 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). The purified Gβ3γ8com-plex was separated on high resolution Tricine-polyacrylamide gels (Schägger and von Jagow, 1987Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar), transblotted onto Immobilon-P membranes (Millipore Corp.), and visualized by staining with Coomassie Blue (Matsudaira, 1987Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). The region of the membranes containing the Gγ8subunit was excised, cleaved with cyanogen bromide, and subjected to gas-phase protein sequence analysis at the Protein Sequencing Facility, University of California, Los Angeles. Degenerate PCR oligonucleotide primers corresponding to residues KKEVKN (5′-primer) and KGIPED (3′-primer) were designed from the partial amino acid sequence of Gγ8. 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 MgCl2, 0.001% gelatin), 1 μM degenerate primers, 50 μM dNTPs, and 0.5 μl (2.5 × 105plaque-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 Gγ8. A [32P]CTP-labeled mRNA probe was then synthesized from the vector and used to screen 3 × 105plaques at 50°C. The Bluescript plasmids containing the desired insert were rescued from the positive phagemid preparation according to the manufacturer's instructions (Stratagene). A fragment of Gγ8cDNA 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-β- D-thiogalactopyranoside induction and extraction of the Gγ8fusion 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 Gγ8fusion 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 CaCl2; and digested with Factor Xa for 4 h at room temperature at a protease/protein ratio of 1:60 (w/w). Immediately following proteolysis, Gγ8was further purified on a Superose-12 column. The resulting Gγ8is structurally similar to the native protein, except for the replacement of N-terminal MA residues with HMDLA residues. In vitro isoprenylation of recombinant Gγ8, 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 MgCl2, 0.5 m M dithiothreitol). The reaction mixture for farnesylation contained 1.5 μM purified protein, 2 μM [3H]farnesyl pyrophosphate (15 Ci/mmol; American Radiolabeled Chemicals), 20 μM geranylgeranyl pyrophosphate, and 4 μl of nuclease-treated rabbit reticulolysate (Promega). Geranylgeranylation was carried out under the same conditions, except that the [3H]farnesyl pyrophosphate and geranylgeranyl pyrophosphate were replaced with [3H]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 Gγ8, Rac2, or Ha-Ras were excised; digested in 20% H2O2at 65°C overnight; and analyzed by liquid scintillation counting. Total RNA from bovine retina was isolated by acid guanidinium thiocyanate/phenol/chloroform extraction (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). RNA from other frozen bovine tissues was prepared by guanidinium thiocyanate extraction and LiCl precipitation (Cathala et al., 1983Cathala G. Savouret J.F. Mendez B. West B.L. Karin M. Martial J.A. Baxter J.D. DNA (N. Y.). 1983; 2: 329-335Crossref PubMed Scopus (1229) Google Scholar), 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 Gγ8cDNA (106cpm/ml) and human Gα11cDNA (106cpm/ml) probes was carried out for 24 h at 38°C in buffer containing 50% formaldehyde, 5 × SSC, 50 m M NaH2PO4, 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. 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 A280 nm= 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. Protein concentrations were determined by Coomassie Blue binding (Bradford, 1976Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using γ-globulin as a standard. SDS-PAGE of proteins was performed by the method of Schägger and von Jagow, 1987Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar (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., 1979Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar. 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.). We have previously reported the purification of a cone Gβγ complex composed of Gβ3and an immunochemically distinct Gγ subunit (Fung et al., 1992Fung B.K.-K. Lieberman B.S. Lee R.H. J. Biol. Chem. 1992; 267: 24782-24788Abstract Full Text PDF PubMed Google Scholar; Lee et al., 1992aLee R.H. Lieberman B.S. Yamane H.K. Bok D. Fung B.K.-K. J. Biol. Chem. 1992; 267: 24776-24781Abstract Full Text PDF PubMed Google Scholar). Furthermore, analysis of this cone Gγ peptide obtained by cyanogen bromide cleavage revealed an internal amino acid sequence that differs from all other known Gγ subunits. To obtain the complete sequence of cone Gγ, 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 Gγ. 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 Gγ. This newly identified Gγ was subsequently named Gγ8. A comparison of the deduced amino acid sequence of Gγ8and other known sequences of Gγ subunits is shown in Fig. 2. As expected for a cone G protein subunit, Gγ8shares the strongest sequence identity (68%) with Gγ1of 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 Gγ8. In contrast, Gγ8exhibits a lesser degree of sequence identity to other forms of Gγ subunits, ranging from 30% (for Gγ5) to 39% (for Gγ2and Gγ7). Like other members of the family, the Gγ8subunit terminates with a C XXX motif (where C = cysteine and X = any amino acid), which has been shown in Gγ1(Fukada et al., 1990Fukada Y. Takao T. Ohguro H. Yoshizawa T. Akino T. Shimonishi Y. Nature. 1990; 346: 658-660Crossref PubMed Scopus (272) Google Scholar; Lai et al., 1990Lai R.K. Perez-Sala D. Canada F.J. Rando R.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7673-7677Crossref PubMed Scopus (178) Google Scholar; Fung et al., 1994Fung B.K.-K. Anant J.S. Lin W.-C. Ong O.C. Yamane H.K. Methods Enzymol. 1994; 237: 509-519Crossref PubMed Scopus (2) Google Scholar) and brain Gγ (Mumby et al., 1990Mumby S.M. Casey P.J. Gilman A.G. Gutowski S. Sternweis P.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5873-5877Crossref PubMed Scopus (201) Google Scholar; Yamane et al., 1990Yamane H.K. Farnsworth C.C. Xie H.Y. Howald W. Fung B.K. Clarke S. Gelb M.H. Glomset J.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5868-5872Crossref PubMed Scopus (202) Google Scholar) to be the signal sequence for multiple post-translational modifications, including isoprenylation, proteolysis, and carboxyl methylation. To examine the tissue distribution of the Gγ8mRNA, 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 Gγ8probe 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 Gγ8message. When the same blot was hybridized to the probe derived from human Gα11cDNA as a control (Jiang et al., 1991Jiang M. Pandey S. Tran V.T. Fong H.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3907-3911Crossref PubMed Scopus (28) Google Scholar), a 3.8-kilobase transcript of Gα11mRNA was readily detected in all preparations of tissue poly(A)+RNA (Fig. 3 B). This result indicates that mRNA of Gγ8is predominantly expressed in retinas. The presence of a CIIS motif at the C terminus of Gγ8suggests that it may be post-translationally modified by isoprenylation. To explore this possibility, we determined the ability of recombinant Gγ8to incorporate radiolabeled isoprenoid groups. Recombinant Gγ8containing 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 Gγ8generated 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 Gγ8was carried out in rabbit reticulolysate in the presence of either [3H]farnesyl pyrophosphate or [3H]geranylgeranyl pyrophosphate. Recombinant Ha-Ras and Rac2 proteins, previously shown to be farnesylated and geranylgeranylated, respectively (Casey et al., 1989Casey P.J. Solski P.A. Der C.J. Buss J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8323-8327Crossref PubMed Scopus (780) Google Scholar; Kinsella et al., 1991aKinsella B.T. Erdman R.A. Maltese W.A. J. Biol. Chem. 1991; 266: 9786-9794Abstract Full Text PDF PubMed Google Scholar), were used as controls to test the labeling reaction. Compared with [3H]geranylgeranyl pyrophosphate, incubation in the presence of [3H]farnesyl pyrophosphate results in the incorporation of 8.3- and 23-fold higher levels of radioactivity into Gγ8and Ha-Ras, respectively (Table I). The lower efficiency in labeling of Gγ8relative to Ha-Ras may be due to dimerization of the Gγ8subunit in the absence of the Gβ3subunit (see Fig. 4 and below). In contrast, the labeling of Rac2 protein with a CSLL motif is 63-fold greater in the presence of [3H]geranylgeranyl pyrophosphate compared with [3H]farnesyl pyrophosphate. Taken together, these results strongly suggest that Gγ8is modified by farnesylation. It is noteworthy that Gγ1, which is highly homologous to Gγ8, is also farnesylated (Fukada et al., 1990Fukada Y. Takao T. Ohguro H. Yoshizawa T. Akino T. Shimonishi Y. Nature. 1990; 346: 658-660Crossref PubMed Scopus (272) Google Scholar; Lai et al., 1990Lai R.K. Perez-Sala D. Canada F.J. Rando R.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7673-7677Crossref PubMed Scopus (178) Google Scholar; Fung et al., 1994Fung B.K.-K. Anant J.S. Lin W.-C. Ong O.C. Yamane H.K. Methods Enzymol. 1994; 237: 509-519Crossref PubMed Scopus (2) Google Scholar). On the other hand, geranylgeranylation has been shown to occur in Gγ2, a less homologous form found in the brain (Mumby et al., 1990Mumby S.M. Casey P.J. Gilman A.G. Gutowski S. Sternweis P.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5873-5877Crossref PubMed Scopus (201) Google Scholar; Yamane et al., 1990Yamane H.K. Farnsworth C.C. Xie H.Y. Howald W. Fung B.K. Clarke S. Gelb M.H. Glomset J.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5868-5872Crossref PubMed Scopus (202) Google Scholar). 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., 1991bKinsella B.T. Erdman R.A. Maltese W.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8934-8938Crossref PubMed Scopus (72) Google Scholar; Reiss et al., 1991Reiss Y. Stradley S.J. Gierasch L.M. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 732-736Crossref PubMed Scopus (311) Google Scholar). In agreement with these findings, both Gγ1and Gγ8, 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 Gγ subunits.Table I:Isoprenylation of Gγ8Table I:Isoprenylation of Gγ8 Specific antiserum was generated against the synthetic peptide corresponding to residues 1-14 of the deduced amino acid sequence of Gγ8. To test the specificity of the antiserum, we purified bovine retinal rod-specific Gβ1γ and cone-specific Gβ3γ 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 Gγ subunit from the Gβ3γ preparation (lane 1), but not the Gγ subunit from the purified Gβ1γ complex (lane 2). The antiserum also recognizes recombinant Gγ8expressed in E. coli, which was used in the isoprenylation experiments. As shown in the immunoblot in Fig. 4B, the Gγ8fusion product containing the N-terminal histidine-rich residues (lane 3) as well as Gγ8generated 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 Gγ8after the histidine-tagged residues were removed. To investigate whether Gγ8is specifically localized to cone photoreceptors, bovine retinal sections were stained with antiserum raised against the N-terminal peptide of Gγ8. 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 Gγ8is specifically localized in cone photoreceptors within the retina and raise the possibility that it is the Gγ subunit of cone transducin involved in phototransduction and color vision. 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, 1986Pugh Jr., E. Cobbs W.H. Vision Res. 1986; 26: 1613-1643Crossref PubMed Scopus (122) Google Scholar). Compared with rods, cones are 100-fold less sensitive to light, but display a faster and shorter response (Pugh and Cobbs, 1986Pugh Jr., E. Cobbs W.H. Vision Res. 1986; 26: 1613-1643Crossref PubMed Scopus (122) Google Scholar; Nakatani and Yau, 1986Nakatani K. Yau K.-W. Invest. Ophthalmol. & Visual Sci. 1986; 27: 300Google Scholar). 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, 1994Yau K.W. Invest. Ophthalmol. Vision Sci. 1994; 35: 9-32PubMed Google Scholar). In rods, a retina-specific G protein (transducin) couples the photoexcitation of rhodopsin to the activation of retinal cGMP phosphodiesterase (Fung, 1986Fung B.K.-K. Prog. Retinal Res. 1986; 6: 151-177Crossref Scopus (27) Google Scholar; Lolley and Lee, 1990Lolley R.N. Lee R.H. FASEB J. 1990; 4: 3001-3008Crossref PubMed Scopus (48) Google Scholar; Stryer, 1991Stryer L. J. Biol. Chem. 1991; 266: 10711-10714Abstract Full Text PDF PubMed Google Scholar). 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, 1986Pugh Jr., E. Cobbs W.H. Vision Res. 1986; 26: 1613-1643Crossref PubMed Scopus (122) Google Scholar). 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, 1985Haynes L. Yau K.W. Nature. 1985; 317: 61-64Crossref PubMed Scopus (155) Google Scholar; Cobbs et al., 1985Cobbs W.H. Barkdoll III, A.E. Pugh Jr., E. Nature. 1985; 317: 64-66Crossref PubMed Scopus (76) Google Scholar). 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, 1986Nakatani K. Yau K.-W. Invest. Ophthalmol. & Visual Sci. 1986; 27: 300Google Scholar). At the molecular level, the cone-specific Gα subunit (Lerea et al., 1986Lerea C.L. Somers D.E. Hurley J.B. Klock I.B. Bunt-Milam A.H. Science. 1986; 234: 77-80Crossref PubMed Scopus (171) Google Scholar), cGMP phosphodiesterase (Gillespie and Beavo, 1988Gillespie P.G. Beavo J.A. J. Biol. Chem. 1988; 263: 8133-8141Abstract Full Text PDF PubMed Google Scholar; Charbonneau et al., 1990Charbonneau H. Prusti R.K. LeTrong H. Sonnenburg W.K. Mullaney P.J. Walsh K.A. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 288-292Crossref PubMed Scopus (103) Google Scholar; Li et al., 1990Li T.S. Volpp K. Applebury M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 293-297Crossref PubMed Scopus (98) Google Scholar), and the cGMP-regulated channel (Bonigk et al., 1993Bonigk W. Altenhofen W. Muller F. Dose A. Illing M. Molday R.S. Kaupp U.B. Neuron. 1993; 10: 865-877Abstract Full Text PDF PubMed Scopus (161) Google Scholar) have been identified, and a Gβγ complex composed of Gβ3and an immunochemically distinct Gγ subunit has also been purified from bovine retinal cones (Fung et al., 1992Fung B.K.-K. Lieberman B.S. Lee R.H. J. Biol. Chem. 1992; 267: 24782-24788Abstract Full Text PDF PubMed Google Scholar; Lee et al., 1992aLee R.H. Lieberman B.S. Yamane H.K. Bok D. Fung B.K.-K. J. Biol. Chem. 1992; 267: 24776-24781Abstract Full Text PDF PubMed Google Scholar). 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 Gγ subunit that associates with Gβ3in retinal cones, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding Gγ8. The predicted protein sequence for Gγ8suggests that it represents a new isoform of the Gγ subunit family. Sequence comparison further reveals that Gγ8shares a relatively higher degree of homology with Gγ1(68% sequence identity) than with other known isoforms of Gγ subunits (ranging from 30 to 39% identity). The similarity in structure between Gγ8and Gγ1strongly implies that, like Gγ1in rods, Gγ8may play a role in cone phototransduction. To address the question of whether Gγ8is differentially localized to one type of photoreceptor in the retina, we generated a rabbit antipeptide antibody against the N-terminal region of Gγ8and performed immunocytochemistry on the sections of bovine retina. The result shows that Gγ8, like its molecular partner Gβ3, is present only in cones. Our immunostaining data also show a distinct distribution of Gγ8in the outer segments, inner segments, cell bodies, axons, and synaptic terminals, with the highest staining intensity in the outer segments. This immunostaining pattern of Gγ8in cones is indistinguishable from the staining pattern obtained by using a Gβ3-specific antipeptide antibody (Lee et al., 1992aLee R.H. Lieberman B.S. Yamane H.K. Bok D. Fung B.K.-K. J. Biol. Chem. 1992; 267: 24776-24781Abstract Full Text PDF PubMed Google Scholar), suggesting that Gβ3and Gγ8form a physiologically functional complex in all of these regions in cones. Peng et al., 1992Peng Y.-W. Robishaw J.D. Levine M.A. Yau K.-W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10882-10886Crossref PubMed Scopus (92) Google Scholar have previously reported that antipeptide antibodies directed against peptides corresponding to residues 2-14 and 36-46 of Gγ2also stained exclusively the cone outer segment, suggesting that Gγ2is also localized in cones. A comparison of the deduced amino acid sequences of Gγ8and Gγ2eliminates the possibility that these antibodies simply cross-reacted with Gγ8. Since Gβ1and Gβ2are both absent in the cone outer segment (Lee et al., 1992aLee R.H. Lieberman B.S. Yamane H.K. Bok D. Fung B.K.-K. J. Biol. Chem. 1992; 267: 24776-24781Abstract Full Text PDF PubMed Google Scholar; Peng et al., 1992Peng Y.-W. Robishaw J.D. Levine M.A. Yau K.-W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10882-10886Crossref PubMed Scopus (92) Google Scholar) and Gβ3and Gγ2do not form a complex in transfected cells (Pronin and Gautam, 1992Pronin A.N. Gautam N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6220-6224Crossref PubMed Scopus (128) Google Scholar), it seems possible that Gγ2may be associated with other forms of Gβ subunits (von Weizsacker et al., 1992von Weizsacker E. Strathmann M.P. Simon M.I. Biochem. Biophys. Res. Commun. 1992; 183: 350-356Crossref PubMed Scopus (78) Google Scholar; Watson et al., 1994Watson A.J. Katz A. Simon M.I. J. Biol. Chem. 1994; 269: 22150-22156Abstract Full Text PDF PubMed Google Scholar). Similar to the Gγ1subunit of rod transducin, Gγ8is found to be post-translationally modified by farnesylation. This is in contrast to the Gγ subunits of the brain G proteins, which are modified by geranylgeranylation (Mumby et al., 1990Mumby S.M. Casey P.J. Gilman A.G. Gutowski S. Sternweis P.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5873-5877Crossref PubMed Scopus (201) Google Scholar; Yamane et al., 1990Yamane H.K. Farnsworth C.C. Xie H.Y. Howald W. Fung B.K. Clarke S. Gelb M.H. Glomset J.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5868-5872Crossref PubMed Scopus (202) Google Scholar). Since the farnesyl groups are less hydrophobic than geranylgeranyl groups, farnesylated Gγ8is likely to associate only loosely with the disc membranes. Consistent with this finding, the Gβ3γ complex can be readily eluted from cone membranes at low ionic strength (Lee et al., 1992aLee R.H. Lieberman B.S. Yamane H.K. Bok D. Fung B.K.-K. J. Biol. Chem. 1992; 267: 24776-24781Abstract Full Text PDF PubMed Google Scholar), and the majority of the protein was found in the supernatant during isolation. In contrast, brain Gβγ protein containing a more hydrophobic geranylgeranyl group can be extracted only in the presence of detergents (Eide et al., 1987Eide B. Gierschik P. Milligan G. Mullaney I. Unson C. Goldsmith P. Spiegel A. Biochem. Biophys. Res. Commun. 1987; 148: 1398-1405Crossref PubMed Scopus (31) Google Scholar). In addition to membrane anchorage, the isoprenyl group of the Gγ subunit may have other biological functions. In the rod phototransduction pathway, the carboxyl-terminal peptides of Gγ1containing a chemically attached farnesyl group appear to stabilize metarhodospin II and to uncouple rhodopsin-transducin interaction (Kisselev et al., 1994Kisselev O.G. Ermolaeva M.V. Gautam N. J. Biol. Chem. 1994; 269: 21399-21402Abstract Full Text PDF PubMed Google Scholar). Methylation of the farnesyl cysteine in Gγ1also facilitates the interaction of transducin with rhodopsin (Fukada et al., 1994Fukada Y. Matsuda T. Kokame K. Takao T. Shimonishi Y. Akino T. Yoshizawa T. J. Biol. Chem. 1994; 269: 5163-5170Abstract Full Text PDF PubMed Google Scholar). Since all other Gγ subunits except those in photoreceptors are shown or predicted to be modified by geranylgeranylation, we speculate that the farnesylation of Gγ8may indicate a specific functional requirement for the phototransduction pathway in cones. With the cloning of the Gγ8subunit 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. 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." @default.
• W2003248941 created "2016-06-24" @default.
• W2003248941 creator A5000506767 @default.
• W2003248941 creator A5016826288 @default.
• W2003248941 creator A5039947475 @default.
• W2003248941 creator A5078119102 @default.
• W2003248941 creator A5087007675 @default.
• W2003248941 creator A5087387856 @default.
• W2003248941 creator A5089313784 @default.
• W2003248941 date "1995-04-01" @default.
• W2003248941 modified "2023-10-16" @default.
• W2003248941 title "Molecular Cloning and Characterization of the G Protein γ Subunit of Cone Photoreceptors" @default.
• W2003248941 cites W1482237901 @default.
• W2003248941 cites W1482911857 @default.
• W2003248941 cites W1490670694 @default.
• W2003248941 cites W1503310668 @default.
• W2003248941 cites W1509626830 @default.
• W2003248941 cites W1514240524 @default.
• W2003248941 cites W1520928812 @default.
• W2003248941 cites W1527306897 @default.
• W2003248941 cites W1550872707 @default.
• W2003248941 cites W1554470867 @default.
• W2003248941 cites W1566050966 @default.
• W2003248941 cites W1593983451 @default.
• W2003248941 cites W1702339793 @default.
• W2003248941 cites W1869250499 @default.
• W2003248941 cites W1932052686 @default.
• W2003248941 cites W1964667616 @default.
• W2003248941 cites W1965404955 @default.
• W2003248941 cites W1966276097 @default.
• W2003248941 cites W1972383232 @default.
• W2003248941 cites W1972760976 @default.
• W2003248941 cites W1975048596 @default.
• W2003248941 cites W1975085160 @default.
• W2003248941 cites W1977118713 @default.
• W2003248941 cites W1986904952 @default.
• W2003248941 cites W2001768785 @default.
• W2003248941 cites W2016002808 @default.
• W2003248941 cites W2017131142 @default.
• W2003248941 cites W2030886393 @default.
• W2003248941 cites W2036405416 @default.
• W2003248941 cites W2045253660 @default.
• W2003248941 cites W2049480397 @default.
• W2003248941 cites W2056221479 @default.
• W2003248941 cites W2058729896 @default.
• W2003248941 cites W2060264934 @default.
• W2003248941 cites W2065664139 @default.
• W2003248941 cites W2075398040 @default.
• W2003248941 cites W2076780529 @default.
• W2003248941 cites W2080875376 @default.
• W2003248941 cites W2080981029 @default.
• W2003248941 cites W2083737798 @default.
• W2003248941 cites W2085801794 @default.
• W2003248941 cites W2086617442 @default.
• W2003248941 cites W2089906643 @default.
• W2003248941 cites W2099375764 @default.
• W2003248941 cites W2101108802 @default.
• W2003248941 cites W2104529835 @default.
• W2003248941 cites W2115588325 @default.
• W2003248941 cites W2131547839 @default.
• W2003248941 cites W2138476193 @default.
• W2003248941 cites W2179950846 @default.
• W2003248941 cites W2423782813 @default.
• W2003248941 cites W33975413 @default.
• W2003248941 cites W4293247451 @default.
• W2003248941 cites W4294216491 @default.
• W2003248941 doi "https://doi.org/10.1074/jbc.270.15.8495" @default.
• W2003248941 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7721746" @default.
• W2003248941 hasPublicationYear "1995" @default.
• W2003248941 type Work @default.
• W2003248941 sameAs 2003248941 @default.
• W2003248941 citedByCount "82" @default.
• W2003248941 countsByYear W20032489412012 @default.
• W2003248941 countsByYear W20032489412013 @default.
• W2003248941 countsByYear W20032489412014 @default.
• W2003248941 countsByYear W20032489412015 @default.
• W2003248941 countsByYear W20032489412016 @default.
• W2003248941 countsByYear W20032489412018 @default.
• W2003248941 countsByYear W20032489412022 @default.
• W2003248941 crossrefType "journal-article" @default.
• W2003248941 hasAuthorship W2003248941A5000506767 @default.
• W2003248941 hasAuthorship W2003248941A5016826288 @default.
• W2003248941 hasAuthorship W2003248941A5039947475 @default.
• W2003248941 hasAuthorship W2003248941A5078119102 @default.
• W2003248941 hasAuthorship W2003248941A5087007675 @default.
• W2003248941 hasAuthorship W2003248941A5087387856 @default.
• W2003248941 hasAuthorship W2003248941A5089313784 @default.
• W2003248941 hasBestOaLocation W20032489411 @default.
• W2003248941 hasConcept C104292427 @default.
• W2003248941 hasConcept C104317684 @default.
• W2003248941 hasConcept C121050878 @default.
• W2003248941 hasConcept C167625842 @default.
• W2003248941 hasConcept C171250308 @default.
• W2003248941 hasConcept C185592680 @default.
• W2003248941 hasConcept C192562407 @default.
• W2003248941 hasConcept C19924922 @default.
• W2003248941 hasConcept C199360897 @default.
• W2003248941 hasConcept C2780841128 @default.

• next