Gene array and expression of mouse retina guanylate cyclase activating proteins 1 and 2.
ABSTRACT To identify gene arrangement, chromosomal localization, and expression pattern of mouse guanylate cyclase activating proteins GCAP1 and GCAP2, retina-specific Ca2+-binding proteins, and photoreceptor guanylate cyclase activators.
The GCAP1 and GCAP2 genes were cloned from genomic libraries and sequenced. The chromosomal localization of the GCAP array was determined using fluorescent in situ hybridization. The expression of GCAP1 and GCAP2 in mouse retinal tissue was determined by immunocytochemistry.
In this study, the mouse GCAP1 and GCAP2 gene array, its chromosomal localization, RNA transcripts, and immunolocalization of the gene products were fully characterized. The GCAP tail-to-tail array is located at the D band of chromosome 17. Each gene is transcribed into a single transcript of 0.8 kb (GCAP1) and 2 kb (GCAP2). Immunocytochemistry showed that both GCAP genes are expressed in retinal photoreceptor cells, but GCAP2 was nearly undetectable in cones. GCAP2 was also found in amacrine and ganglion cells of the inner retina. Light-adapted and dark-adapted retinas showed no significant difference in the distribution of the most intense GCAP2 staining within the outer segment and outer plexiform layers.
Identical GCAP gene structures and the existence of the tail-to-tail gene array in mouse and human suggest an ancient gene duplication-inversion event preceding mammalian diversification. Identification of both GCAPs in synaptic regions, and of GCAP2 in the inner retina suggest roles of these Ca-binding proteins in addition to regulation of phototransduction.
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ABSTRACT: In vertebrate rods and cones, photon capture by rhodopsin leads to the destruction of cyclic GMP (cGMP) and the subsequent closure of cyclic nucleotide gated ion channels in the outer segment plasma membrane. Replenishment of cGMP and reopening of the channels limit the growth of the photon response and are requisite for its recovery. In different vertebrate retinas, there may be as many as four types of membrane guanylyl cyclases (GCs) for cGMP synthesis. Ten neuronal Ca(2+) sensor proteins could potentially modulate their activities. The mouse is proving to be an effective model for characterizing the roles of individual components because its relative simplicity can be reduced further by genetic engineering. There are two types of GC activating proteins (GCAPs) and two types of GCs in mouse rods, whereas cones express one type of GCAP and one type of GC. Mutant mouse rods and cones bereft of both GCAPs have large, long lasting photon responses. Thus, GCAPs normally mediate negative feedback tied to the light-induced decline in intracellular Ca(2+) that accelerates GC activity to curtail the growth and duration of the photon response. Rods from other mutant mice that express a single GCAP type reveal how the two GCAPs normally work together as a team. Because of its lower Ca(2+) affinity, GCAP1 is the first responder that senses the initial decrease in Ca(2+) following photon absorption and acts to limit response amplitude. GCAP2, with a higher Ca(2+) affinity, is recruited later during the course of the photon response as Ca(2+) levels continue to decline further. The main role of GCAP2 is to provide for a timely response recovery and it is particularly important after exposure to very bright light. The multiplicity of GC isozymes and GCAP homologs in the retinas of other vertebrates confers greater flexibility in shaping the photon responses in order to tune visual sensitivity, dynamic range and frequency response.Frontiers in Molecular Neuroscience 06/2014; 7:45.
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ABSTRACT: Summary The complex sensation of vision begins with the relatively simple photoisomerization of the visual pig- ment chromophore 11-cis-retinal to its all-trans config- uration. This event initiates a series of biochemical reactions that are collectively referred to as phototrans- duction, which ultimately lead to a change in the electrochemical signaling of the photoreceptor cell. To operate in a wide range of light intensities, however, the phototransduction pathway must allow for adjustments to background light. These take place through physio- logical adaptation processes that rely primarily on Ca2á ions. While Ca2á may modulate some activities directly, it is more often the case that Ca2á-binding proteins mediate between transient changes in the concentration of Ca2á and the adaptation processes that are asso- ciated with phototransduction. Recently, combined genetic, physiological, and biochemical analyses have yielded new insights about the properties and functions of many phototransduction-specific components, in- cluding some novel Ca2á-binding proteins. Understand- ing these Ca2á-binding proteins will provide a more complete picture of visual transduction, including the mechanisms associated with adaptation, and of related degenerative diseases. BioEssays 22:337-350, 2000. fl 2000 John Wiley & Sons, Inc.BioEssays 01/2000; 22(4):337-350. · 4.84 Impact Factor
- Gastroenterology 01/2011; 140(5). · 12.82 Impact Factor
Gene Array and Expression of Mouse Retina Guanylate
Cyclase Activating Proteins 1 and 2
Kim Howes,1'6 J. Darin Bronson,1'6 Yan Li Dang,2 Ning Li,1 Kai Zhang,1 Claudia Ruiz,2
Bharati Helekar,2 Muriel Lee,3 Iswari Subbaraya,2 Helga Kolb,1 Jeannie Chen,43 and
PURPOSE. TO identify gene arrangement, chromosomal localization, and expression pattern of mouse
guanylate cyclase activating proteins GCAP1 and GCAP2, retina-specific Ca2+-binding proteins, and
photoreceptor guanylate cyclase activators.
METHODS. The GCAP1 and GCAP2 genes were cloned from genomic libraries and sequenced. The
chromosomal localization of the GCAP array was determined using fluorescent in situ hybridization. The
expression of GCAP1 and GCAP2 in mouse retinal tissue was determined by immunocytochemistry.
RESULTS. In this study, the mouse GCAP1 and GCAP2 gene array, its chromosomal localization, RNA
transcripts, and immunolocalization of the gene products were fully characterized. The GCAP
tail-to-tail array is located at the D band of chromosome 17. Each gene is transcribed into a single
transcript of 0.8 kb (GCAP1) and 2 kb (GCAP2). Immunocytochemistry showed that both GCAP
genes are expressed in retinal photoreceptor cells, but GCAP2 was nearly undetectable in cones.
GCAP2 was also found in amacrine and ganglion cells of the inner retina. Light-adapted and
dark-adapted retinas showed no significant difference in the distribution of the most intense GCAP2
staining within the outer segment and outer plexiform layers.
CONCLUSIONS. Identical GCAP gene structures and the existence of the tail-to-tail gene array in mouse
and human suggest an ancient gene duplication-inversion event preceding mammalian diversifi-
cation. Identification of both GCAPs in synaptic regions, and of GCAP2 in the inner retina suggest
roles of these Ca-binding proteins in addition to regulation of phototransduction. (Invest Ophthal-
To date, two GCAPs (GCAP1 and GCAP2) have been identi-
uanylate cyclase activating proteins (GCAPs) are
Ca2+-binding proteins of the calmodulin gene fami-
ly1 that contain four EF hand Ca2+-binding motifs.
From the 'Moran Eye Center, University of Utah Health Science
Center, Salt Lake City; the 2Department of Ophthalmology, Baylor
College of Medicine, Houston, Texas; the 3MRC Human Genetics Unit,
Western General Hospital, Edinburgh, United Kingdom; and the ''De-
partment of Cell and Neurobiology and the 5Department of Ophthal-
mology, University of Southern California, School of Medicine, Los
Angeles;6 these authors contributed equally to the work presented
here and should therefore be regarded as equivalent senior authors.
Present address: Claudia Ruiz, MD-PhD program, University of
Texas Medical School, Houston, TX 77030.
Present address: Bharati Helekar, Department of Dermatology,
University of Texas Medical School, Houston, TX 77030.
Supported by grants from the National Eye Institute (EY08123,
WB; EYO3323O, HK; R29EY11268, IS), Bethesda, MD; from the Foun-
dation Fighting Blindness, Bethesda, MD (WB); from the Ruth and
Milton Steinbach Fund GO; Fight for Sight, Inc., New York, NY,
Research Division of Prevent Blindness America, in memory of Dr.
Charles A. Perera (KH); and from the Knights Templar Eye Foundation,
Chicago, IL (KH); and by an award from Research to Prevent Blindness,
New York, NY, to the Department of Ophthalmology at the University
of Utah. JC is the recipient of a Research to Prevent Blindness research
career development award. WB is the recipient of a Senior Investigator
Award from Research to Prevent Blindness, New York, NY.
Submitted for publication June 26, 1997; revised September 30,
1997 and January 6, 1998; accepted January 23, 1998.
Proprietary interest category: N.
Reprint requests: Wolfgang Baehr, John A. Moran Eye Center, 75
N. Medical Drive, Salt Lake City, UT 84132.
fied in mammalian retina.2"6 Both GCAPs activate photore-
ceptor guanylate cyclase in the presence of low [Ca2+], a
regulatory mechanism that promotes accelerated synthesis
of cGMP, the internal messenger of phototransduction, after
photobleaching. The human GCAP genes are arranged in a
compact tail-to-tail gene array in which both genes are
transcribed from opposite strands.7 Biochemical and immu-
nologic studies to determine the cellular and subcellular
distribution of GCAPs in the retina have produced conflict-
ing results. RNA expression studies show nearly identical
expression patterns of GCAP1 and GCAP2 mRNAs in rod and
cone photoreceptors.5'8'9 Bovine GCAP1 was isolated from
rod outer segment membranes,310 but GCAP2 could5 or
could not be isolated610 from this source. Immunocyto-
chemistry using GCAP 1-specific antibodies has revealed that
GCAP1 is present in rod and cone outer segments and in
synaptic regions.6 GCAP2 was seen in outer and inner seg-
ments of rods but not of cones by one group of investiga-
tors.5 It was seen in the inner segments of rods and cones
but not in outer segments by another group.9 The reasons
for these discrepancies in GCAP2 distribution are unknown.
In this study, we characterized the GCAP genes and their
expression patterns in the retina of the mouse, an animal amena-
ble to genetic manipulation. The ultimate goal of this research
was to prevent expression of GCAP genes by targeted gene
replacement (gene knockout) and to analyze the consequences of
this manipulation on the physiology and morphology of photore-
ceptor cells. As a first step toward this goal, we describe the GCAP
Investigative Ophthalmology & Visual Science, May 1998, Vol. 39, No. 6
Copyright © Association for Research in Vision and Ophthalmology
868 Howes et al.
IOVS, May 1998, Vol. 39, No. 6
gene arrangement and chromosomal localization in mouse, and
discuss expression patterns of both GCAPs in the retina.
MATERIALS AND METHODS
Cloning of Mouse GCAP2 cDNA and DNA
Mouse GCAP1 cDNA clones were described previously.8 To
isolate mouse GCAP2 cDNA clones, a mouse retina cDNA
library was screened with a bovine GCAP2 cDNA insert. Two
clones were isolated, mG2-6 and mG2-7. The inserts of AzapII
bacteriophage (Invitrogen, San Diego, CA) clones were excised
according to the manufacturer's protocol. Supercoiled plasmid
DNA was sequenced using the double-stranded procedure de-
scribed previously1' or using an automatic sequencer (LI-COR
Model 4000L) and universal primers labeled with an infrared
fluorescent tag. The coding portions of all clones were com-
pletely sequenced on both strands. mG2-6 was 5'-truncated in
exon 1. Clone mG2-7 contained the complete coding sequence
but had an extended 5'-untranslated region (UTR) deviating
from the gene sequence.
Northern Blot Analysis
Mouse retina mRNA was isolated (Fasttrack; Invitrogen), was
separated on 0.43 M formaldehyde agarose gels,12 and was trans-
ferred to maximum strength nylon filters (Nytran; Schleicher and
Schuell), as described previously.'*' '3 The GCAP1 probe was
mGl-4, the GCAP2 probe mG2-6 (Fig. 1). The mouse opsin cDNA
probe used as a control has been described previously.M
Mouse GCAP Genomic Clones
A nick-translated GCAP1 fragment was used to screen a mouse
genomic library (mouse 129SVJ strain, prepared from liver of a
4 - 8-week-old female; Stratagene, La Jolla, CA). Three genomic
clones (AMG1, AMG2, and AMG3) with inserts of 15 to 18 kb were
identified and characterized by subcioning. .EcoRI fragments of
AMG2 corresponded to the two major GCAP1 genomic fragments
seen on Southern blots,* and were subcloned into pUC13 to yield
pUC-bot and pUC-top (Fig. 2). Puc-bot and pUC-top contained
introns b and c and most of intron a of the GCAP1 gene. The
remainder of intron a was amplified with sequence-specific prim-
ers. Introns of the GCAP2 gene were amplified from genomic
DNA with exon-specific primers (Fig. 2), using either Tag poly-
merase according to the Cetus (Berkeley, CA)/Perkin-Elmer (Nor-
walk, CT) protocol, or Taq/Pwo polymerases (Expand Long PCR
system, Boehringer-Mannheim, Indianapolis, IN), and cloned into
Polyclonal antibody UW14 was raised against bacterially ex-
pressed, truncated bovine GCAP I.6 Polyclonal UW50 was
raised against bovine GCAP2 expressed in bacteria.9 The anti-
human growth hormone antibody (anti-hGH) was character-
Chromosomal Localization by Fluorescent In Situ
DNA from clone AMG1 was labeled with bio-16-dUTP (Boe-
hringer-Mannheim) by nick translation and hybridized to
mouse embryonic stem cells and to mouse embryonic liver
FIGURE 1. Northern blot of normal adult mouse retina mRNA
probed with GCAP (guanylate cyclase activating protein) 1 and
GCAP2. Two micrograms of retina mRNA were loaded. Lane 1,
the blot was probed with mouse GCAP1. Lane 2, the blot was
stripped and reprobed with mouse GCAP2. Lane 3, stripped
and reprobed with mouse opsin (mOPS) cDNA as a control.
The mOPS gene is transcribed into five mRNA species differing
in polyadenylation sites.14 Known size standards are indicated
on the right in kilobases.
cells as described.16 Hybridization signals were detected
with successive layers of avidin Texas red (Vector Labora-
tories, Burlingame CA), biotinylated antiavidin (Vector), and
avidin Texas Red. The initial localization was confirmed by
using a mouse chromosome 17 paint (Cambio, La Jolla, CA).
Normal C57BL/6 and transgenic mice of both sexes were killed
under normal illumination. For dark-adapted experiments,
three females of one litter were killed at night (12 midnight)
under red light after 6 hours of dark adaptation. Once the eyes
were removed, an incision was made through the anterior
chamber to facilitate fixation in 4% paraformaldehyde in 0.1 M
phosphate buffer (PB; pH 7.4). After fixation for 6 hours at 4°C,
the lenses were removed, and the eyes were rinsed and cryo-
protected in 30% buffered sucrose overnight at 4°C. Eight-
micrometer-thick cryosections were incubated in 10% normal
goat serum for 30 minutes to inhibit nonspecific binding of the
antibodies. In some cases, the tissue sections were also perme-
abilized with 0.1 mg/ml proteinase K for 2 minutes before
blocking with normal goat serum. Primary and secondary an-
tibodies were diluted with 0.1 M PB and 0.3% Triton X-100 for
all reactions. Sections were rinsed with 0.1 M PB after each
incubation. Sections were incubated overnight at 4°C with
1:2000 UW50 and 1:3000 UW14 antibodies. Fluorescein iso-
thiocyanate (FITC) or Texas red-conjugated goat antirabbit
IgG (Vector) was used at a dilution of 1:100 for 1 hour at room
IOVS, May 1998, Vol. 39, No. 6
Mouse GCAP Gene Array 869
direction of transcription
direction of transcription
mG1-2 " " '
FIGURE 2. Physical map of mouse cDNA and genomic clones, and diagram of the GCAP
(guanylate cyclase activating protein) 2-GCAP1 gene array. The 5'-UTRs of the GCAP2 (left)
and GCAP1 (right) genes are flanking the gene array, which is depicted as an 18-kb contig.
Exons are shown as boxes; the coding portions are filled. Introns (length in kilobases) and
flanking sequences are shown as lines. Boxes within introns depict dinucleotide repeats of
various lengths. EcoBl restriction sites identified in the gene sequence and cloned fragments
(pUC-bot, pUC-top) are shown. Bars underneath and above the exons indicate the extent of
cloned cDNA for both genes (mGCAPI, clones mGl-4, and mGl-2; and mGCAP2, clones
mG2-6 and mG2-7, respectively). Lines under the GCAP2 gene marked by a, b, and c symbolize
intron clones generated by amplification with exon-specinc primers. Large arrows indicate
the direction of transcription. AMG1, AMG2, and AMG3 are genomic AfixII clones. Vertical
broken lines identify their starting and ending points, if known, determined by direct
sequencing of ADNA (for details, see the Genbank submission).
temperature. For double-labeling experiments, sections were
processed by one of two methods. In the first, sections were
incubated with 10 /xg/ml FITC- or Texas red-peanut agglutinin
(PNA; Sigma, St. Louis, MO) and UW50 or UW14 antibodies,
then by FITC- conjugated or Texas red- conjugated goat anti-
rabbit IgG. In the second method, sections were sequentially
FIGURE 3. Chromosomal localization of the GCAP (guanylate cyclase activating protein) gene
array. (A) Localization of the GCAP gene array by fluorescent in situ hybridization to chromo-
some 17. (B) Ideogram of chromosome 17 and location of the gene array at 17D.
870 Howes et al.
IOVS, May 1998, Vol. 39, No. 6
*^ Blue Cone
FIGURE 4. Immunolocalization of GCAPl in normal and transgenic human growth hormone
(hGH) mice, (a) GCAP (guanylate cyclase activating protein) 1 signal is most intense within the
outer segments of rods and cones and in the outer plexiform layer, (b) Colocalization of Texas
red-labeled GCAPl with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin for
cones (note the yellow signal obtained in the outer segments of cones), (c) FITC-labeled
GCAPl outer segments are contiguous with inner segments of blue cones (Texas red) detected
with the hGH antibody in transgenic mouse sections, (d) Preincubation of the GCAPl-specific
antibody UW14 with 25 /xg/ml of GCAPl protein results in loss of outer segment and outer
plexiform layer staining. Only staining of cones with FITC-conjugated peanut agglutinin is
observed. Scale bars, 12.5 fim.
incubated with the hGH primary antibody, and the goat anti-
rabbit secondary antibody and then the UW14 or UW50 pri-
mary antibody and fiuorochrome-conjugated goat antirabbit
secondary antibody. Immunofluorescence was photographed
with an inverted laser scanning confocal microscope (LSM410;
Carl Zeiss, Oberkochen, Germany).
GCAPl and GCAP2 mJRNA
All GCAPl and GCAP2 clones were truncated at the 5'-UTR,
the 3'-UTR, or both. None contained a polyA tail, and only
mouse GCAPl appeared to be alternatively spliced at very low
levels.8 Northern blots of mouse retina mRNA revealed single
transcripts of 0.8 kb (GCAPl) and 2 kb (GCAP2; Fig. 1). The
open reading frames of the GCAPl and GCAP2 mRNA predict
polypeptides of nearly identical size (202 and 201 amino acids,
respectively) and identical domain structure (see Fig. 7). The
predicted polypcptide sequences are very similar (90%) to
those of corresponding human and bovine GCAPs (Fig. 7 and
Tail-to-Tail GCAP Gene Array
To elucidate the gene arrangement in the mouse, we isolated
three overlapping genomic clones AMG1, AMG2, and AMG3 (Fig.
2). Subcloning of genomic EcoRl fragments, polymerase chain
reaction amplification of the intergenic region, and direct se-
quencing showed that the tail-to-tail gene array is preserved in the
mouse and that AMG3 contained the entire GCAPl gene, whereas
AMG1 and AMG2 contained the GCAP2 gene and only portions of
the GCAPl gene (Fig. 2). The GCAPl and GCAP2 coding regions
were each interrupted by three introns whose respective posi-
tions were identical (Figs. 2, 7). The sizes of the introns varied,
ranging from 35 kb to 280 bp, and no sequence similarity was
found among corresponding introns of die two genes. Transcrip-
tion start points of the GCAP genes have not been determined,
but the size of the mRNA for GCAPl (800 bp) would predict
transcription start and polyadenylation sites within 100 bp of the
borders of the coding region of this gene. No consensus polyad-
IOVS, May 1998, Vol. 39, No. 6
Mouse GCAP Gene Array 871
FIGURE 5. Tmmunolocalization of GCAP (guanylate cyclase activating protein) 2 in retinal
sections from normal and transgenic human growth hormone (hGH) mice, (a) GCAP2 staining
is detectable in outer segments, inner segments, and soma of photoreceptors; the outer
plexiform layer; amacrine cells (a) of the inner nuclear layer; and ganglion cells, (b) Proteinase
K treatment produces a staining pattern identical with that shown in (a) for all layers, with an
increase in intensity of staining of the signal within the inner segments of photoreceptors. (c)
GCAP2 immunostaining is indicated by fluorescein isotliiocyanate (FITC) staining of the outer
and inner segments of rods, rod soma, and synaptic termini. Weak double-staining of cones
with Texas red- conjugated peanut agglutinin indicates the presence of GCAP2. (d), blue cone
soma and inner segments labeled with Texas Red also show a relatively weak continuation of
labeling of the outer segments with GCAP2 (FITC). (e) lower magnification of c. Scale bars,
12.5 /Am. BC, bipolar cell; CS, cone somata.
enylation signals could be identified close to the translation stop
codons of either gene, but AATAAA signals17 are present farther
downstream. Thus, die presence of untranslated exons in die
3'-UTR cannot be excluded. The distance between the translation
stop codons of the two mouse GCAP genes is 2.65 kb, approxi-
mately one half of the intergenic distance (4.5 kb) observed in
The GCAP Array Is Located on Mouse
We predicted by synteny with human chromosome structure,
that the mouse GCAP I gene should be located on chromosome
17.s To verify the location of the GCAP gene array, we used
biotinylated AMG3 as a probe for fluorescent in situ hybridiza-
tion (FISH) studies of mouse chromosomes. The initial local-
ization to chromosome 17 was made on 4,6-diamidino-2-phe-
nylindole (DAI3!)-banded chromosomes and was confirmed
using mouse chromosome 17 paint (Fig. 3). No significant
labeling was observed on other chromosomes, indicating ab-
sence of pseudogenes or other closely related GCAP genes, a
result consistent with those obtained by fluorescent in situ
hybridization in human chromosomes.
Immunolocalization of GCAP1
We performed inimunocytochemical analyses using monospe-
cific antibodies for GCAP1 and GCAP2 in sections of normal
mouse retina, in combination with fluorochrome-conjugated PNA
to identify cones. To distinguish among cone types, we also
analyzed sections of transgenic mouse retinas that express hGH
specifically in blue cones and bipolar cells.15 For GCAP1 immu-
nolocalization, we used UW14, a monospecinc polyclonal anti-
body raised against bacterially expressed GCAP.6 When applied to
sections from normal mouse retina, the most intense response
was seen in the outer segments and synaptic termini of rods and
cones (Fig. 4a), a distribution consistent with that reported earlier
in the bovine retina.6 The signal was completely abolished by
872 Howes et al.
IOVS, May 1998, Vol. 39, No. 6
FIGURE 6. Immunocytochemical localization of GCAP (guanylate cyclase activating protein) 2
in dark-adapted (a) versus light-adapted (b) mice. Color-enhanced fluorescein-labeled section
superimposed on the bright-field background. Predominant staining for GCAP2 resides within
outer segments and the outer plexiform layer for light- and dark-adapted mice. A slight increase
in GCAP2 signal is visible in the inner segment and outer nuclear layer of dark-adapted mouse
retinas. Bar, 25 jxm. IS, inner segment; OS, outer segments; ONL, outer nuclear layer; OPL,
outer plexiform layer.
preabsorption of the UW14 antibody with 25 Mg/ml bovine
GCAP1 (Fig. 4d). Sections double stained with PNA (FITC) and
GCAP1 (Texas red) revealed the presence of numerous cone
photoreceptors, the outer segments of which were double
stained (Fig. 4b). When transgenic retinas expressing hGH were
double labeled with UW14 and anti-hGH antibody, blue cone
inner segments were positive for hGH, blue cone outer segments
for GCAP1, and rods for only GCAP1 (Fig. 4c). As expected,
bipolar cells were stained only with hGH. The results show that
GCAPl is present at high levels in mouse rod and cone outer
segments in agreement with results in bovine.6
Immunolocalization of GCAP2
For GCAP2 localization experiments, we used a monospe-
cific polyclonal antibody raised against bacterially expressed
GCAP2.9 In normal adult mouse retinas, GCAP2 responses
were seen in the outer segments and, to a lesser extent, in
the inner segments of rods and in the synaptic terminals
(Fig. 5a). Pretreatment of sections with proteinase K re-
sulted in a substantial increase in GCAP2 immunolabeling
only in the inner segments (Fig. 5b). Proteinase K treatment
did not alter GCAPl or hGH staining (data not shown). The
intensity of GCAP2 staining of the rod inner segment was
much stronger than that observed in GCAPl (Fig. 4a). In
addition, immunostaining was seen in the inner retina in
amacrine and ganglion cell types (but not in bipolar cells),
whereas GCAPl staining had been limited to photoreceptors
(Fig. 4a). When cones were double labeled with PNA (Texas
red) and UW50 (FITC), only relatively weak GCAP2 immu-
nostaining was detected in cone outer and inner segments
(Figs. 5c, 5e). Blue cones, specifically identified with anti-
hGH antibody, also only weakly costained with UW50 (Fig.
5d). In monkey and human, GCAP2 was strongly detected by
immunocytochemistry in cone inner segments.9 In bovine,
GCAP2 staining of the cone inner segments is less evident
than that seen in monkey and human.
Light-Dark Dependence of GCAP2 Staining
When GCAP2 staining was performed in parallel with light-
and dark-adapted retinas (Fig. 6), no significant differences
were observed in regard to predominant staining for GCAP2
in the outer segments of rods and the outer plexiform layer.
The only differences are a slight increase in GCAP2 staining
in the inner segment region as well as in the outer nuclear
layer of dark-adapted mice, possibly indicating a replenish-
ment mechanism for GCAP2 during the scotophase. We
conclude that in mouse, GCAP2 staining is strong in rods,
including synaptic termini, but is nearly undetectable in
cones. In contrast to GCAPl, GCAP2 can also be detected in
the inner retina, particularly in amacrine and ganglion cells
in which its function is unknown. There is no significant
effect of light- dark adaptation on the apparent distribution
of GCAP2 in the mouse retina.
IOVS, May 1998, Vol. 39, No. 6
Mouse GCAP Gene Array 873
FIGURE 7. Alignment of GCAP (guanylate cyclase activating protein) 1 and GCAP2 amino acid
sequences from various species. The sequences were divided into two subgroups, GCAP1 and
GCAP2. In the GCAP1 subgroup, the mouse GCAPl sequence8 was aligned with GCAP1 sequences
from bovine, human, frog,4 and chicken.20 In the GCAP2 subgroup, the mouse GCAP2 sequence
was aligned with the GCAP2 sequences from human,7 bovine,6 and chicken.20 L=I=V=M; Y=F;
E=D; R=K; A=T=S are considered conservative substitutions. For best fit, several gaps were
introduced (shown by hyphens). Residues conserved in all GCAP sequences are shown on black
background. Residues identical in only one of the groups are lightly shaded. Predicted EF hand
Ca2+-binding domains (EF2-EF4) are boxed, EF1 (presumably not functional for Ca2+-binding) is
boxed by a broken line. The identical positions of introns a- c in mouse, human, and bovine
GCAPs are shown by triangles above the alignments. Domains conserved in all GCAPs shown are
identified by a shaded bar above the alignment (CD1-CD3), and variable domains are shown by
VD1-VD4. A dendrogram (PC-GENE, IntelliGenetics; Mountain View, CA) calculated on the basis
of the amino acid sequences is shown at the top left.
GCAPs are members of a subfamily of Ca2+-binding proteins
belonging to the large superfamily of calmodulin-like Ca2+-
binding proteins,18 which characteristically contain four EF
hand helix-loop-helix motifs.19 Five GCAPl and GCAP2
genes- cDNA have been cloned to date from various verte-
brate species.4"6'8'20 A consensus is emerging concerning
conserved and variable domains and concerning domains
involved in membrane association and GC stimulation. Vari-
able domains include the N- and C-terminal ends and spacer
domains between EF1-EF2 and EF3-EF4 (Fig. 7). Function-
ally indispensable domains include regions surrounding the
four EF hand motifs. The exact stoichiometry of bound
Ca2+/M r;rAP has not been determined, but EF1 is thought
to be nonfunctional, whereas EF2, EF3 and EF4 conform to
the EF hand consensus sequence and most likely are fully
functional.1 Inactivation of EF hand Ca2+binding domains
in GCAP2 by mutagenesis produces a constitutive activator
of GC lacking Ca2+ sensitivity.21 Similar effects have been
seen with mutant GCAPl.22 For GCAPl we also showed that
the N-terminal domain, although variant in the GCAPs, is
indispensable for GC stimulation and membrane associa-
tion.23 All GCAPs appear to be myristoylated at the N ter-
minus of the processed proteins (Gly-2; Fig. 7). The fatty
acid side chain, however, is not directly involved in mem-
brane association, because a GCAPl-G2A mutant, in which
the myr anchor Gly-2 was replaced by Ala, sediments with
rod outer segment membranes.23 Membrane association of
GCAPl was only abolished when the N-terminal 25 amino
acids were deleted, a domain that does not contain an
obvious motif for protein-membrane interaction.
In the present study, we attempted to identify the cell
types and the subcellular compartments of cells expressing
GCAPs in the mouse retina. The mouse retina has no fovea, but
874 Howes et al.
IOVS, May 1998, Vol. 39, No. 6
it contains a substantial number of cones (3%-9% of all photo-
receptors).24'25 The cones can be identified, among other tech-
niques, by staining with peanut agglutinin.26 Our study shows
apparent distribution of GCAP2 mostly in rods and certain cell
types of the inner retina but near absence of GCAP2 in cones.
In bovine retina,9 the GCAP2 signal is generally more intense in
the inner segments of rods and is weaker in cones, but in
monkey and human retinas, the GCAP2 signal is more intense
in cone inner segments and is much weaker in rods. The
reason for this apparent discrepancy among species is un-
known. The specificity of the antibodies used in this study was
examined by early experiments in which a complete loss of
immunocytochemical signal in the mouse retina occurred
when UW14 and UW50 antibodies were preabsorbed with
bovine GCAP1 and GCAP2 proteins, respectively (data not
shown). One possible reason for the discrepancy in GCAP2
staining among species could be the partial masking of the
epitope recognized by the anti-GCAP2 antibody UW50. To
investigate this possibility, mouse retina sections were sub-
jected to limited proteinase K treatment before immunocyto-
chemistry. GCAP2 staining in inner segments of rods was
substantially increased (compare Fig. 5A with 5B), consistent
with partial removal of masking protein antigens. Limited pro-
teolysis, however, did not significantly improve GCAP2 stain-
ing in mouse cones. Furthermore, comparison of dark- and
light-adapted mouse retinas did not reveal significant differ-
ences in GCAP2 distribution. Based on these results and re-
ports by others on GCAP2 distribution in other animals, we
conclude that there is an apparent species-dependent variation
of GCAP2 levels in cones.
In contrast to most other calmodulin-like Ca2+-binding
proteins, the function of GCAPs has been unambiguously
established: Both GCAPs stimulate photoreceptor GC in low
free [Ca2+].' Important questions that remain concern the
specific roles that GCAPs play in regulating the function of
photoreceptors and in pathways unrelated to phototrans-
duction in photoreceptors or in other retinal cells. Common
to both GCAPs is their presence in the synaptic region of
photoreceptors, a region remote from phototransduction.
cGMP-gated channels have been observed in these subcel-
lular compartments, which suggests that a GC-GCAP system
unrelated to phototransduction may exist in synaptic termi-
nals of photoreceptors. A major difference is the presence of
GCAP2 and absence of GCAP1 in the inner retina, particu-
larly in amacrine and ganglion cells. The function of GCAP2
in the inner retina, or the pathways in which it participates,
Distinct cellular or subcellular expression of GCAPs would
be consistent with the tail-to-tail arrangements of their genes.
Such arrangements of related genes occur in a variety of mam-
malian genes.7 Gene duplication mechanisms that lead to tail-
to-tail orientations require an inversion in addition to nonho-
mologous breakage. The consequence of inversion is that the
5' regulatory elements governing tissue specificity are located
on opposite ends of the gene arrangement, thus allowing for
divergent evolution and differential tissue or subcellular ex-
pression. The presence of GCAP tail-to-tail arrangements in
human and mouse suggests that the GCAP duplication-inver-
sion event occurred before mammalian diversification, more
than 300 million years ago.
Based on the tail-to-tail gene arrangement and similar ex-
pression patterns of the GCAPs, we conclude that the mouse
will provide a unique model for genetic manipulation. The
gene array will allow for double-knockout constructs, and the
consequence of absence of both GCAPs on retinal develop-
ment and function can be examined. In addition, single knock-
outs will answer the question of whether the GCAPs provide
redundant or unique functions in photoreceptor cells. Eventu-
ally, genetically altered mouse models will most likely resolve
unanswered questions on localization and function of GCAPs
in the retina.
The authors thank Krzysztof Palczewski for GCAPl-specific and
GCAP2-specinc antibodies and for many helpful suggestions during
this work, Sue Semple-Rowland and Jeanne Frederick for critical
comments and careful review of the manuscript, and Trish Goede for
help with questions concerning the generation of photomicrographs
and the use of the computer software.
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