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• W2076644311 abstract "The G protein-coupled receptor kinases (GRKs) are critical enzymes in the desensitization of activated G protein-coupled receptors. Six members of the GRK family have been identified to date. Among these enzymes, GRK1 (rhodopsin kinase) is involved in phototransduction and is the most specialized of the family. GRK1 phosphorylates photoactivated rhodopsin, initiating steps in its deactivation. In this study, we found that human retina expressed all GRKs except GRK4. Based on results of molecular cloning and immunolocalization, it appears that both rod and cone photoreceptors express GRK1. This conclusion was supported by the cloning of only GRK1 from cone-dominated chicken retina. Human photoreceptors also transcribe a splice variant of GRK1, which differs in its C-terminal region next to the catalytic domain. This novel variant, GRK1b, is produced by retention of the last intron. mRNA encoding GRK1b is exported to the cytosol; however, the level of the protein is relatively low compared with GRK1 (now called GRK1a), and GRK1b appears to have very low catalytic activity. Thus, these studies suggest that rods and cones, express the same form of GRK1. The G protein-coupled receptor kinases (GRKs) are critical enzymes in the desensitization of activated G protein-coupled receptors. Six members of the GRK family have been identified to date. Among these enzymes, GRK1 (rhodopsin kinase) is involved in phototransduction and is the most specialized of the family. GRK1 phosphorylates photoactivated rhodopsin, initiating steps in its deactivation. In this study, we found that human retina expressed all GRKs except GRK4. Based on results of molecular cloning and immunolocalization, it appears that both rod and cone photoreceptors express GRK1. This conclusion was supported by the cloning of only GRK1 from cone-dominated chicken retina. Human photoreceptors also transcribe a splice variant of GRK1, which differs in its C-terminal region next to the catalytic domain. This novel variant, GRK1b, is produced by retention of the last intron. mRNA encoding GRK1b is exported to the cytosol; however, the level of the protein is relatively low compared with GRK1 (now called GRK1a), and GRK1b appears to have very low catalytic activity. Thus, these studies suggest that rods and cones, express the same form of GRK1. Desensitization of G protein-coupled receptors is mediated, at least in part, by a family of Ser/Thr kinases called GRKs 1The abbreviations used are: GRK, G protein-coupled receptor kinase; Rho, rhodopsin; Rho*, photolyzed Rho; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; GCAP, guanylate cyclase-activating protein; bp, base pair(s); kb, kilobase pair(s). (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Google Scholar). Distinct properties set these enzymes apart from other protein kinases, including (a) broad and overlapping substrate specificities that are, however, restricted to ligand-activated G protein-coupled receptors and (b) complex interactions with the receptor that involve low affinity binding of GRKs to the region of the receptor that is phosphorylated and high affinity, multipoint interactions of GRKs with cytoplasmic loops of the receptor. To date, six members of the GRK family have been cloned from vertebrate species and Drosophila. Based on sequence homology, they are divided into three subgroups. Group I contains GRK1 (Rho kinase), group II contains GRK2 and GRK3 (β-adrenergic receptor kinase 1 and 2) and Drosophila GPRK1, and group III contains newly identified members GRK4, GRK5, GRK6, and Drosophila GPRK2. The overall protein sequence similarities among these kinases are 53–93%, with the lowest sequence homology between group I and group II (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Google Scholar, 2Palczewski K. Eur. J. Biochem. 1997; 248: 261-269Google Scholar). In addition, four splice variants (α, β, γ, and δ) of GRK4 with different N- or C-terminal regions were found primarily in the testis (3Sallese M. Mariggio S. Collodel G. Moretti E. Piomboni P. Baccetti B. De Blasi A. J. Biol. Chem. 1997; 272: 10188-10195Google Scholar, 4Premont R.T. Macrae A.D. Stoffel R.H. Chung N. Pitcher J.A. Ambrose C. Inglese J. MacDonald M.E. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 6403-6410Google Scholar), and GRK6 may exist in two splice forms (5Firsov D. Elalouf J-M. Am. J. Physiol. 1997; 273: C953-C961Google Scholar). In vitro, of the four variants of GRK4, only the longest form, GRK4, phosphorylates the model substrate, Rho* (3Sallese M. Mariggio S. Collodel G. Moretti E. Piomboni P. Baccetti B. De Blasi A. J. Biol. Chem. 1997; 272: 10188-10195Google Scholar). This suggests that alternative splicing may be one of the mechanisms for generating GRK isoforms with different specificities. This alternative splicing among the members of the GRK family might be an important diversification mechanism, because only six members have been found so far, whereas hundreds of G protein-coupled receptors are subject to receptor phosphorylation. Diverse mRNA species are produced by alternative splicing. Splice variants can be generated by several mechanisms, including exon skipping, alternative selection of exons, differential usage of splicing sites, and intron retention. Many splice variants have different tissue or cellular localizations, perform different physiological functions, and are differently regulated. Some of the variants have different sequences in the protein coding region, whereas others differ in their 5′- or 3′-untranslated regions. These untranslated regions frequently contain regulatory elements for transcription, translation, and mRNA stability (6Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Google Scholar). In rod photoreceptors, Rho* triggers a phototransduction cascade through the activation of a G protein (Gt, also called transducin), leading to an increase in cGMP phosphodiesterase activity. The hydrolysis of intracellular cGMP by phosphodiesterase leads to the closure of cGMP-gated channels in the plasma membrane and hyperpolarization of the photoreceptor cells. The quenching of Rho* is initiated by its phosphorylation, catalyzed by GRK1, and is followed by the binding of the regulatory protein, arrestin, to the phosphorylated Rho* (2Palczewski K. Eur. J. Biochem. 1997; 248: 261-269Google Scholar). The role of GRK1 in the regulation of phototransduction was further defined by its role in Oguchi's disease, a special form of congenital night blindness (7Fuchs S. Nakazawa M. Maw M. Tamai M. Oguchi Y. Gal A. Nat. Genet. 1995; 10: 360-362Google Scholar, 8Yamamoto S. Sippel K.C. Berson E.L. Dryja T.P. Nat. Genet. 1997; 15: 175-178Google Scholar, 9Cideciyan A.V. Zhao X. Nielsen L. Khani S.C. Jacobson S.G. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 328-333Google Scholar). The effects on human vision of a mutation in the GRK1 gene causing Oguchi's disease, was recently investigated in detail. A slowing of rod and cone deactivation kinetics in the homozygote was detected by electroretinography. However, phosphorylation of Rho* appears not to be involved in the regulation of the initial catalytic properties of Rho*. Cones may rely mainly on regeneration for the inactivation of photolyzed visual pigment, but GRK1 (or its cone homolog) also contributes to cone recovery (9Cideciyan A.V. Zhao X. Nielsen L. Khani S.C. Jacobson S.G. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 328-333Google Scholar). Phototransduction in rods and cones differs in electrophysiological response kinetics and sensitivity partly because of the differences in cell-specific subsets of phototransduction proteins. Due to the paucity of cones and the difficulties in their isolation from mammalian retina, cone phototransduction is less well understood at the biochemical level. Molecular cloning of cone phototransduction proteins has been successful, including cloning of the red/green/blue visual pigments (10Nathans J. Thomas D. Hogness D.S. Science. 1986; 232: 193-202Google Scholar), cone Gtα, β, and γ subunits (11Lerea C.L. Somers D.E. Hurley J.B. Klock I.B. Bunt-Milam A.H. Science. 1986; 234: 77-80Google Scholar, 12Lee R.H. Lieberman B.S. Yamane H.K. Bok D. Fung B.K. J. Biol. Chem. 1992; 267: 24776-24781Google Scholar, 13Ong O.C. Yamane H.K. Phan K.B. Fong H.K. Bok D. Lee R.H. Fung B.K. J. Biol. Chem. 1995; 270: 8495-8500Google Scholar), cone phosphodiesterase α and γ subunits (14Li T.S. Volpp K. Applebury M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 293-297Google Scholar, 15Hamilton S.E. Prusti R.K. Bentley J.K. Beavo J.A. Hurley J.B. FEBS Lett. 1993; 318: 157-161Google Scholar), cone arrestin (16Craft C.M. Whitmore D.H. Wiechmann A.F. J. Biol. Chem. 1994; 269: 4613-4619Google Scholar), and the α subunit of the cGMP-gated cation channel (17Bonigk W. Altenhofen W. Müller F. Dose A. Illing M. Molday R.S. Kaupp U.B. Neuron. 1993; 10: 865-877Google Scholar). Several phototransduction proteins are present in both rods and cones, including retinal guanylate cyclase 1 (18Dizhoor A.M. Lowe D.G. Olshevskaya E.V. Laura R.P. Hurley J.B. Neuron. 1994; 12: 1345-1352Google Scholar), guanylate cyclase-activating proteins (GCAP1 and GCAP2) (19Gorczyca W.A. Polans A.S. Surgucheva I.G. Subbaraya I. Baehr W. Palczewski K. J. Biol. Chem. 1995; 270: 22029-22036Google Scholar, 20Otto-Bruc A. Fariss R.N. Haeseleer F. Huang J. Buczylko J. Surgucheva I. Baehr W. Milam A.H. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4727-4732Google Scholar), and recoverin (21Polans A.S. Burton M.D. Haley T.L. Crabb J.W. Palczewski K. Investig. Ophthalmol. Visual Sci. 1993; 34: 81-90Google Scholar). GRK1 has also been localized in both bovine rods and cones using polyclonal antibodies, suggesting that cones may contain either GRK1, its splice form, or a closely related homolog (22Palczewski K. Buczylko J. Lebioda L. Crabb J.W. Polans A.S. J. Biol. Chem. 1993; 268: 6004-6013Google Scholar). In this study, using a combination of biochemical and immunocytochemical methods, we found that GRK1 is expressed in rods and cones and that human and chicken retinas contain GRK1 and an alternative spliced form, GRK1b, which retains the last intron. GRK1b is not a cone-specific splice variant and appears to have low catalytic activity. A chicken retinal cDNA library was provided by Dr. S. Semple-Rowland (University of Florida, Gainesville, FL). Human eyes were obtained from the Lions' Eye Bank (University of Washington, Seattle, WA), and chicken eyes were obtained from Mr. Wendell Luse of ACME Poultry Co., Inc. (Seattle, WA). Human fovea tissue punches were taken from 22 human retinas using an 18-gauge needle. Messenger RNA was isolated (FastTrackTM, Invitrogen), and reverse transcription-PCR was performed as described (23Zhao X. Haeseleer F. Fariss R.N. Huang J. Baehr W. Milam A.H. Palczewski K. Visual Neurosci. 1997; 14: 225-232Google Scholar). The degenerate oligonucleotide primers were designed according to the conserved sequences in the catalytic regions of GRKs (2Palczewski K. Eur. J. Biochem. 1997; 248: 261-269Google Scholar). The primer pairs used in the first round of PCR were as follows: XZ-1 (forward, 5′-TACGAATTCAC(A/C/T/G)GG(A/C/T/G)AA(A/G)CT(A/C/T/G)TA(T/C)GC-3′) and XZ-2 (reverse, 5′-ATCAAGCTT(T/C)TC(A/C/T/G)GG(A/C/T/G)GCCAT(A/G)AA(A/C/T/G)C-3′); XZ-3 (forward, 5′-GG(A/C/T/G)GG(A/C/T/G)TT(C/T)GG(A/T/C/G)GA(A/G)GT-3′) and XZ-4 (reverse, 5′-AG(A/C/T/G)CC(A/C)AGGTC(A/C/T/G)GA(A/T/G)AT-3′); XZ-1 and XZ-4; or XZ-3 and XZ-2. The first-round of PCR contained 10 mm Tris/HCl (pH 9.0), 50 mm KCl, 0.1% Triton X-100, 1.5 mm MgCl2, 0.2 mmdNTP, ∼10 ng of cDNA, and 1 μm primers. The samples were heated to 94 °C for 5 min, followed by the addition of 5 units of Taq DNA polymerase (Promega). The reactions were first cycled 5 times at low stringency (94 °C for 1 min, 40 °C or 50 °C for 2 min, and 72 °C for 3 min) and then cycled 35 times at high stringency (94 °C for 1 min, 60 °C or 65 °C for 2 min, and 72 °C for 3 min). The PCR products were separated on a 1% agarose gel, and DNA bands corresponding to the predicted size were excised and extracted using a Qiax Gel Extraction kit (Qiagen). This DNA was then used as a template in the second round of amplification reactions using XZ-1 and XZ-4 primers. The PCR conditions were similar to those described above but without the initial 5 cycles at the lower annealing temperature. The products from the first and the second round of PCR were cloned into pCRTMII (Invitrogen) and sequenced either manually (Sequenase 2.0; U. S. Biochemical Corp.) or using an automated Taq dideoxy terminator cycle sequencing kit (ABI-prism, Perkin-Elmer) at the University of Washington Molecular Pharmacology Facility. Human GRK1 genomic clone containing exons 4–7 in pBluescript SK(−) (Stratagene, Inc.) was provided by Dr. T. Dryja (24Khani S.C. Abitbol M. Yamamoto S. Maravic-Magovcevic I. Dryja T.P. Genomics. 1996; 35: 571-576Google Scholar). To obtain the size of introns 4, 5, and 6, the following primers were used in PCR: primer b (forward, from exon 4, 5′-AAGACCAAGGGCTACGCAGGGA-3′); primer c (forward, from exon 6, 5′-AGAAGGACCCGGAGAAGCGCCT-3′); XZ-57 (forward, from exon 5, 5′-GACTTCTCCGTGGACTACTTTGC-3′); primer PA8 (reverse, from exon 5, 5′-TTCTCTCCACGGGCTCGGAA-3′); primer XZ-54 (reverse, from exon 6, 5′-GCCTCCAGCTGCCTCCAGTTAAG-3′); primer d (reverse, from intron 6, 5′-TCAAGCAAGTGCTGGTGGGTGGA-3′); and primer e (reverse, from exon 7, 5′-CTAGGAAACCAGACACATCCCTGA-3′). The identities of the products were verified by Southern blot analyses, using [γ-32P]dCTP-labeled probe encompassing the catalytic region, 3′ region, or intron 6 of human GRK1a. Human retinas were dissected 2–15 h post mortem from human eyes 2Human donor post-mortem retinas and mRNA were generated from experiments described by Zhao et al. (23Zhao X. Haeseleer F. Fariss R.N. Huang J. Baehr W. Milam A.H. Palczewski K. Visual Neurosci. 1997; 14: 225-232Google Scholar).and stored at −80 °C until needed. Total RNA was isolated using guanidinium isothiocyanate as described previously (25MacDonald R.J. Swift G.H. Przybyla A.E. Chirgwin J.M. Methods Enzymol. 1987; 152: 219-227Google Scholar). cDNA used in PCR was prepared by reverse transcription with oligo(dT) primer (Life Technologies, Inc. (23Zhao X. Haeseleer F. Fariss R.N. Huang J. Baehr W. Milam A.H. Palczewski K. Visual Neurosci. 1997; 14: 225-232Google Scholar). The 3′ region of GRK1b was cloned by the rapid amplification of cDNA end (3′-RACE) using a MarathonTM DNA amplification kit (CLONTECH Laboratories, Inc.) as described previously (23Zhao X. Haeseleer F. Fariss R.N. Huang J. Baehr W. Milam A.H. Palczewski K. Visual Neurosci. 1997; 14: 225-232Google Scholar). To verify that the GRK1b transcript was not from genomic DNA contamination, genomic DNA and cDNA were amplified using primers derived from different exon sequences as shown in Fig. 4. The PCR conditions and primers b–e were the same as for the genomic PCR experiments. Primer a is 5′-GATGGATTTCGGGTCTTTGGAGAC-3′. To determine the relative amounts of GRK1a and GRK1b, quantitative PCR was performed as described previously (26Jia G.Q. Gutierrez-Ramos J.C. Eur. Cytokine Netw. 1995; 6: 253-255Google Scholar). Briefly, each PCR contained 10 mm Tris/HCl (pH 9.0), 50 mm KCl, 0.1% Triton X-100, 1.5 mmMgCl2, 0.2 mm dNTP, 0.5 μl of cDNA, 0.5 μm each of primers, and 0.5 μCi of [α-32P]dCTP (300 cpm/pmol; NEN Life Science Products). The samples were heated to 94 °C for 2 min, followed by the addition of 2.5 units of Taq DNA polymerase and 0.05 units of Tli DNA polymerase (Promega). The reactions were cycled 30 times (94 °C for 45 s, 65 °C for 1 min, and 72 °C for 1 min) to amplify human GCAP1 as an internal control, or in separate experiments, the reactions were cycled 30 times (94 °C for 45 s, 68 °C for 1 min, and 72 °C for 1 min) to amplify GRK1a and GRK1b at the same time. The products were separated on a 1.5% agarose gel. Bands corresponding to GRK1a, GRK1b, and GCAP1 were excised, dissolved in 6 m sodium perchlorate, and counted in a scintillation counter. The relative amounts of GRK1a versusGRK1b was calculated as the ratio of the radioactivity associated with the GRK1a band to the radioactivity associated with the GRK1b band, taking the molecular weight differences of the PCR products into consideration. Primer b (as in genomic cloning) and primer e were used for GRK1a, primer b and primer d were used for GRK1b, and primers FH-13 (5′-ATCGATGTCAATCTTGGAGAACACTGTATC-3′) and FH-17 (5′-AGCCTGGTCCTCAAGGGGAAG-3′) were used for GCAP1. Full-length sequences of GRK1a (1,692 bp) and GRK1b (3.6 kb, containing intron 6) were cloned into pGEM-T Easy (Promega). The plasmid DNA was purified through several steps under RNase-free conditions as described below. DNA was isolated using a Qiagen spin miniprep kit (Qiagen), passed through a CentiflexTM-AG column (Advanced Genetic Technologies, Corp.), precipitated by ethanol, then resuspended in diethyl pyrocarbonate-treated water. The in vitrotranscription/translation reaction was carried out using a TnT T7-coupled reticulocyte lysate system (Promega) according to the manufacturer's protocol. Briefly, equal molar amounts of circular template DNA of GRK1a (1 μg) and GRK1b (1.8 μg) were added to the reaction mixture (total 50 μl) containing 25 μl of rabbit reticulocyte lysate, 1 μl of provided amino acid mixture, 1 μl of RNase inhibitor, and 1 μl of T7 RNA polymerase (Promega). After 2 h at 30 °C, the samples were mixed with 1% SDS and 2 μl of β-mercaptoethanol, heated to 100 °C for 5 min, and centrifuged at 86,000 × g for 30 min. The proteins were separated on a 10%, 1.5-mm thick SDS-polyacrylamide electrophoresis gel and transferred to an Immobilon membrane (Millipore) at 90 V for 1.5 h. The translational products were detected by immunoblotting using D11 anti-GRK1 monoclonal antibodies (1.5 mg/ml at diluted 1:10,000). GRK1 activity was measured as described previously (22Palczewski K. Buczylko J. Lebioda L. Crabb J.W. Polans A.S. J. Biol. Chem. 1993; 268: 6004-6013Google Scholar). Human retinas were fixed for 6 h and stored at −20 °C in methanol until use (20Otto-Bruc A. Fariss R.N. Haeseleer F. Huang J. Buczylko J. Surgucheva I. Baehr W. Milam A.H. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4727-4732Google Scholar). The transcription template was a cDNA fragment encompassing bases 1,500–1,890 of the human GRK1b sequence cloned into pBluescript. The digoxigenin-labeled probes were generated from linearized plasmid DNA using T3 RNA polymerase for the antisense probe and T7 RNA polymerase for the sense probe (Ambion). Both probes were hydrolyzed with 60 mmNa2CO3, 40 mm NaHCO3, and 80 mm dithiothreitol at 60 °C for 40 min to reduce the probe length to 150–250 bases. In situ hybridization was performed as described previously (20Otto-Bruc A. Fariss R.N. Haeseleer F. Huang J. Buczylko J. Surgucheva I. Baehr W. Milam A.H. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4727-4732Google Scholar). Partial or full-length sequences of GRK1a and GRK1b cDNAs were cloned into pQE30 (Qiagen). The plasmid DNA was transformed into Escherichia coli strain M15 (Qiagen) for protein expression. Protein expression and purification were carried out according to the protocol provided by the manufacturer (Qiagen). The purity in SDS-polyacrylamide gel electrophoresis of His-tagged recombinant proteins was greater than 80%. The bacterially expressed, full-length human GRK1a was dialyzed against 70 mm sodium phosphate buffer (pH 7.5) and injected into mice with Ribi adjuvant (Ribi ImmunoChem Research, Inc.). Two monoclonal antibodies were produced according to standard procedures (27Campbell A.M. Burdon R.H. van Knippenberg P.H. Laboratory Techniques in Biochemistry and Molecular Biology 13. Elsevier Science Publishing Co., Inc., New York1984Google Scholar): G8 (C-terminal specificity; see Fig. 1) and D11 (N-terminal specificity; see Fig. 1). Monoclonal antibodies were purified using protein A-Sepharose (Pharmacia Biotech Inc.). A bacterially expressed C-terminal fragment of GRK1b (residues 463–598) was used to immunize rabbits to generate a polyclonal antibody (Cocalico Biologicals, Inc.). The anti-human GRK1b polyclonal antibody (UW54) was purified using antigen coupled to CNBr-Sepharose. The human sections were processed as in the single labeling experiments as described previously (20Otto-Bruc A. Fariss R.N. Haeseleer F. Huang J. Buczylko J. Surgucheva I. Baehr W. Milam A.H. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4727-4732Google Scholar). For double labeling, tissue sections were first incubated with primary antibodies to GRK1 (G8) and red/green cone opsin (JH492) or blue cone opsin (JH455) (28Chiu M.I. Nathans J. Visual Neurosci. 1994; 11: 773-780Google Scholar), followed by secondary Cy-3-conjugated goat anti-rabbit IgG and Cy-2-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.). Several approaches were employed to explore the presence of different forms of GRKs in human retina, especially the existence of cone-specific kinases. For example, a combination of oligonucleotide primers and PCR using freshly prepared cDNA as well as screening of human and bovine retinal cDNA libraries with the bovine GRK1 probe yielded only GRK1 (Rho kinase). Because the human retina is rod-dominant, with 95% rod and 5% cone photoreceptors (29Curcio C.A. Sloan K.R. Kalina R.E. Hendrickson A.E. J. Comp. Neurol. 1990; 292: 497-523Google Scholar), these methods could have inherent problems in detecting rare cone kinase in the presence of relatively large amounts of rod GRK1. To enrich with cDNA encoding putative cone kinase, 18-gauge punches were taken from human retinas around the fovea that contained a higher ratio of cone to rod cells in addition to the cells of the neuronal retina. mRNA was isolated and reverse-transcribed and followed by amplification of the cDNA from highly conserved catalytic regions using degenerate oligonucleotide primers designed to hybridize with all GRK. Among the 41 clones sequenced, 22 matched the published sequence of GRK1 (2Palczewski K. Eur. J. Biochem. 1997; 248: 261-269Google Scholar, 24Khani S.C. Abitbol M. Yamamoto S. Maravic-Magovcevic I. Dryja T.P. Genomics. 1996; 35: 571-576Google Scholar, 30Lorenz W. Inglese J. Palczewski K. Onorato J.J. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8715-8719Google Scholar), 15 matched GRK2/3, which have identical sequence in the chosen fragment of the catalytic region (31Benovic J.L. DeBlasi A. Stone W.C. Caron M.G. Lefkowitz R.J. Science. 1989; 246: 235-240Google Scholar,32Benovic J.L. Onorato J.J. Arriza J.L. Stone W.C. Lohse M. Jenkins N.A. Gilbert D.J. Copeland N.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1991; 266: 14939-14946Google Scholar), 2 matched the sequence of GRK5 (33Kunapuli P. Benovic J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5588-5592Google Scholar, 34Premont R.T. Koch W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6832-6841Google Scholar), and 2 encoded GRK6 (35Benovic J.L. Gomez J. J. Biol. Chem. 1993; 268: 19521-19527Google Scholar). No GRK4 sequence (4Premont R.T. Macrae A.D. Stoffel R.H. Chung N. Pitcher J.A. Ambrose C. Inglese J. MacDonald M.E. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 6403-6410Google Scholar, 36Ambrose C. James M. Barnes G. Lin C. Bates G. Altherr M. Duyao M. Groot N. Church D. Wasmuth J.J. Lehrach H. Housman D. Buckler A. Gusella J.F. MacDonald M.E. Hum. Mol. Genet. 1992; 1: 697-703Google Scholar) was identified in this cDNA. Despite the fact that different GRKs could be amplified from this cDNA, we were unable to detect any homolog of GRK1, suggesting that the human retina contains one visual pigment kinase. Alternatively, rod and putative cone kinases are identical in the catalytic regions defined by the degenerate oligonucleotide primers. To localize GRK1 in the human retina, monoclonal antibodies were raised against bacterially expressed kinase. Two antibodies were selected for their recognition of the N- (D11) and C-terminal (G8) sites (Fig. 1). Retinal flat mount immunolocalization with G8 monoclonal antibody showed intense staining of cone and rod outer segments throughout the retina (Fig. 2). The immunostaining was blocked by preincubation of the antibody with recombinant kinase. Immunofluorescence microscopy of human retina with the monoclonal antibody against the C-terminal domain of the kinase revealed that GRK1 was present mainly in the cone outer segments and, to a lesser degree, in rod outer segments. Weak labeling was found in somata and synaptic terminals of the cones and the inner segments of rods (Fig. 3). The immunolabeling was abolished by preincubation of the antibody with bacterially expressed GRK1 (Fig. 3 D). In double-labeled sections of human retina, GRK1 was localized to cone outer segments (Fig. 3, A and D), including those whose outer segments were reactive with anti-red/green (Fig. 3, B and C) and blue cone opsins (Fig. 3, E and F). Identical localization of GRK1 was obtained using D11 monoclonal antibody with a specificity toward the N-terminal GRK1 (data not shown), polyclonal antibodies raised against recombinant GRK1 (data not shown), and native GRK1 (22Palczewski K. Buczylko J. Lebioda L. Crabb J.W. Polans A.S. J. Biol. Chem. 1993; 268: 6004-6013Google Scholar). Thus, the immunostaining was indistinguishable using antibodies of different specificity. These results support the idea that the same kinase may be present in rod and cone cells. It appears that GRK1 is highly abundant in all classes of cone cells.Figure 3Immunofluorescence localization of GRK1 in human retina. A, GRK1 immunolabeling using G8 monoclonal antibody was strongest in cone (arrows pointing down) and rod (arrowheads pointing down) outer segments. Immunolabeling was also present in cones (arrows pointing up) and rods (arrowheads pointing up). B, addition of bacterially expressed GRK1 (20 μg/ml) to anti-GRK monoclonal antibodies abolishes GRK1 immunoreactivity. C, sections preincubated with buffer without anti-GRK1 showed weak autofluorescence. Panels D, E, and F, localization of GRK1 and red/green cone opsin (anti-red/green cone opsin polyclonal antibodies, JH492). D, the cones and rods were immunolabeled with anti-GRK1, with the strongest labeling in the cone outer segments. E, anti-red/green cone opsin labeled a majority of cones. F, double labeling with anti-GRK1 (green) and anti-red/green cone opsin (red) showed that red/green cones are immunopositive for GRK1. Panels G, H, and I, localization of GRK1 and blue cone opsin (blue cone opsin pAb JH455 from Dr. Jeremy Nathans). G, the cones and rods were immunolabeled with anti-GRK1. H, anti-blue cone opsin labeled a single cone. I, double labeling with anti-GRK1 (green) and anti-red/green cone opsin (red) showed that the blue cone is immunopositive for GRK1. Bar = 50 μm.View Large Image Figure ViewerDownload (PPT) To identify novel forms of GRK1 from human retinal cDNA, RACE PCR and primers derived from the catalytic region were used to amplify the 3′ and 5′ regions of the kinase. The RACE products were cloned into pCR2.1 and sequenced. 5′-RACE PCR yielded identical clones to human GRK1 (23Zhao X. Haeseleer F. Fariss R.N. Huang J. Baehr W. Milam A.H. Palczewski K. Visual Neurosci. 1997; 14: 225-232Google Scholar). From 24 clones derived from the 3′-RACE PCR, 16 clones hybridized with the catalytic region but not with the C-terminal region of GRK1 probes on Southern blots. Since there is only one GRK1 gene in the genome (24Khani S.C. Abitbol M. Yamamoto S. Maravic-Magovcevic I. Dryja T.P. Genomics. 1996; 35: 571-576Google Scholar), this latter product, named GRK1b (the original GRK1 is now named GRK1a) might be a splice variant of GRK1. This form was observed not only by reverse transcription-PCR, but it was found also by screening the retinal cDNA library (data not shown and Ref. 24Khani S.C. Abitbol M. Yamamoto S. Maravic-Magovcevic I. Dryja T.P. Genomics. 1996; 35: 571-576Google Scholar). To investigate the molecular structure of the GRK1b transcript, human GRK1 genomic DNA was analyzed using a genomic clone containing exon 4 to 7 (G2) (24Khani S.C. Abitbol M. Yamamoto S. Maravic-Magovcevic I. Dryja T.P. Genomics. 1996; 35: 571-576Google Scholar). The sizes of introns 4, 5, and 6 were identified using a PCR technique (4Premont R.T. Macrae A.D. Stoffel R.H. Chung N. Pitcher J.A. Ambrose C. Inglese J. MacDonald M.E. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 6403-6410Google Scholar) (Fig. 4 A). Employing PCR primers residing at different exons and introns, it was determined that the GRK1b transcript was identical to GRK1a, except that it retained the last intron, intron 6 (Fig. 4 B). In addition, the sequence of intron 6 was identical with the 3′-end of GRK1b. Within the intron 6 sequence, there was a stop codon found ∼300 bp from the catalytic region (Fig. 5). GRK1b was not an amplification artifact of genomic DNA because the PCR primer pair b and e amplified an 11-kb fragment from the genomic DNA but only 650 bp (corresponding to GRK1a) and 2.4 kb (corresponding to GRK1b) fragments from cDNA (Fig. 4 B). Using PCR and pairs of primers a and e and a and d, we have amplified the full-length coding sequence of both GRK1a and GRK1b (Fig. 4, lower panel). All the PCR products from cDNA were sequenced, and their identity to the PCR products from genomic DNA was established by Southern blotting. These results demonstrate that human GRK1a has a splice variant, GRK1b, which retains the last intron in its mRNA. Radiometric quantitative PCR was performed on cDNA derived from four human retinas to investigate the abundance and prevalence of GRK1b. GRK1a and GRK1b (650- and 740-bp produc" @default.
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• W2076644311 title "Molecular Forms of Human Rhodopsin Kinase (GRK1)" @default.
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