(Received for publication, October 21, 1994; and in revised form, November 23, 1994)
From the
The cytoplasmic loops of rhodopsin, the rod cell photoreceptor,
play important regulatory roles in the activation of both rhodopsin
kinase and the rod cell G protein, G. A number of studies
have identified domains in rhodopsin that are important for the
activation of G
. However, less is known concerning the
cytoplasmic regions that regulate phosphorylation of the photoreceptor
by rhodopsin kinase. To identify regions that participate in these
processes, a series of alanine mutations were generated in the three
cytoplasmic loops of rhodopsin and transiently expressed in HEK-293
cells. Membranes prepared from these cells were reconstituted with the
opsin chromophore, 11-cis-retinal, and characterized for their
ability to undergo light-dependent phosphorylation by rhodopsin kinase
and to catalyze GTP
S (guanosine
5`-O-(3-thiotriphosphate)) binding to G
. We have
identified mutants that fall into three distinct categories: 1) those
that show altered phosphorylation but normal G
activation,
such as T62A/V63A/Q64A and R147A/F148A/G149A in Loops I and II,
respectively; 2) mutants that have reduced ability to activate G
but are phosphorylated normally, including T242A/T243A and
V250A/T251A/R252A in Loop III; and 3) mutants that affect both
phosphorylation and G
activation, including
A233G/A234G/A235G and A233N/A234N/A235N in Loop III. The use of these
two assays in parallel have allowed us to distinguish the presence of
distinct functional domains within the cytoplasmic loops which are
specific for interaction with rhodopsin kinase or G
.
G protein-coupled receptors are members of a large multigene
family having seven transmembrane-spanning domains that regulate
intracellular signaling pathways in response to external stimuli (Fig. 1). Their function is the activation of heterotrimeric G
proteins, which regulate a growing list of effector enzymes important
in cell growth control and metabolism. One mechanism to limit
continuous G protein activation is homologous desensitization, which is
mediated by phosphorylation of the receptors followed by the binding of
a member of the arrestin family of proteins. These events result in the
generation of receptors unable to interact with their G proteins. The
phosphorylation is mediated by a class of serine/threonine kinases,
known as G protein-coupled receptor kinases (GRKs), ()which
bind only the ligand-activated form of the
receptors(1, 2) . Rhodopsin, the photoreceptor of the
mammalian rod cell, has been used extensively as a model for defining
the molecular interactions between receptors, their G proteins, and the
GRKs that mediate desensitization.
Figure 1: Amino acid sequence of the cytoplasmic domains of bovine rhodopsin. I, II, and III designate the three intracellular loops. IV represents the fourth cytoplasmic loop formed by the palmitoylation of cysteines 322 and 323 and their attachment to the plasma membrane. K296 is the lysine that forms the attachment site for 11-cis-retinal. The asterisks (*) represent the sites of palmitoylation.
Studies of several different G protein-coupled receptors, including rhodopsin, have suggested that the cytoplasmic loops (Fig. 1) play important roles in their interactions with G proteins(3, 4, 5, 6, 7, 8, 9, 10, 11) . In contrast, far less is known concerning the critical sites of interaction between these receptors and GRKs. A series of 7 serines and threonines on the C terminus of rhodopsin serve as phosphoacceptor sites for rhodopsin kinase, the rod cell GRK, although only one or two are thought to be physiologically relevant(4, 12, 13) . Peptide competition studies have suggested that the cytoplasmic loops of the photoreceptor protein are involved in phosphorylation of rhodopsin by rhodopsin kinase(14, 15) . In addition, the efficient phosphorylation of a synthetic peptide corresponding to the C terminus requires activation of the kinase by the cytoplasmic loops of rhodopsin(16, 17, 18) . Mastoparan, which can activate several G proteins in place of their receptors, also potentiates the phosphorylation of the synthetic peptide substrate by rhodopsin kinase (18) . These data suggest that the cytoplasmic loops of rhodopsin participate in the light-dependent activation of the kinase, allowing it to phosphorylate its substrate sites on the C terminus.
Using a series of alanine mutations in the three
cytoplasmic loops of rhodopsin, we have examined the phosphorylation of
rhodopsin by rhodopsin kinase. To control for mutations that may cause
extensive conformational changes in the cytoplasmic loops and therefore
disrupt the ability of light to activate rhodopsin, we have also
measured the activation of the rod cell G protein, G. We
have identified mutants that fall into three distinct categories: 1)
those that affect phosphorylation by rhodopsin kinase, 2) those that
affect G
activation, and 3) mutations that affect both of
these processes. The parallel use of these two assays has allowed us to
begin to discriminate domains on the cytoplasmic surface of rhodopsin
that participate in the phosphorylation of rhodopsin by rhodopsin
kinase from those that are involved in the activation of G
.
Figure 2:
Time course
of phosphorylation of wild-type rhodopsin expressed in HEK-293 cells. A, autoradiogram of time course of phosphorylation. Membranes
reconstituted with 11-cis-retinal were phosphorylated in the
light or in the dark for the indicated times, as described under
``Experimental Procedures,'' and chromatographed on a 10%
polyacrylamide gel, followed by autoradiography. B,
quantification by phosphorimage analysis. The dried gel from A containing samples phosphorylated in the light () and in the
dark (
) was exposed to a Molecular Dynamics phosphorimage screen
and subjected to phosphorimage analysis using a Molecular Dynamics
PhosphorImager.
Figure 3:
Phosphorylation and G
activation of Loop I mutants. A, phosphorylation by rhodopsin
kinase. Membranes from transfected HEK-293 cells were harvested,
reconstituted with 11-cis-retinal, and assayed for
phosphorylation in the light and in the dark according to procedures
described under ``Experimental Procedures.'' After
subtracting the dark values from the phosphorylation in the light, the
results were expressed as a fraction of the level of light-dependent
phosphorylation for wild-type rhodopsin normalized to a value of 1.0.
The results represent the averages of duplicates from at least two
transfections. The error bars represent S.E. B, time course of
phosphorylation of T62A/V63A/Q64A compared to wild-type rhodopsin.
Membranes were phosphorylated and quantified as described under
``Experimental Procedures'' and in the legend to Fig. 2. Phoshorylation was performed in the light (open
symbols) or in the dark (closed symbols).
and
, wild-type rhodopsin;
and
, T62A/V63A/Q64A. C, activation of G
. Membranes expressing mutant
rhodopsins were reconstituted with 11-cis-retinal and assayed
for the light-dependent binding of GTP
S to G
purified
from rod outer segments as described under ``Experimental
Procedures.'' Rates of activation were calculated from the slopes
of the reaction curves. After subtracting the rates of samples
incubated in the dark, light-dependent GTP
S binding was expressed
as a fraction of wild-type rhodopsin normalized to a value of 1.0. The
results represent the average of at least two independent transfections
performed in duplicate. The error bars represent S.E. The average rate
of GTP
S binding for wild-type rhodopsin was 0.1
mol/s
mol.
The Loop I mutants were also
assayed for their ability to activate G (Fig. 3C). Initial rates of activation comparing
the mutants with wild-type rhodopsin expressed in HEK-293 cells suggest
that T62A/V63A/Q64A is normal in its ability to catalyze the binding of
GTP
S to G
, despite its enhanced rate of
phosphorylation described above. L72A/N73A also showed a small
(approximately 27%) decrease in G
activation although it
was phosphorylated normally by rhodopsin kinase. The other four mutants
demonstrated no significant differences in either G
activation or phosphorylation compared to the wild-type protein.
Figure 4:
Phosphorylation and G
activation of Loop II mutants. A, phosphorylation by rhodopsin
kinase. Membranes from transfected HEK-293 cells were harvested and
assayed for phosphorylation as described under ``Experimental
Procedures'' and the legend to Fig. 3. The results are
expressed as a fraction of the level of light-dependent phophorylation
for wild-type rhodopsin normalized to a value of 1.0 and represent the
averages of duplicates from at least two transfections. The error
bars represent S.E. B, time course of phosphorylation of
R147A/F148A/G149A compared to wild-type rhodopsin. Membranes were
phosphorylated and quantified as described under ``Experimental
Procedures'' and in the legend to Fig. 2. Phosphorylation
was performed in the light (open symbols) and in the dark (closed symbols).
and
, wild-type rhodopsin;
and
, R147A/F148A/G149A. C, activation of
G
. Membranes expressing mutant rhodopsins were
reconstituted with 11-cis-retinal and assayed for the
light-dependent binding of GTP
S to G
as described
under ``Experimental Procedures'' and the legend to Fig. 3. Rates of activation calculated from the slopes of the
lines are expressed as a fraction of wild-type rhodopsin normalized to
a value of 1.0. The results represent the average of at least two
independent transfections performed in duplicate. The error bars
represent S.E. The average rate of GTP
S binding for wild-type
rhodopsin was 0.1 mol/s
mol.
Figure 5:
Phosphorylation and G
activation of Loop III mutants. A, phosphorylation by
rhodopsin kinase. Membranes from transfected HEK-293 cells were
harvested and assayed for phosphorylation as described under
``Experimental Procedures'' and the legend to Fig. 3.
The results are expressed as a fraction of the level of light-dependent
phophorylation for wild-type rhodopsin normalized to a value of 1.0 and
represent the averages of duplicates from at least two transfections.
The error bars represent S.E. B, activation of
G
. Membranes expressing mutant rhodopsins were
reconstituted with 11-cis-retinal and assayed for the
light-dependent binding of GTP
S to G
as described
under ``Experimental Procedures'' and the legend to Fig. 3. Rates of activation calculated from the slopes of the
lines are expressed as a fraction of wild-type rhodopsin normalized to
a value of 1.0. The results represent the average of at least two
independent transfections performed in duplicate. The error bars
represent S.E. The average rate of GTP
S binding for wild-type
rhodopsin was 0.1 mol/s
mol.
Figure 6:
Phosphorylation and G
activation of the Loop III mutants A233N/A234N/A235N and
A233G/A234G/A235G. A, phosphorylation by rhodopsin kinase.
Membranes from transfected HEK-293 cells were harvested and assayed for
phosphorylation as described under ``Experimental
Procedures'' and the legend to Fig. 3. The results are
expressed as a fraction of the level of light-dependent phophorylation
for wild-type rhodopsin normalized to a value of 1.0 and represent the
averages of duplicates from at least two transfections. The error bars
represent S.E. B, activation of G
. Membranes
expressing mutant rhodopsins were reconstituted with
11-cis-retinal and assayed for the light-dependent binding of
GTP
S to G
as described under ``Experimental
Procedures'' and the legend to Fig. 3. Rates of activation
calculated from the slopes of the lines are expressed as a fraction of
wild-type rhodopsin normalized to a value of 1.0. a, the
results represent the average of two independent transfections
performed in duplicate. Error bars represent S.E. b, error
bars represent the range of duplicates from a single transfection. The
average rate of GTP
S binding for wild-type rhodopsin was 0.05
mol/s
mol.
A233G/A234G/A235G also showed a dramatic loss in
ability to activate G (Fig. 5B). Therefore,
this mutant displays a reduced coupling to its G protein as well as
reduced phosphorylation. Interestingly, A233N/A234N/A235N seems to be
less severely affected for G
activation (Fig. 6B) than the glycine mutant of this sequence.
Measurements of absorbance for A233G/A234G/A235G and A233N/A234N/A235N
indicated that retinal binding is equivalent to wild-type (Fig. 7). Therefore, the integrity of the transmembrane domains
in these mutants is preserved.
Figure 7: Subtraction spectra of the Loop III mutants A233G/A234G/A235G and A233N/A234N/A235N. Membranes were reconstituted with 11-cis-retinal and assayed for retinal binding in the dark and in the light as described under ``Experimental Procedures.'' The spectra generated in the light were subtracted from the spectra generated in the dark to define the absorption peak of membranes from nontransfected, wild-type, and mutant-transfected cells.
The other Loop III mutants were also
assayed for their ability to catalyze GTPS binding to G
(Fig. 5B). Two of these mutants, T242A/T243A and
V250A/T251A/R252A, demonstrated 57% and 69% reduced ability to activate
G
, respectively, despite normal levels of phosphorylation.
These data suggest that multiple domains within Loop III are important
specifically for the regulation of G
activation.
This report describes the use of alanine-scanning mutagenesis
to define the participation of the cytoplasmic loops in the interaction
of rhodopsin with rhodopsin kinase and with its G protein,
G. Previously, this technique has been used to uncover the
functions of specific domains for a variety of proteins, including the
sites of interaction between human growth hormone and its
receptor(35) , the effector binding region of
G
(36) , and muscarinic acetylcholine receptor
sequence required for G protein activation(37) . Although the
role of amino acids similar to alanines may not be successfully
evaluated by this technique, it has the advantage of being less
disruptive than other methods of analysis. Palczewski et al.(18) demonstrated that a proteolytic fragment of rhodopsin
missing the C-terminal phosphorylation sites is required for maximal
phosphorylation of a synthetic peptide substrate by rhodopsin kinase.
Therefore, cytoplasmic domains distinct from the phosphorylation sites
participate in the activation of the kinase. Rhodopsin kinase and
G
have been shown to compete for interaction with
rhodopsin, suggesting the possibility of overlapping binding
sites(38, 39) . We have examined the phosphorylation
of rhodopsin by rhodopsin kinase and the activation of G
in
parallel in order to identify regions of the cytoplasmic surface that
participate specifically in the activation of these two proteins.
In
Loops I and II we were able to define regions that affect
phosphorylation when they are mutated to alanines. We detected a
significant increase in phosphorylation in the Loop I mutant
T62A/V63A/Q64A, although G activation was similar to
wild-type rhodopsin. Several potential mechanisms may account for this
observation, including a release of steric restraint or an increase in
affinity for the kinase. In contrast, R147A/F148A/G149A in Loop II
showed reduced ability to be phosphorylated by rhodopsin kinase but
normal activation of G
. These data suggest for the first
time that domains that participate in the activation of rhodopsin
kinase reside in these two loops. None of the mutations in Loop II
described in this study appeared to significantly affect G
activation. Mutation of the highly conserved Arg
(found at the border between the third transmembrane domain and
Loop II of most G protein-coupled receptors) to uncharged amino acids
abolishes the binding and activation of G
(8, 9, 10) . Therefore this amino acid,
which was not included in our present studies, is critical for
interaction with G
. It would be interesting to determine
the effects of mutations in Arg
on phosphorylation by
rhodopsin kinase in order to fully understand the role of this
important amino acid.
In Loop III we have detected a significant
loss of both phosphorylation and G activation by mutation
of Ala
-Ala
-Ala
to either
glycines or asparagines. Interestingly, A233N/A234N/A235N was less
severely affected for G
activation than the glycine mutant
of this sequence. These observations suggest that the conformational
requirements may be moderately different for phosphorylation and G
protein activation, as demonstrated by Robinson et
al.(40) . It is not clear from our data whether these
mutations have affected just the
Ala
-Ala
-Ala
region or whether
they have affected the secondary structure of the entire loop. Loop III
is predicted by Fourier transform infrared spectroscopy to be an
helix(41) . Computer analysis, based on Chou-Fasman
algorithms(42) , predicts that either glycines or asparagines
substituted for the alanines will introduce turns in the Loop III
sequence (data not shown). Previously, substitution of glycines, which
are known to disrupt
helices, at Thr
-Thr
(located in the center of Loop III) resulted in a 90% loss in
GTPase activity compared to a 66% loss when these amino acids were
substituted with valines(9) . In addition, proteolytic cleavage
of Loop III between Glu
and Ser
destroys
both the ability of the photoreceptor to activate rhodopsin kinase (18) and disrupts activation of G
(43) .
These data, taken together with the present report, strongly suggest
that the secondary structure of a large segment of this loop is
critical for the ability of rhodopsin to couple to G
as
well as to promote phosphorylation of the C terminus by rhodopsin
kinase. Loop III may be important for the conformational changes that
must occur in response to light to convert rhodopsin to an active
protein. Nevertheless, we cannot rule out through our present studies
that Ala
-Ala
-Ala
alone may be
the required sequence for these light-dependent conformational changes.
Nor can we rule out the possibility that G
and rhodopsin
kinase have partially overlapping sites of interaction. Future studies
will focus on additional mutations using amino acids that are less
disruptive to secondary structure to distinguish between these
possibilities.
Previously, the use of peptides as competitive
inhibitors suggested that the C-terminal region of Loop III is
important for phosphorylation of the C terminus by rhodopsin
kinase(14, 15) . However, none of the mutants in that
region affected phosphorylation in our assay. In contrast, we did
observe reduced activation of G in the Loop III mutants
T242A/T243A and V250T251R252. T242A/T243A was reduced by approximately
55%, similar to the results by Franke et al.(9) where
these amino acids were substituted with valines instead of alanines, as
described above. Val
-Thr
-Arg
,
adjacent to the sixth transmembrane domain, has been identified for the
first time by our studies as a critical site for G
activation. Since both mutants showed normal phosphorylation,
they appear to be important specifically for G
activation.
Franke et al. also demonstrated that a mutant containing a
deletion of the central region of Loop III that includes
Thr
-Thr
(amino acids #237-249) can
bind G
but is unable to catalyze guanine nucleotide
exchange(8) , suggesting that this region may be needed not for
binding, but for the catalysis of guanine nucleotide exchange. Whether
Val
-Thr
-Arg
is involved in
the binding or the activation of G
remains to be determined
in future experiments. Two amino acid mutations in Loop III (S240A and
K348L) reported previously to be important in the activation of G
(9) were not detected in our studies. The reasons for
these differences between the two laboratories are unclear. Perhaps
they are due to differences in the assay conditions, such as the use of
membranes in our case compared to detergent-extracted proteins in the
earlier report, and/or the use of different mutations at these sites.
Little is known concerning the involvement of the cytoplasmic loops
in the regulation of phosphorylation of other G protein-coupled
receptors by GRKs. In contrast, there have been a number of studies
implicating Loops II and III in the activation of various G proteins.
For example, a hydrophobic amino acid located in Loop II, leucine in
the M1 muscarinic acetylcholine receptor and phenylalanine in the
-adrenergic receptor, is required for G protein-mediated signal
transduction in these two receptors(37) . Replacement of either
amino acid with alanine led to a greater than 85% loss in coupling. In
the present study, replacement of the equivalent amino acid in
rhodopsin, Met
, had no effect on either G
activation or phosphorylation by rhodopsin kinase. Therefore, the
critical domains in rhodopsin may be different from those in some
closely-related receptors. Both Loops II and III, as well as the
``fourth cytoplasmic loop'' (Loop IV, Fig. 1), have
been implicated in the selectivity of coupling of specific G proteins
to receptors such as the metabotropic glutamate, muscarinic
acetylcholine, and
-adrenergic
receptors(31, 44) . The participation of the rhodopsin
cytoplasmic domains in the selectivity of coupling to G proteins
remains to be determined.
In summary, we have, for the first time,
defined domains in Loops I and II of rhodopsin that specifically affect
the activation of rhodopsin kinase and that are distinct from regions
involved in G activation represented by mutations in Loop
III. In addition, mutation of
Ala
-Ala
-Ala
in the N-terminal
domain of Loop III disrupted both G protein activation and
phosphorylation. Additional experiments will be needed to separate and
define the specific amino acids that play a role in these two pathways
and to understand their significance in signaling events mediated by
rhodopsin.