From the Departments of Ophthalmology and
§ Pharmacology, University of Washington, Seattle,
Washington 98195 and the ¶ Department of Ophthalmology, State
University of New York, Buffalo, New York 14215
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ABSTRACT |
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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.
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INTRODUCTION |
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Desensitization of G protein-coupled receptors is mediated, at
least in part, by a family of Ser/Thr kinases called
GRKs1 (1). 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 (-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 (1, 2). In
addition, four splice variants (
,
,
, and
) of GRK4 with
different N- or C-terminal regions were found primarily in the testis
(3, 4), and GRK6 may exist in two splice forms (5). In
vitro, of the four variants of GRK4, only the longest form,
GRK4, phosphorylates the model substrate, Rho* (3). 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 (6).
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* (2). 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 (7-9). 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 (9).
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
(10), cone Gt,
, and
subunits (11-13), cone phosphodiesterase
and
subunits (14, 15), cone arrestin (16),
and the
subunit of the cGMP-gated cation channel (17). Several
phototransduction proteins are present in both rods and cones,
including retinal guanylate cyclase 1 (18), guanylate cyclase-activating proteins (GCAP1 and GCAP2) (19, 20), and recoverin
(21). 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 (22).
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.
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EXPERIMENTAL PROCEDURES |
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Materials-- 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).
Cloning of GRKs from the Fovea Region of the Human Retina-- 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 (23). The degenerate oligonucleotide primers were designed according to the conserved sequences in the catalytic regions of GRKs (2). 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 mM dNTP, ~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.
Determination of the Size of Human GRK1 Introns 4, 5, and 6 by
PCR--
Human GRK1 genomic clone containing exons 4-7 in pBluescript
SK() (Stratagene, Inc.) was provided by Dr. T. Dryja (24). 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
[
-32P]dCTP-labeled probe encompassing the catalytic
region, 3' region, or intron 6 of human GRK1a.
Cloning of Human GRK1b--
Human retinas were dissected 2-15 h
post mortem from human eyes2
and stored at 80 °C until needed. Total RNA was isolated using guanidinium isothiocyanate as described previously (25). cDNA used
in PCR was prepared by reverse transcription with oligo(dT) primer
(Life Technologies, Inc. (23). 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 (23). 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'.
Relative Amounts of GRK1a and GRK1b mRNA in Human
Retina--
To determine the relative amounts of GRK1a and GRK1b,
quantitative PCR was performed as described previously (26). Briefly, each PCR contained 10 mM Tris/HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.5 mM
MgCl2, 0.2 mM dNTP, 0.5 µl of cDNA, 0.5 µM each of primers, and 0.5 µCi of
[-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 versus
GRK1b 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.
In Vitro Translation of GRK1a and GRK1b--
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 vitro
transcription/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
-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 (22).
In Situ Hybridization--
Human retinas were fixed for 6 h
and stored at 20 °C in methanol until use (20). 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 mM
Na2CO3, 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 (20).
Expression and Purification of Human GRK1 in Bacteria-- 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%.
Anti-human GRK1a and GRK1b Antibodies-- 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 (27): 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.
Immunocytochemistry-- The human sections were processed as in the single labeling experiments as described previously (20). 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) (28), followed by secondary Cy-3-conjugated goat anti-rabbit IgG and Cy-2-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.).
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RESULTS |
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GRKs in Human Retinal Fovea-- 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 (29), 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 (2, 24, 30), 15 matched GRK2/3, which have identical sequence in the chosen fragment of the catalytic region (31, 32), 2 matched the sequence of GRK5 (33, 34), and 2 encoded GRK6 (35). No GRK4 sequence (4, 36) 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.
Localization of GRK1 in Human Retina-- 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. 3D). 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 (22). 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.
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A Splice Variant of Human GRK1-containing Intronic Sequence-- 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 (23). 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 (24), 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. 24). 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) (24). The sizes of introns 4, 5, and 6 were identified using a PCR technique (4) (Fig. 4A). 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. 4B). 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. 4B). 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.
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Localization of mRNA Encoding GRK1b-- In situ hybridization using human tissue and digoxigenin-labeled antisense and sense probes encoding the sequence of the 5'-terminal part of intron 6 was employed to determine if the mRNA of GRK1b has nuclear or ribosomal localization. In human retina, cells in the outer nuclear layer were specifically labeled with the antisense probe (Fig. 7A), whereas no hybridization signal was produced by the sense probe (Fig. 7B). The most intense staining was found in the cone and rod inner segments. Due to the size of the probes (300 bp), however, some nuclear DNA was also nonspecifically stained. This result shows that the mRNA for GRK1b is exported from the nucleus.
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DISCUSSION |
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GRKs in the Human Retina-- Among the GRK genes, GRK1, GRK2/3, GRK5, and GRK6 were found to be transcribed in human retina. In double immunolocalization experiments, GRK1 was found to be present in all photoreceptors, including red/green and blue opsin-containing cones. Similar results were found using two anti-GRK1 monoclonal antibodies specific for N- or C-terminal regions (Figs. 1-3). In chicken retina, GRK1 immunolocalizes to all photoreceptors, suggesting that the same kinase is present in these cells. The localization of other GRKs in the retina was not investigated.
Although we cannot completely exclude the existence of a novel cone-specific kinase, our data suggest, however, that both rods and cones of human and chicken retinas express the same photoreceptor-specific kinase, GRK1. This conclusion is based on the following evidence. (a) Screening human and chicken retinal libraries yielded one form of the enzyme, (b) PCR with degenerate oligonucleotides derived from the catalytic region of GRKs yielded only one form of GRK1 even though distantly related GRK2/3 was detected, (c) freshly prepared mRNA from cone-rich retina (chicken) and cone-enriched fovea of human retina yielded one GRK1 with reverse transcription-PCR and RACE methods, (d) one photoreceptor kinase is present in chicken and mammalian pineal gland (23), (e) the kinase was immunolocalized to both rod and cone cells using specific antibodies (this study and Ref. 22), and (f) lack of a novel sequence derived from human retina deposited in the EST data base. However, it is possible that lower vertebrates have more than one kinase, as they have more than one recoverin, for example (39).Splice Form of GRK1--
Gene expression is controlled in part by
mRNA processing. Alternative splicing of nuclear mRNA
(pre-mRNA) occurs in at least one out of 20 genes (6). Sequences
that are essential for intron removal are limited to the intron/exon
borders (40). In some cases, intron retention is believed to result
from suppression of the utilization of both 5' and 3' splice sites on
pre-mRNA. The consensus sequences of the 5' splice site is
(C/A)AGGU(A/G)AGU (the splice site is denoted by a
,
invariant nucleotides are underlined) and of the 3' splice site is
(T/C)AG
GU. These consensus sequences are well conserved
within eukaryotic species from yeast to human (41). During the past
several years, intron retention has been shown to be a facet of normal
mRNA splicing, and intron-containing mRNA is associated with
many cellular functions. For example, a fraction (0.1-20%) of bovine
growth hormone cytosolic mRNA retains the last intron, intron D, in
bovine anterior pituitary somatotrophs (6). An alternative mRNA of
human nontransmembrane phosphotyrosine phosphatase (PTP-1B) retains the
last intron and encodes a protein with a different C-terminal region.
The amount of intron-retaining mRNA was increased upon growth
factor stimulation (42). In some cases, such as mouse tyrosinase,
intron retention serves as a negative regulator for either functional
mRNA production (43) or functional protein synthesis as found for
the kinase-deficient splice variants of Janus kinase 3 (44). Intron
retention has also been shown to cause several types of genetic
diseases; for example, the retention of intron 10 in the
phosphofructokinase gene causes Tauri disease (45), and retention of
intron 9 in CD44 causes certain cases of urinary bladder cancer
(46).
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ACKNOWLEDGEMENTS |
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We offer special thanks to Dr. Ann H. Milam (University of Washington, Department of Ophthalmology) for support, access to tissue processing instrumentation, and helpful discussion during the course of this study. We thank Dr. T. Dryja for the genomic clone used in this study, and Dr. Jack Saari for comments. We thank J. Preston Van Hooser and Toni Haun for help during the course of these studies and D. Possin for assistance with tissue processing.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grant EY08061 and awards from Research to Prevent Blindness, Inc. to the Department of Ophthalmology at the University of Washington.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) AF019766 for chicken GRK1 and AFO19764 and AF019765 for intron 6 of human GRK1.
A recipient of a Jules and Doris Stein Professorship from
Research to Prevent Blindness, Inc. and to whom correspondence should be addressed: Dept. of Ophthalmology, University of Washington, Box
356485, Seattle, WA 98195-6485. Tel.: 206-543-9074; Fax: 206-543-4414; E-mail: palczews{at}u.washington.edu.
1 The 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).
2 Human donor post-mortem retinas and mRNA were generated from experiments described by Zhao et al. (23).
3 K. Palczewski, X. Zhao, and M. Gelb, manuscript in preparation.
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REFERENCES |
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