Each rhodopsin molecule binds its own arrestin.
Proceedings of the National Academy of Sciences of the United States of America (2007)
- DOI: 10.1073/pnas.0610886104
- PubMed: 17360618
Available from www.pubmedcentral.nih.gov
or
Abstract
Arrestins (Arrs) are ubiquitous regulators of the most numerous family of signaling proteins, G protein-coupled receptors. Two models of the Arr-receptor interaction have been proposed: the binding of one Arr to an individual receptor or to two receptors in a dimer. To determine the binding stoichiometry in vivo, we used rod photoreceptors where rhodopsin (Rh) and Arr are expressed at comparably high levels and where Arr localization in the light is determined by its binding to activated Rh. Genetic manipulation of the expression of both proteins shows that the maximum amount of Arr that moves to the Rh-containing compartment exceeds 80%, but not 100%, of the molar amount of Rh present. In vitro experiments with purified proteins confirm that Arr "saturates" Rh at a 1:1 ratio. Thus, a single Rh molecule is necessary and sufficient to bind Arr. Remarkable structural conservation among receptors and Arrs strongly suggests that all Arr subtypes bind individual molecules of their cognate receptors.
Author-supplied keywords
Available from www.pubmedcentral.nih.gov
Page 1
Each rhodopsin molecule binds its own arrestin.
Each rhodopsin molecule binds its own arrestin
Susan M. Hanson, Eugenia V. Gurevich, Sergey A. Vishnivetskiy, Mohamed R. Ahmed, Xiufeng Song,
and Vsevolod V. Gurevich*
Department of Pharmacology, Vanderbilt University, 2200 Pierce Avenue, PRB, Room 418, Nashville, TN 37232
Communicated by John H. Exton, Vanderbilt University School of Medicine, Nashville, TN, January 4, 2007 (received for review September 19, 2006)
Arrestins (Arrs) are ubiquitous regulators of the most numerous
family of signaling proteins, G protein-coupled receptors. Two models
of the Arr–receptor interaction have been proposed: the binding of
one Arr to an individual receptor or to two receptors in a dimer. To
determine the binding stoichiometry in vivo, we used rod photore-
ceptors where rhodopsin (Rh) and Arr are expressed at comparably
high levels and where Arr localization in the light is determined by its
binding to activated Rh. Genetic manipulation of the expression of
both proteins shows that the maximum amount of Arr that moves to
the Rh-containing compartment exceeds 80%, but not 100%, of the
molar amount of Rh present. In vitro experiments with purified
proteins confirm that Arr ‘‘saturates’’ Rh at a 1:1 ratio. Thus, a single
Rh molecule is necessary and sufficient to bind Arr. Remarkable
structural conservation among receptors and Arrs strongly suggests
that all Arr subtypes bind individual molecules of their cognate
receptors.
protein–protein interactions signal shutoff vision receptor
dimerization
G protein-coupled receptors (GPCRs) are the most numerousfamily of signaling proteins. Arrestins (Arrs) bind to phos-
phorylated activated receptors terminating G protein activation
and redirecting the signaling to alternative pathways, in which Arr
serves as an adaptor for protein kinase Src, ubiquitin ligase Mdm2,
phosphodiesterase PDE4, or as a scaffold forMAP kinase cascades
(1, 2). The dimerization of some GPCRs (3, 4) led to the idea that
one Arr needs both receptors in a dimer to bind (5). The stoichi-
ometry of the Arr–receptor complex has profound implications for
the mechanism of Arr-mediated desensitization of GPCRs and
receptor trafficking. The composition of the complex, i.e., the size
and structure of the ‘‘signalosome,’’ also determines its scaffolding
potential and the ability to initiate the ‘‘second round’’ of signaling
by organizing downstream proteins, such as Src, MAP kinases,
ubiquitin ligase, etc.
Rod photoreceptors provide a unique model where the stoi-
chiometry of the Arr–receptor interaction can be determined in
vivo because they express comparable amounts of the receptor
(Rh) and Arr at very high levels unparalleled in any other cell
type (6–8). In dark-adapted rods, Arr is predominantly localized
in the inner segment, where it is held by its low-affinity binding
to microtubules that are abundant in this compartment (9). Light
induces Arr translocation to the outer segment (OS), where it is
retained in bright light through binding to light-activated Rh (8,
9). The expression of Rh and Arr can be genetically manipulated
independently, and the extent of Arr translocation can be used
to determine the stoichiometry of their interaction.
Results
Hemizygous Rh (Rh/) and Arr (Arr/) mice express about half
of these respective proteins as compared with wild-type animals
(10, 11). To obtain mice expressing different ratios of Arr and Rh,
we bred Rh/, Arr/, and Rh//Arr/ animals and compared
the content of Rh and Arr in their retinas with that of wild-type
mice (Table 1). Both proteins were measured by quantitative
Western blot in the homogenates of whole eyecups by using the
corresponding purified proteins to construct calibration curves
(Fig. 1). The results indicate that in both cases the elimination of
one allele reduces the expression by half, so that the Arr/Rh ratio
in wild-type and doubly hemizygous animals is similar (0.8:1 and
0.94:1, respectively), whereas in Arr/ and Rh/ mice it is
significantly shifted in the expected direction, to 0.38:1 and 1.74:1,
respectively (Table 1). The absolute levels of Rh andArr expression
in wild-type retinas obtained by these measurements are in good
agreement with previous reports (6–8).
If two Rh molecules bind one Arr, virtually complete Arr
translocation to theOS in the lightwould only be expected inArr/
animals but not in wild-type mice. In contrast, in the case of a 1:1
interaction, the translocation would be incomplete only in Rh/
animals that express Arr in excess of Rh. We invariably observed
virtually complete Arr translocation in the light-adapted retinas of
mouse lines expressing more Rh than Arr (Fig. 1). Quantitative
image analysis shows that in wild-type mice and the other two lines
that express excess Rh, 81–89% of Arr translocates to the OS in the
light (Table 1). Not surprisingly, in Rh/ mice, only about half of
that amount of Arr moves to the OS (Table 1). These data are
consistent with 1:1 binding and cannot be reconciledwith themodel
of Rh dimer interacting with just one Arr molecule. Based on the
1:1 model, the extent of Arr translocation, and the absolute
expression levels of both proteins, we calculated that in the light-
adapted retinas of these mice, 65–83% of Rh is occupied at steady
state by bound Arr, with the exception of Arr/ mice, where Arr
is limiting and Rh occupancy is about half that in wild type (33%)
(Table 1). The Rh occupancy in both Rh/ lines (75–83%; Table
1) exceeds the theoretical maximum for a 1:2model up to 1.66-fold.
To further extend the range of Arr/Rh ratios in vivo we
also analyzed transgenic mice expressing mutant mouse
Arr(L374A,V375A,F376A) under the control of the Rh promoter.
Earlier. we showed that this mutant demonstrates light-dependent
translocation that is qualitatively similar to wild-type Arr but
proceeds more slowly (9). However, after 60 min, the translocation
of both WT and mutant Arr reaches its maximum (9). Here, we
used the transgenic line expressing thisArr at 240%ofwild type and
compared Arr translocation in tgArr/ and tgArr/ mice that
have Arr/Rh ratios of 2.4 and 2.7, respectively (Table 1). As
expected, in animals with a large excess of Arr, the extent of its
light-dependent translocation to the OS was much lower (29% and
23%, respectively, which is equivalent to Rh occupancy of 65–72%)
(Fig. 1). Importantly, the percentage of Arr that moves to the OS
in the light progressively decreases with an increasing excess of Arr
over Rh, but the absolute amount of Arr in the OS is proportional
to Rh expression and remains essentially the same: 0.23–0.26 nmol
in Rh/ mice, and 0.13–0.15 nmol in both Rh/ lines. These
numbers correspond to 0.65–0.83mol of Arr per 1mol of Rh. Thus,
Author contributions: S.M.H. and V.V.G. designed research; S.M.H., E.V.G., S.A.V., M.R.A.,
X.S., and V.V.G. performed research; S.M.H., E.V.G., S.A.V., M.R.A., X.S., and V.V.G. analyzed
data; and S.M.H., E.V.G., and V.V.G. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: GPCR, G protein-coupled receptor; OS, outer segment; Arr, arrestin; Rh,
rhodopsin.
*To whom correspondence should be addressed. E-mail: vsevolod.gurevich@vanderbilt.
edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0610886104/DC1.
© 2007 by The National Academy of Sciences of the USA
www.pnas.orgcgidoi10.1073pnas.0610886104 PNAS February 27, 2007 vol. 104 no. 9 3125–3128
BI
O
CH
EM
IS
TR
Y
Susan M. Hanson, Eugenia V. Gurevich, Sergey A. Vishnivetskiy, Mohamed R. Ahmed, Xiufeng Song,
and Vsevolod V. Gurevich*
Department of Pharmacology, Vanderbilt University, 2200 Pierce Avenue, PRB, Room 418, Nashville, TN 37232
Communicated by John H. Exton, Vanderbilt University School of Medicine, Nashville, TN, January 4, 2007 (received for review September 19, 2006)
Arrestins (Arrs) are ubiquitous regulators of the most numerous
family of signaling proteins, G protein-coupled receptors. Two models
of the Arr–receptor interaction have been proposed: the binding of
one Arr to an individual receptor or to two receptors in a dimer. To
determine the binding stoichiometry in vivo, we used rod photore-
ceptors where rhodopsin (Rh) and Arr are expressed at comparably
high levels and where Arr localization in the light is determined by its
binding to activated Rh. Genetic manipulation of the expression of
both proteins shows that the maximum amount of Arr that moves to
the Rh-containing compartment exceeds 80%, but not 100%, of the
molar amount of Rh present. In vitro experiments with purified
proteins confirm that Arr ‘‘saturates’’ Rh at a 1:1 ratio. Thus, a single
Rh molecule is necessary and sufficient to bind Arr. Remarkable
structural conservation among receptors and Arrs strongly suggests
that all Arr subtypes bind individual molecules of their cognate
receptors.
protein–protein interactions signal shutoff vision receptor
dimerization
G protein-coupled receptors (GPCRs) are the most numerousfamily of signaling proteins. Arrestins (Arrs) bind to phos-
phorylated activated receptors terminating G protein activation
and redirecting the signaling to alternative pathways, in which Arr
serves as an adaptor for protein kinase Src, ubiquitin ligase Mdm2,
phosphodiesterase PDE4, or as a scaffold forMAP kinase cascades
(1, 2). The dimerization of some GPCRs (3, 4) led to the idea that
one Arr needs both receptors in a dimer to bind (5). The stoichi-
ometry of the Arr–receptor complex has profound implications for
the mechanism of Arr-mediated desensitization of GPCRs and
receptor trafficking. The composition of the complex, i.e., the size
and structure of the ‘‘signalosome,’’ also determines its scaffolding
potential and the ability to initiate the ‘‘second round’’ of signaling
by organizing downstream proteins, such as Src, MAP kinases,
ubiquitin ligase, etc.
Rod photoreceptors provide a unique model where the stoi-
chiometry of the Arr–receptor interaction can be determined in
vivo because they express comparable amounts of the receptor
(Rh) and Arr at very high levels unparalleled in any other cell
type (6–8). In dark-adapted rods, Arr is predominantly localized
in the inner segment, where it is held by its low-affinity binding
to microtubules that are abundant in this compartment (9). Light
induces Arr translocation to the outer segment (OS), where it is
retained in bright light through binding to light-activated Rh (8,
9). The expression of Rh and Arr can be genetically manipulated
independently, and the extent of Arr translocation can be used
to determine the stoichiometry of their interaction.
Results
Hemizygous Rh (Rh/) and Arr (Arr/) mice express about half
of these respective proteins as compared with wild-type animals
(10, 11). To obtain mice expressing different ratios of Arr and Rh,
we bred Rh/, Arr/, and Rh//Arr/ animals and compared
the content of Rh and Arr in their retinas with that of wild-type
mice (Table 1). Both proteins were measured by quantitative
Western blot in the homogenates of whole eyecups by using the
corresponding purified proteins to construct calibration curves
(Fig. 1). The results indicate that in both cases the elimination of
one allele reduces the expression by half, so that the Arr/Rh ratio
in wild-type and doubly hemizygous animals is similar (0.8:1 and
0.94:1, respectively), whereas in Arr/ and Rh/ mice it is
significantly shifted in the expected direction, to 0.38:1 and 1.74:1,
respectively (Table 1). The absolute levels of Rh andArr expression
in wild-type retinas obtained by these measurements are in good
agreement with previous reports (6–8).
If two Rh molecules bind one Arr, virtually complete Arr
translocation to theOS in the lightwould only be expected inArr/
animals but not in wild-type mice. In contrast, in the case of a 1:1
interaction, the translocation would be incomplete only in Rh/
animals that express Arr in excess of Rh. We invariably observed
virtually complete Arr translocation in the light-adapted retinas of
mouse lines expressing more Rh than Arr (Fig. 1). Quantitative
image analysis shows that in wild-type mice and the other two lines
that express excess Rh, 81–89% of Arr translocates to the OS in the
light (Table 1). Not surprisingly, in Rh/ mice, only about half of
that amount of Arr moves to the OS (Table 1). These data are
consistent with 1:1 binding and cannot be reconciledwith themodel
of Rh dimer interacting with just one Arr molecule. Based on the
1:1 model, the extent of Arr translocation, and the absolute
expression levels of both proteins, we calculated that in the light-
adapted retinas of these mice, 65–83% of Rh is occupied at steady
state by bound Arr, with the exception of Arr/ mice, where Arr
is limiting and Rh occupancy is about half that in wild type (33%)
(Table 1). The Rh occupancy in both Rh/ lines (75–83%; Table
1) exceeds the theoretical maximum for a 1:2model up to 1.66-fold.
To further extend the range of Arr/Rh ratios in vivo we
also analyzed transgenic mice expressing mutant mouse
Arr(L374A,V375A,F376A) under the control of the Rh promoter.
Earlier. we showed that this mutant demonstrates light-dependent
translocation that is qualitatively similar to wild-type Arr but
proceeds more slowly (9). However, after 60 min, the translocation
of both WT and mutant Arr reaches its maximum (9). Here, we
used the transgenic line expressing thisArr at 240%ofwild type and
compared Arr translocation in tgArr/ and tgArr/ mice that
have Arr/Rh ratios of 2.4 and 2.7, respectively (Table 1). As
expected, in animals with a large excess of Arr, the extent of its
light-dependent translocation to the OS was much lower (29% and
23%, respectively, which is equivalent to Rh occupancy of 65–72%)
(Fig. 1). Importantly, the percentage of Arr that moves to the OS
in the light progressively decreases with an increasing excess of Arr
over Rh, but the absolute amount of Arr in the OS is proportional
to Rh expression and remains essentially the same: 0.23–0.26 nmol
in Rh/ mice, and 0.13–0.15 nmol in both Rh/ lines. These
numbers correspond to 0.65–0.83mol of Arr per 1mol of Rh. Thus,
Author contributions: S.M.H. and V.V.G. designed research; S.M.H., E.V.G., S.A.V., M.R.A.,
X.S., and V.V.G. performed research; S.M.H., E.V.G., S.A.V., M.R.A., X.S., and V.V.G. analyzed
data; and S.M.H., E.V.G., and V.V.G. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: GPCR, G protein-coupled receptor; OS, outer segment; Arr, arrestin; Rh,
rhodopsin.
*To whom correspondence should be addressed. E-mail: vsevolod.gurevich@vanderbilt.
edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0610886104/DC1.
© 2007 by The National Academy of Sciences of the USA
www.pnas.orgcgidoi10.1073pnas.0610886104 PNAS February 27, 2007 vol. 104 no. 9 3125–3128
BI
O
CH
EM
IS
TR
Y
Page 2
Arr translocation in every genetic background is only consistent
with the 1:1 model of Arr–Rh binding in vivo.
To further test this model and ascertain that other proteins
present in live photoreceptors do not affect the translocation of
Arr to the OS, we reproduced similar Arr/Rh ratios (0.5, 1.1, 2.2,
and 3.3) in vitro with purified proteins carefully quantified by
amino acid analysis. In these experiments, we mixed recombi-
nant bovine Arr with bovine Rh in native disk membranes that
was phosphorylated with endogenous Rh kinase (12) and fully
regenerated with 11-cis-retinal, illuminated the samples for 5
min at 37°C, and then pelleted Rh along with bound Arr. Control
samples contained the same amounts of Arr and no Rh. Equal
aliquots of the original samples, pellets, and supernatants were
resolved by SDS/PAGE and stained with Coomassie blue (Fig.
2). In addition, we measured the amounts of Rh and Arr in the
original samples, pellets, and supernatants by quantitative West-
ern blot with appropriate standards (Fig. 2). We found that Rh
pellets quantitatively, whereas the amount of Arr pelleted in the
absence of Rh does not exceed 4.5%. Under these conditions we
observed clear saturation of Rh by increasing concentrations of
Arr, reaching up to 0.9 mol of specifically bound Arr per 1 mol
of Rh (Fig. 2). These results are in perfect agreement with our
in vivo data (Fig. 1) and can only be rationalized in the context
of the model of one Arr molecule binding one Rh molecule.
Discussion
Because of their mechanism of action, a molecule of agonist or a
photon of light can activate only a single GPCR or visual pigment,
respectively. Therefore, until recently, it was implicitly assumed that
a singleGPCR is a signaling unit that activates its cognateGprotein
and becomes subsequently inactivated by Arr binding (recently
reviewed in ref. 13). The observations by indirect and direct
methods that many GPCRs (14), including Rh (4), dimerize, along
with the reports that GPCR heterodimerization affects the phar-
macological profile of their signaling (14, 15), challenged this view.
The diameter of the cytoplasmic tip of the onlyGPCR for which the
crystal structure has been determined, dark (inactive) Rh (16, 17),
is40 Å, which is about half the length of the long axis of the two
Table 1. Light-dependent translocation of arrestin to the outer segment as a function of arrestin and rhodopsin expression
Genotype
Rh content,
nmol per retina
Arr content,
nmol per retina
Arr/Rh
molar ratio
Arr, % in
OS (dark)
Arr, % in
OS (light)
Arr in OS
(light), nmol
Rh occupied
by Arr, %
WT 0.40 0.05 (7) 0.32 0.04 (7) 0.80 1.8 0.8 81.4 1.3 0.26 65
A/ 0.40 0.03 (5) 0.15 0.02 (5) 0.38 3.1 1.1 88.8 0.5 0.13 33
A/Rh/ 0.18 0.02 (4) 0.17 0.02 (4) 0.94 1.4 0.5 89.1 1.0 0.15 83
Rh/ 0.19 0.01 (4) 0.33 0.02 (4) 1.74 1.1 0.4 43.5 6.5 0.14 75
tg A/ 0.32 0.02 (4) 0.77 0.04 (4) 2.41 1.2 0.2 29.3 6.2 0.23 72
tg A/ 0.37 0.05 (5) 1.01 0.03 (5) 2.70 1.3 0.7 23.4 3.4 0.24 65
Mice with the indicated genotypes were dark-adapted overnight (dark) or exposed to 2,700 lux for 1 h (light). One eyecup from each mouse was fixed and
processed for immunohistochemistry (9), whereas the other was homogenized for Rh and Arr quantification by Western blot as described (7, 37), by using the
corresponding purified proteins to construct calibration curves. The proportion of Arr localized in the OS was quantified by the intensity of Arr immunostaining
in 10 images per animal from 3–5 animals per genotype per light condition. Means SD are shown. The data were analyzed by one-way ANOVA with genotype
as a main factor. The Rh content of A/Rh/ and Rh/ and the arrestin content of A/ and A/Rh/ were statistically different from all other genotypes
(P 0.0001) but were not different from each other. Arr expression in tg A/ and tg A/ animals was statistically different from all other genotypes (P
0.0001). Statistical significance of the differences in the percent of Arr in the OS in the light are indicated in Fig.1. The amount of Arr in the OS (nanomoles) was
calculated by multiplying the Arr content by the percentage of Arr in the OS in the light. The percentage of Rh occupied was determined by dividing this value
by the total Rh content.
Fig. 1. The extent of light-dependent Arr translocation is determined by the Arr/Rh expression ratio. (A) Mice with the indicated genotypes were dark-adapted
overnight (DARK) or exposed to 2,700 lux for 1 h (LIGHT). One eyecup was fixed and processed for Arr immunohistochemistry (9). The positions of the outer (OS)
and inner segments (IS) and of the outer nuclear layer (ONL) are indicated. (B) The proportion of Arr localized in the OS was quantified by the intensity of Arr
immunostaining (green) in 10 images per animal from 3–5 animals per genotype per light condition. Means SD are shown. The data for light- and dark-adapted
mice were analyzed separately by one-way ANOVA with genotype as a main factor. Statistical significance of the differences is indicated above the corresponding
bars: *, P 0.001; **, P 0.0001. (C) Typical Western blots for Arr and Rh (Rh) in mice with the indicated genotypes and corresponding standards.
3126 www.pnas.orgcgidoi10.1073pnas.0610886104 Hanson et al.
with the 1:1 model of Arr–Rh binding in vivo.
To further test this model and ascertain that other proteins
present in live photoreceptors do not affect the translocation of
Arr to the OS, we reproduced similar Arr/Rh ratios (0.5, 1.1, 2.2,
and 3.3) in vitro with purified proteins carefully quantified by
amino acid analysis. In these experiments, we mixed recombi-
nant bovine Arr with bovine Rh in native disk membranes that
was phosphorylated with endogenous Rh kinase (12) and fully
regenerated with 11-cis-retinal, illuminated the samples for 5
min at 37°C, and then pelleted Rh along with bound Arr. Control
samples contained the same amounts of Arr and no Rh. Equal
aliquots of the original samples, pellets, and supernatants were
resolved by SDS/PAGE and stained with Coomassie blue (Fig.
2). In addition, we measured the amounts of Rh and Arr in the
original samples, pellets, and supernatants by quantitative West-
ern blot with appropriate standards (Fig. 2). We found that Rh
pellets quantitatively, whereas the amount of Arr pelleted in the
absence of Rh does not exceed 4.5%. Under these conditions we
observed clear saturation of Rh by increasing concentrations of
Arr, reaching up to 0.9 mol of specifically bound Arr per 1 mol
of Rh (Fig. 2). These results are in perfect agreement with our
in vivo data (Fig. 1) and can only be rationalized in the context
of the model of one Arr molecule binding one Rh molecule.
Discussion
Because of their mechanism of action, a molecule of agonist or a
photon of light can activate only a single GPCR or visual pigment,
respectively. Therefore, until recently, it was implicitly assumed that
a singleGPCR is a signaling unit that activates its cognateGprotein
and becomes subsequently inactivated by Arr binding (recently
reviewed in ref. 13). The observations by indirect and direct
methods that many GPCRs (14), including Rh (4), dimerize, along
with the reports that GPCR heterodimerization affects the phar-
macological profile of their signaling (14, 15), challenged this view.
The diameter of the cytoplasmic tip of the onlyGPCR for which the
crystal structure has been determined, dark (inactive) Rh (16, 17),
is40 Å, which is about half the length of the long axis of the two
Table 1. Light-dependent translocation of arrestin to the outer segment as a function of arrestin and rhodopsin expression
Genotype
Rh content,
nmol per retina
Arr content,
nmol per retina
Arr/Rh
molar ratio
Arr, % in
OS (dark)
Arr, % in
OS (light)
Arr in OS
(light), nmol
Rh occupied
by Arr, %
WT 0.40 0.05 (7) 0.32 0.04 (7) 0.80 1.8 0.8 81.4 1.3 0.26 65
A/ 0.40 0.03 (5) 0.15 0.02 (5) 0.38 3.1 1.1 88.8 0.5 0.13 33
A/Rh/ 0.18 0.02 (4) 0.17 0.02 (4) 0.94 1.4 0.5 89.1 1.0 0.15 83
Rh/ 0.19 0.01 (4) 0.33 0.02 (4) 1.74 1.1 0.4 43.5 6.5 0.14 75
tg A/ 0.32 0.02 (4) 0.77 0.04 (4) 2.41 1.2 0.2 29.3 6.2 0.23 72
tg A/ 0.37 0.05 (5) 1.01 0.03 (5) 2.70 1.3 0.7 23.4 3.4 0.24 65
Mice with the indicated genotypes were dark-adapted overnight (dark) or exposed to 2,700 lux for 1 h (light). One eyecup from each mouse was fixed and
processed for immunohistochemistry (9), whereas the other was homogenized for Rh and Arr quantification by Western blot as described (7, 37), by using the
corresponding purified proteins to construct calibration curves. The proportion of Arr localized in the OS was quantified by the intensity of Arr immunostaining
in 10 images per animal from 3–5 animals per genotype per light condition. Means SD are shown. The data were analyzed by one-way ANOVA with genotype
as a main factor. The Rh content of A/Rh/ and Rh/ and the arrestin content of A/ and A/Rh/ were statistically different from all other genotypes
(P 0.0001) but were not different from each other. Arr expression in tg A/ and tg A/ animals was statistically different from all other genotypes (P
0.0001). Statistical significance of the differences in the percent of Arr in the OS in the light are indicated in Fig.1. The amount of Arr in the OS (nanomoles) was
calculated by multiplying the Arr content by the percentage of Arr in the OS in the light. The percentage of Rh occupied was determined by dividing this value
by the total Rh content.
Fig. 1. The extent of light-dependent Arr translocation is determined by the Arr/Rh expression ratio. (A) Mice with the indicated genotypes were dark-adapted
overnight (DARK) or exposed to 2,700 lux for 1 h (LIGHT). One eyecup was fixed and processed for Arr immunohistochemistry (9). The positions of the outer (OS)
and inner segments (IS) and of the outer nuclear layer (ONL) are indicated. (B) The proportion of Arr localized in the OS was quantified by the intensity of Arr
immunostaining (green) in 10 images per animal from 3–5 animals per genotype per light condition. Means SD are shown. The data for light- and dark-adapted
mice were analyzed separately by one-way ANOVA with genotype as a main factor. Statistical significance of the differences is indicated above the corresponding
bars: *, P 0.001; **, P 0.0001. (C) Typical Western blots for Arr and Rh (Rh) in mice with the indicated genotypes and corresponding standards.
3126 www.pnas.orgcgidoi10.1073pnas.0610886104 Hanson et al.
Sign up today - FREE
Mendeley saves you time finding and organizing research. Learn more
- All your research in one place
- Add and import papers easily
- Access it anywhere, anytime
Start using Mendeley in seconds!
Readership Statistics
12 Readers on Mendeley
by Discipline
by Academic Status
33% Post Doc
17% Professor
17% Ph.D. Student
by Country
42% United States
25% Germany
8% South Korea