Proc. Nati. Acad. Sci. USA Vol. 88, pp. 3907-3911, May 1991 Cell Biology
Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells (signal transduction/photoreceptor/pertussis toxin)
MEISHENG JIANG*, SUJAY PANDEYt, VINH T. TRANt,
AND
HENRY K. W. FONG**§
Departments of *Microbiology and tOphthalmology, University of Southern California School of Medicine, Los Angeles, CA 90033; and tDoheny Eye Institute, Los Angeles, CA 90033
Communicated by Gordon H. Sato, January 25, 1991 (received for review October 31, 1990)
ABSTRACT The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein a subunits (Ga) including Gsa, G.1la, Gi.2a, Gi.3a, and Gza (or Gxa), where G, and G1 are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensitive events. Other Ga-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to Gi.2a and Gsa protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the Ga gene family, at least two other Ga subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional Gca subunits were isolated and characterized. The results indicate that this Ga subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.
The retinal pigment epithelium (RPE) forms a highly differentiated monolayer of polarized cells situated between the photoreceptors and the choroidal capillaries. The RPE controls transport of ions, nutrients, macromolecules, and fluid between the blood supply and the photoreceptors (1, 2). It is vital in maintaining the visual process by removing through phagocytosis the shed tips of photoreceptor outer segments (3), and it plays an essential role in the regeneration of the visual pigments (4). Conversion of the chromophore alltrans-retinal to 11-cis-retinal by retinoid isomerase occurs in the RPE, which also stores vitamin A as retinyl esters (5, 6). Phagocytosis and shedding of photoreceptor outer segments are associated with a diurnal cycle (7, 8). These regulatory functions of the RPE, if perturbed, may lead to degeneration of the photoreceptors and to.a variety of visual disorders, such that the two cell types, RPE and photoreceptor, can be considered a functional interactive unit. The molecular regulation of RPE function is not well understood. It has been reported that cAMP in RPE cells affects ion transport and transepithelial fluid movement (9, 10). RPE cells have been shown to possess muscarinic, adrenergic, and other pharmacologic receptors (11-16). Acetylcholine or carbachol increases cytoplasmic free Ca2+ in human RPE cells (11), and 8-adrenergic agonists stimulate intracellular cAMP (12, 13). By linking membrane surface receptors to effectors for intracellular signaling, GTP-binding regulatory proteins (G proteins) are involved in regulation of a remarkable variety of biological processes, including phototransduction, olfaction, neurotransmission, secretion, chemotaxis, cell proliferation, The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
3907
and cell differentiation (17, 18). In studying G proteinmediated signal transduction and regulation in RPE, we have analyzed the expression of G protein a subunits (Ga) in cultured RPE cells using molecular genetic techniques. Here we describe also the cloning of an additional Ga that may transduce signals via a pertussis toxin-insensitive mechanism.
MATERIALS AND METHODS RPE Cell Cultures. Human adult RPE cell cultures were provided by Nino Sorgente (Doheny Eye Institute). Human fetal RPE cell cultures, RPE-30 (repository no. AG05338), were obtained from the Coriell Institute for Medical Research (Camden, NJ). Bovine RPE cells were isolated and cultured as described (19). RNA Blot Hybridization. RNA samples were isolated, size-fractionated by agarose/formaldehyde gel electrophoresis, transferred directly to nylon membranes, and hybridized to radiolabeled probes as described (20). Hybridization under standard conditions was carried out at 420C in buffer containing 50% formamide, 5 x SSC, 50 mM NaH2PO4 (pH 7.0), 2x Denhardt's solution, 0.1% SDS, and 50 gg of denatured salmon sperm DNA per ml (1 x SSC = 0.15 M NaCl/15 mM sodium citrate; and 1x Denhardt's solution = 0.02% bovine serum albumin/0.02% polyvinylpyrrolidone/0.02% Ficoll). The filters were washed as indicated in the figure legends. Hybridization Probes. Complete protein-coding cDNA probes for rat Ga species that mediate stimulation (Gs) and inhibition (G1) of adenylyl cyclase-specifically Gsa, Gi-1a, Gi-2a, and Gi-3a-subcloned in the pGEM-2 plasmid, were provided by David T. Jones and Randall R. Reed (Johns Hopkins University School of Medicine, Baltimore, MD). The human Gza probe, a 1.2-kilobase (kb) cDNA fragment subcloned in pBluescript, consisted of the 3' untranslated region of human Gza cDNA from the Nae I site at nucleotide 1491 to the poly(A) tract ending at nucleotide 2684, as numbered by Fong et al. (21); Gza is a Ga that may mediate pertussis toxin-insensitive events (also called Gxa). The bovine s, subunit cDNA has been described (22). A complete protein-coding cDNA probe for human Gi-2a was isolated from a human retinal cDNA library. The cDNA plasmid probes were labeled with [a-32P]dATP by nick-translation. cDNA Amplification by the Polymerase Chain Reaction (PCR). First-strand cDNA was synthesized in 100-,l reaction mixtures containing 10-30 ,g of total RNA, 20 mM Tris HCl (pH 8.4), 50 mM KCI, 2.5 mM MgCl2, 0.5 mM dithiothreitol, 50 ,ug of bovine serum albumin per ml, 4 mM sodium Abbreviations: G protein, GTP-binding regulatory protein; Ga, G protein a subunit(s); G, and G0, G proteins that mediate stimulation and inhibition of adenylyl cyclase, respectively; Gz, a G protein that may mediate pertussis toxin-insensitive events; RPE, retinal pigment epithelium; PCR, polymerase chain reaction. §To whom reprint requests should be addressed.
3908
Cell
Biology: Jiang et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
pyrophosphate, 40 units of RNasin ribonuclease inhibitor (Promega), 250 ,uM of each dNTP, S Ag of oligo(dT)12.18 primer, and 40 units of AMV reverse transcriptase. The first-strand cDNA was passed through a Sephadex G-50 column equilibrated in 20 mM Tris1HCl, pH 8.4/50 mM KCI; 20-40o of the cDNA sample was used for amplification, which was performed in a 100-lI reaction mixture containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100, 200 ttM of each dNTP, 5 Ag each of the primers aA and aC, and 5 units of Thermus aquaticus (Taq) polymerase (Promega). The reaction was carried out in steps of 1 min at 94°C, 2 min at 55°C, and 3 min at 72°C for 40 cycles in an Eppendorf microcycler (Eppendort). The primer aA consists of a mixture of 64 oligonucleotides, each 31 nucleotides in length (GGAATTCAARAGYACYATYGTSAARCAGATG), and primer aC consists of a mixture of 128 oligonucleotides, each 30 nucleotides in length (GGAGCTCYCSTYRAARCASTGRATCCACTT), where Y is T or C, S is G or C, and R is G or A. A restriction site was incorporated in the oligonucleotides to facilitate subcloning. Isolation of cDNA Clones and DNA Sequencing. Clones were isolated from a human retinal AgtlO cDNA library that was provided by Jeremy Nathans (Johns Hopkins University School of Medicine). Isolated cDNA inserts were subcloned into the pBluescript vector, and DNA sequencing was performed with single-stranded DNA by the dideoxynucleotide chain-termination method as described (20).
RESULTS Expression of G Protein mRNA in RPE Cells. G protein mRNA in human RPE cells was analyzed by RNA blot hybridizations using cDNA probes for known members of the G protein family. Total RNA preparations from RPE cell cultures and, for comparison, from brain tissue were hybridized to a 61 subunit and to various Ga cDNA probes. Like many other cell types with diverse functions, the RPE cells were found to express multiple Ga subunits (Figs. 1-3). Adult RPE cells contain mRNA for Gsa, G-1a, Gi-2a, Gi-3a, and Gza. For each Ga subunit, the probes hybridized to one or more mRNA transcripts that were of the expected size consistent with results of published reports (23). Fetal RPE cells also express mRNA for Gsa, G, 2a, Gi3a, and Gza. Although G1j1a mRNA transcripts were expressed in adult
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Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells (signal transduction/photoreceptor/pertussis toxin)
MEISHENG JIANG*, SUJAY PANDEYt, VINH T. TRANt,
AND
HENRY K. W. FONG**§
Departments of *Microbiology and tOphthalmology, University of Southern California School of Medicine, Los Angeles, CA 90033; and tDoheny Eye Institute, Los Angeles, CA 90033
Communicated by Gordon H. Sato, January 25, 1991 (received for review October 31, 1990)
ABSTRACT The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein a subunits (Ga) including Gsa, G.1la, Gi.2a, Gi.3a, and Gza (or Gxa), where G, and G1 are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensitive events. Other Ga-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to Gi.2a and Gsa protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the Ga gene family, at least two other Ga subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional Gca subunits were isolated and characterized. The results indicate that this Ga subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.
The retinal pigment epithelium (RPE) forms a highly differentiated monolayer of polarized cells situated between the photoreceptors and the choroidal capillaries. The RPE controls transport of ions, nutrients, macromolecules, and fluid between the blood supply and the photoreceptors (1, 2). It is vital in maintaining the visual process by removing through phagocytosis the shed tips of photoreceptor outer segments (3), and it plays an essential role in the regeneration of the visual pigments (4). Conversion of the chromophore alltrans-retinal to 11-cis-retinal by retinoid isomerase occurs in the RPE, which also stores vitamin A as retinyl esters (5, 6). Phagocytosis and shedding of photoreceptor outer segments are associated with a diurnal cycle (7, 8). These regulatory functions of the RPE, if perturbed, may lead to degeneration of the photoreceptors and to.a variety of visual disorders, such that the two cell types, RPE and photoreceptor, can be considered a functional interactive unit. The molecular regulation of RPE function is not well understood. It has been reported that cAMP in RPE cells affects ion transport and transepithelial fluid movement (9, 10). RPE cells have been shown to possess muscarinic, adrenergic, and other pharmacologic receptors (11-16). Acetylcholine or carbachol increases cytoplasmic free Ca2+ in human RPE cells (11), and 8-adrenergic agonists stimulate intracellular cAMP (12, 13). By linking membrane surface receptors to effectors for intracellular signaling, GTP-binding regulatory proteins (G proteins) are involved in regulation of a remarkable variety of biological processes, including phototransduction, olfaction, neurotransmission, secretion, chemotaxis, cell proliferation, The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
3907
and cell differentiation (17, 18). In studying G proteinmediated signal transduction and regulation in RPE, we have analyzed the expression of G protein a subunits (Ga) in cultured RPE cells using molecular genetic techniques. Here we describe also the cloning of an additional Ga that may transduce signals via a pertussis toxin-insensitive mechanism.
MATERIALS AND METHODS RPE Cell Cultures. Human adult RPE cell cultures were provided by Nino Sorgente (Doheny Eye Institute). Human fetal RPE cell cultures, RPE-30 (repository no. AG05338), were obtained from the Coriell Institute for Medical Research (Camden, NJ). Bovine RPE cells were isolated and cultured as described (19). RNA Blot Hybridization. RNA samples were isolated, size-fractionated by agarose/formaldehyde gel electrophoresis, transferred directly to nylon membranes, and hybridized to radiolabeled probes as described (20). Hybridization under standard conditions was carried out at 420C in buffer containing 50% formamide, 5 x SSC, 50 mM NaH2PO4 (pH 7.0), 2x Denhardt's solution, 0.1% SDS, and 50 gg of denatured salmon sperm DNA per ml (1 x SSC = 0.15 M NaCl/15 mM sodium citrate; and 1x Denhardt's solution = 0.02% bovine serum albumin/0.02% polyvinylpyrrolidone/0.02% Ficoll). The filters were washed as indicated in the figure legends. Hybridization Probes. Complete protein-coding cDNA probes for rat Ga species that mediate stimulation (Gs) and inhibition (G1) of adenylyl cyclase-specifically Gsa, Gi-1a, Gi-2a, and Gi-3a-subcloned in the pGEM-2 plasmid, were provided by David T. Jones and Randall R. Reed (Johns Hopkins University School of Medicine, Baltimore, MD). The human Gza probe, a 1.2-kilobase (kb) cDNA fragment subcloned in pBluescript, consisted of the 3' untranslated region of human Gza cDNA from the Nae I site at nucleotide 1491 to the poly(A) tract ending at nucleotide 2684, as numbered by Fong et al. (21); Gza is a Ga that may mediate pertussis toxin-insensitive events (also called Gxa). The bovine s, subunit cDNA has been described (22). A complete protein-coding cDNA probe for human Gi-2a was isolated from a human retinal cDNA library. The cDNA plasmid probes were labeled with [a-32P]dATP by nick-translation. cDNA Amplification by the Polymerase Chain Reaction (PCR). First-strand cDNA was synthesized in 100-,l reaction mixtures containing 10-30 ,g of total RNA, 20 mM Tris HCl (pH 8.4), 50 mM KCI, 2.5 mM MgCl2, 0.5 mM dithiothreitol, 50 ,ug of bovine serum albumin per ml, 4 mM sodium Abbreviations: G protein, GTP-binding regulatory protein; Ga, G protein a subunit(s); G, and G0, G proteins that mediate stimulation and inhibition of adenylyl cyclase, respectively; Gz, a G protein that may mediate pertussis toxin-insensitive events; RPE, retinal pigment epithelium; PCR, polymerase chain reaction. §To whom reprint requests should be addressed.
3908
Cell
Biology: Jiang et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
pyrophosphate, 40 units of RNasin ribonuclease inhibitor (Promega), 250 ,uM of each dNTP, S Ag of oligo(dT)12.18 primer, and 40 units of AMV reverse transcriptase. The first-strand cDNA was passed through a Sephadex G-50 column equilibrated in 20 mM Tris1HCl, pH 8.4/50 mM KCI; 20-40o of the cDNA sample was used for amplification, which was performed in a 100-lI reaction mixture containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100, 200 ttM of each dNTP, 5 Ag each of the primers aA and aC, and 5 units of Thermus aquaticus (Taq) polymerase (Promega). The reaction was carried out in steps of 1 min at 94°C, 2 min at 55°C, and 3 min at 72°C for 40 cycles in an Eppendorf microcycler (Eppendort). The primer aA consists of a mixture of 64 oligonucleotides, each 31 nucleotides in length (GGAATTCAARAGYACYATYGTSAARCAGATG), and primer aC consists of a mixture of 128 oligonucleotides, each 30 nucleotides in length (GGAGCTCYCSTYRAARCASTGRATCCACTT), where Y is T or C, S is G or C, and R is G or A. A restriction site was incorporated in the oligonucleotides to facilitate subcloning. Isolation of cDNA Clones and DNA Sequencing. Clones were isolated from a human retinal AgtlO cDNA library that was provided by Jeremy Nathans (Johns Hopkins University School of Medicine). Isolated cDNA inserts were subcloned into the pBluescript vector, and DNA sequencing was performed with single-stranded DNA by the dideoxynucleotide chain-termination method as described (20).
RESULTS Expression of G Protein mRNA in RPE Cells. G protein mRNA in human RPE cells was analyzed by RNA blot hybridizations using cDNA probes for known members of the G protein family. Total RNA preparations from RPE cell cultures and, for comparison, from brain tissue were hybridized to a 61 subunit and to various Ga cDNA probes. Like many other cell types with diverse functions, the RPE cells were found to express multiple Ga subunits (Figs. 1-3). Adult RPE cells contain mRNA for Gsa, G-1a, Gi-2a, Gi-3a, and Gza. For each Ga subunit, the probes hybridized to one or more mRNA transcripts that were of the expected size consistent with results of published reports (23). Fetal RPE cells also express mRNA for Gsa, G, 2a, Gi3a, and Gza. Although G1j1a mRNA transcripts were expressed in adult
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