From the
The photoreceptor rhodopsin is a seven-transmembrane helix
receptor that activates the G protein transducin in response to light.
Several site-directed rhodopsin mutants have been reported to be
defective in transducin activation. Two of these mutants bound
transducin in response to light, but failed to release the bound
transducin in the presence of GTP (Franke, R. R., König, B.,
Sakmar, T. P., Khorana, H. G., and Hofmann, K. P. (1990) Science 250, 123-125). The present study was carried out to
determine the nucleotide-binding state of transducin as it interacts
with rhodopsin mutants. Five mutant bovine opsin genes were prepared by
site-specific mutagenesis. Three mutant genes had deletions from one
cytoplasmic loop each: AB
Rhodopsin, the visual photoreceptor of the rod cell, is a member
of the family of seven-transmembrane helix receptors that activate
guanine nucleotide-binding regulatory proteins (G
proteins).
The postulated secondary
structure of rhodopsin and its topology with respect to the membrane
bilayer
(1) has been supported by a projection structure of
rhodopsin obtained by cryoelectron microscopy
(2) and by
comparison with other G protein-coupled receptors
(3) . Three
cytoplasmic loops, AB, CD, and EF link successive transmembrane helices
Fig. 1
). A fourth putative loop is formed by a region of the
carboxyl-terminal tail between the seventh helix and two palmitoylated
cysteines (Cys
In this report, five rhodopsin
mutants with amino acid replacements or deletions in either the
cytoplasmic loop AB, CD, or EF were characterized. Three types of
assays were carried out on the mutant pigments: 1) assay of
R*-dependent GTP
Illumination of each pigment yielded a
species with a
The aim of this work was to further characterize rhodopsin
mutants with alterations of their cytoplasmic loops. These mutants were
chosen because of their particular properties with respect to
transducin interaction. Mutant AB
Mutants CD
r140-152, EF
According to the
transducin activation pathway (Fig. 2), an inability of these
mutants to catalyze G
In the present study, a set of five rhodopsin mutants
was studied to elucidate the particular step impaired in the activation
pathway of transducin. A fluorometric assay was used to measure the
accumulation of G
Direct binding of transducin to
photoactivated pigments reconstituted into phosphatidylcholine vesicles
was measured in a light-scattering assay (Fig. 5). Both
transducin-dependent binding and dissociation signals can be obtained
using the assay. Compared with the extra MII assay this has the
advantage that stable binding need not be inferred from an effect on
the MI/MII equilibrium between pigment photoproducts. The
light-scattering assay is therefore not dependent on the effects of a
particular mutation on the MI-like/MII-like equilibrium for a
particular mutant pigment. Mutant pigments have been described with
markedly altered photoactivation pathways. For example, a mutant
rhodopsin photoproduct with a protonated Schiff base and a
Recombinant rhodopsin and mutants EF
A
nucleotide-release assay (Fig. 3 B) measured the ability
of each pigment to catalyze the release of GDP from
G
The photoproducts of
mutants E134R/R135E and CD
Based upon the
results discussed above, the mutant pigments can be grouped according
to defects in discrete transducin activation steps depicted in
Fig. 2
. Mutants E134R/R135E and CD
The relationship
between the properties of mutants CD
Detailed information is now
available about the role of the conserved residue Glu
-We thank T. Zvyaga, M. Heck, K. Fahmy, C. Min,
and R. R. Franke for assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
70-71; CD
143-150; and
EF
237-249. Two additional loop CD mutant genes were
prepared: E134R/R135E had a reversal of a conserved charge pair, and CD
r140-152 had a 13-amino acid sequence replaced by a sequence
derived from the amino-terminal tail. Three types of assays were
carried out: 1) a fluorescence assay of photoactivated rhodopsin
(R*)-dependent guanosine 5`- O-(3-thiotriphosphate) uptake by
transducin, 2) an assay of R*-dependent release of labeled GDP from the
-subunit of transducin holoenzyme (G
)
GDP,
and 3) a light-scattering assay of R*
G
complex
formation and dissociation. We show that the mutant pigments, which are
able to bind transducin in a light-dependent manner but lack the
ability to activate transducin, most likely form
R*
G
GDP complexes that are
impaired in GDP release.
(
)
The opsin apoprotein and its
chromophore 11 -cis-retinal are covalently linked via a
protonated Schiff base at Lys
. Photoisomerization of the
chromophore to all- trans-retinal is followed by several
conformational changes and results in the formation of metarhodopsin II
(MII), the active photoproduct which catalyzes nucleotide exchange in
the rod cell G protein, transducin. The photoactivated form of
rhodopsin can be referred to as R*.
and Cys
). The cytoplasmic
surface of the receptor interacts with transducin, which is
peripherally bound to the membrane surface in the GDP-bound state, and
with other cytoplasmic proteins such as rhodopsin kinase and arrestin
(4) .
Figure 1:
Schematic
representation of rhodopsin mutants studied. The seven putative
transmembrane helices are depicted as cylinders ( A-G).
According to secondary structure predictions (1) and a projection
structure (2), the helices are successively connected by loops (3).
Interactions with other proteins of the signaling cascade occur at the
cytoplasmic surface of the receptor toward the top of the
figure. The cytoplasmic loops are designated AB, CD,
and EF. A fourth cytoplasmic loop is formed by a portion of
the carboxyl-terminal tail between helix G and a pair of palmitoylated
cysteines. Five mutant rhodopsins are shown schematically. Three
mutants contained partial deletions of one of the loops ( AB
70-71; CD
143-150; EF
237-249). Deleted residues are boxed. A fourth
mutant ( E134R/R135E) contained a reversal of two amino acids
of the CD loop. In the fifth mutant ( CD r140-152) the
original sequence of residues 140-152 was replaced by the amino
acid sequence GTEGPNFYVPFTS (see Table I). The amino termini of the
recombinant rhodopsins may be acetylated as is the case in bovine
rhodopsin.
The assignment of the transducin-interacting domains of
rhodopsin has been largely based upon analysis of site-directed mutants
(5, 6, 7, 8) . In addition, biochemical
(9) , peptide competition
(10) , and antibody competition
(11) approaches have been employed. Cytoplasmic loops CD, EF,
and the fourth loop were proposed to be the interacting sites between
rhodopsin and transducin
(5, 6, 7, 8, 10) . Cytoplasmic
loop AB also may be involved in rhodopsin-transducin interaction
(11, 12) . In general, all of the assays used to define
rhodopsin-transducin interaction in recombinant pigments have relied
upon a loss of the ability of R* to catalyze guanine-nucleotide
exchange in transducin. However, according to the transducin activation
scheme shown in Fig. 2, it is clear that an inability to form
GGTP
S could result from a defect at any one
of four steps: 1) R* binding to G
GDP,
2) GDP dissociation from the
R*
G
GDP complex, 3) GTP
S
uptake by the R*
G
(empty) complex, or
4) dissociation of G
GTP
S and
G
from R*.
Figure 2:
Experimental reaction scheme for
light-induced transducin activation by rhodopsin. Photoactivated
rhodopsin ( R*) binds to transducin
(G) and induces GDP release followed by
GTP
S uptake by the nucleotide-depleted
R*
G
(empty) complex leading to
dissociation of the R*, G
GTP
S, and
G
. R*-dependent GDP release ( step 3) is
studied by a nucleotide release assay. Nucleotide uptake of the
nonhydrolyzable GTP analogue GTP
S ( step 4) is studied by
the fluorescence assay.
Two rhodopsin mutants have been
reported that bound transducin in response to light but failed to
release the bound transducin in the presence of GTP
(6) . These
rhodopsin mutants formed spectrally normal R* species, which bound
transducin. However, it was not determined whether the mutant
R*G
complex contained GDP, GTP, or no
nucleotide. It is important for a detailed understanding of the
mechanism of receptor-mediated G-protein activation to be able to
develop methods to study receptor mutants that are inactive in standard
GTPase or filter-binding assays.
S uptake by transducin, 2) assay of R*-dependent
GDP release from G
GDP, and 3) assay of
R*
G
complex formation and dissociation. The mutants,
which are able to bind transducin in a light-dependent manner but lack
the ability to activate transducin, are shown to form
R*
G
GDP complexes that are
impaired in GDP release.
Materials
Sources of most materials have been
previously reported
(7, 8, 13) . Detergents were
purchased from Anatrace, Inc. (Cleveland, OH). Radionuclides were from
DuPont NEN and BA85 nitrocellulose filters were from Schleicher &
Schuell. Nucleotides were purchased from Boehringer Mannheim.
Preparation of Rhodopsin Mutants
The five mutants
studied are shown schematically in Fig. 1. The mutant opsin genes
were prepared by cassette mutagenesis of a synthetic gene for bovine
rhodopsin
(8, 14) that had been cloned into the
expression vector as described previously
(5) . Individual
mutant genes were prepared as described previously: AB 70-71
(12) ; CD
143-150
(7) ; EF
237-249
(6) ; E134R/R135E
(8) ; CD r140-152
(6) .
Opsin genes were expressed in COS-1 cells following transient
transfection by a DEAE-dextran procedure as described
(15) . COS
cells expressing the mutant apoproteins were harvested and then
incubated in the presence of 11- cis-retinal under dim red
light to reconstitute pigments as described elsewhere
(7, 8) . The purification procedure employed was based
on the immunoaffinity procedure of Oprian et al.(15) ,
which was modified as previously described
(7, 8, 16) . Pigments were generally prepared in
10 mM BTP, pH 7.0, 130 mM NaCl, 1 mM
MgCl
, and 0.02% (w/v) DM or 1.5% (w/v) OG detergent. For
some experiments, pigments AB
70-71 and CD r140-152 in
DM detergent buffer were concentrated slightly using Microcon-30
filters (Amicon). All pigments prepared in OG detergent buffer were
concentrated 2-3-fold using Centricon-30 filters. Each pigment
displayed a visible
value of 500 nm as previously
reported
(6, 7, 8, 12) . Each pigment
concentration was determined based on its absorbance at 500 nm
(
= 42,700 M
cm
).
Fluorescence Assay of G
Bovine transducin was purified from rod outer segment
extracts by hexyl-agarose chromatography
(17) . A stock solution
of transducin (approximately 25 µM) was stored at
-20 °C in 10 mM MOPS, pH 7.5, 2 mM
MgClGTP
S
Formation
, 1 mM DTT, 5 µM GDP, 50%
glycerol. Active transducin concentration was determined precisely by
fluorometric titration
(18) . Fluorescence assay of
G
GTP
S formation rate catalyzed by rhodopsin
and mutant pigments in light was carried out as described previously
(18) except as noted below. Briefly, 1.5 ml of a mixture of 2
nM pigment and 200 nM transducin (10 mM BTP,
pH 7.0, 130 mM NaCl, 1 mM MgCl
, 1
mM DTT, 0.01% (w/v) DM) was continuously stirred at 10 °C
and continuously illuminated in the cuvette through a fiber optic guide
with 543.5-nm light from a HeNe laser (Melles-Griot). The sample was
excited at 300 nm and fluorescence emission at 345 nm was recorded.
After 7 min, 50 µl of GTP
S was injected into the cuvette to
give a final concentration of 5 µM, and fluorescence
increase over time was recorded.
Nucleotide Release Assay
A nucleotide-release
assay was employed to measure the ability of rhodopsin and of mutant
pigments to catalyze GDP release by GGDP.
Transducin used for this assay was purified as described previously
(19) and was stored at -80 °C at concentrations of
6-8 µM in storage buffer (10 mM BTP, pH
7.0, 130 mM NaCl, 1 mM MgCl
, and 1
mM DTT). Transducin concentration was precisely determined by
fluorometric titration
(18) . The intrinsic GTPase activity of
transducin was used to prepare
G
[
-
P]GDP from
transducin pre-loaded with [
-
P]GTP.
Transducin samples were thawed and mixed with a catalytic amount of
rhodopsin and [
-
P]GTP to give a final
solution of 1 µM transducin, 1.5 nM rhodopsin,
and approximately 60 or 150 pM
[
-
P]GTP (3200 or 800 Ci/mmol, respectively)
in storage buffer. To ensure uptake of the labeled GTP and its
hydrolysis to GDP, the reaction mixture was incubated for 1 h at room
temperature under continuous illumination (495 nm long-pass filter).
During this incubation, the R* present decayed to opsin and free
all- trans-retinal. The resulting
[
-
P]GDP-loaded transducin was kept on ice,
and the assay was carried out immediately as described. An assay
mixture, containing 200 nM
[
-
P]GDP-loaded transducin and 400
nM rhodopsin or mutant pigment in a total volume of 100
µl, was illuminated for 5 min. The light source was a 150-watt
projector lamp fitted with a 495-nm long-pass filter (Oriel, Inc.). The
assay was carried out in parallel with a reference sample in dim-red
light (dark sample). The dark sample measures the total amount of
GDP-loaded transducin present under the conditions of the assay. After
5 min of illumination, aliquots (20 µl
4) were removed from
each of the two (light and dark) samples and vacuum filtered through a
nitrocellulose membrane. The filters were rapidly washed (6 ml) and
then air dried. The amount of bound [
-
P]GDP
was quantitated using the PhosphorImager system (Molecular Dynamics).
The average value of four measurements from the reference (dark)
experiment was normalized to 100%. The standard error was in the range
of ±5%. The mean of the four values obtained from the
illuminated sample was then expressed as percentage of the reference
(dark) sample. The photolyzed rhodopsin sample catalyzed the release of
96.6 ± 0.7% of the labeled GDP from transducin during the course
of the assay. This corresponds to
3.4%
G
[
-
P]GDP bound.
Measurement of Mutant Pigment Schiff Base
Stability
An acid denaturation method was used to determine the
extent of Schiff base hydrolysis
(8) . Under acidic conditions,
rhodopsin denatures, but the chromophore-opsin protonated Schiff base
linkage is stable, resulting in a broad peak with a value of 440 nm. Free all- trans-retinal gives a peak
with a
of about 380 nm. After an initial dark
spectrum was recorded on a 90-µl aliquot, the remaining sample was
illuminated for 20 s. After 5 min, 10 µl of 2 M
hydrochloric acid was added to denature the pigment, and a spectrum was
recorded.
Reconstitution of Rhodopsin and Mutant Pigments into
Egg-Lecithin Vesicles
Purified rhodopsin or mutant pigment
solubilized in OG detergent buffer was reconstituted into phospholipid
vesicles in a 1:350 (pigment:phospholipid) molar ratio. All
manipulations were carried out under dim red light. A suspension of 5
mg/ml egg-yolk phosphatidylcholine (Fluka) in deionized water was added
to pigment (1.5-1.7 µM) in OG solution. The sample
was dialyzed in a microdialysis unit (Pierce) against 2 liters of
buffer (10 mM BTP, pH 7.0, 130 mM NaCl, and 1
mM MgCl) for 20 h at 4 °C. Vesicles were
subjected to UV-visible spectroscopy to determine the concentration of
reconstituted pigment. Freshly prepared vesicles were used for
light-scattering measurements.
Light-Scattering Assay
Light-scattering
measurements were performed using an apparatus previously described
(20) that recently has been used to study rhodopsin
kinase/rhodopsin
(21) and transducin/PDE
(19) interaction. The samples contained 700 nM
recombinant pigment in reconstituted vesicles and 4 µM
transducin in 10 mM BTP, pH 7.0, 130 mM NaCl, 1
mM MgCl, and 0.3 mM DTT in a final volume
of 260 µl. Transducin used in this assay was prepared as described
previously
(19) and stored in 10 mM BTP, pH 7.0, 130
mM NaCl, 1 mM MgCl
, 1 mM DTT at
-80 °C. Measurements were carried out in a 10-mm path length
cuvette. Light-scattering changes were monitored in an angular range of
16 ± 2 degrees by a continuous 840-nm incident light beam. To
achieve a stable baseline, samples were incubated for up to 20 min. For
measurements in the presence of GTP, 100 µM hydroxylamine
was present during the 20-min incubation period
(22) . When
required, GTP was added immediately prior to measurement. Binding
signals were recorded after photolysis of samples with a 500 ±
20-nm flash. The light-scattering signals are interpreted as binding
signals and dissociation signals of transducin from the photoactivated
pigment
(23) . The physical correlate of the light-scattering
signals is the light-induced gain or loss of protein mass by the
pigment-phospholipid vesicle
(20) .
Preparation of Recombinant Pigments
Five mutant
bovine opsin genes were prepared by site-specific mutagenesis as shown
schematically in Fig. 1. Three mutant genes had deletions from
one cytoplasmic loop: AB 70-71; CD
143-150, and
EF
237-249 (numbers indicate deleted amino acid residues).
Two additional loop CD mutant genes were prepared: E134R/R135E had a
reversal of a conserved charge pair and CD r140-152 had a
13-amino acid sequence replaced by a sequence derived from the
amino-terminal tail region (). The genes were expressed by
transient transfection of COS-1 cells and the expressed opsins were
regenerated with 11- cis-retinal chromophore. The resulting
pigments were purified in detergent solution and showed absorption
maxima identical to rod outer segment rhodopsin (
= 500 nm).
value of 380 nm characteristic of
MII. The decay of the Schiff base linkage in the MII-like species for
each mutant was identical to that measured for native MII. In DM
detergent solution at room temperature, less than 5% of the Schiff base
decayed to opsin plus all- trans-retinal after 5 min. As
reported previously, the expression level and regeneration efficiency
for mutants AB
70-71
(12) and CD r140-152
(6) were significantly lower than for rhodopsin (not shown).
Three types of assays were carried out on recombinant rhodopsin and on
the mutant pigments as described below.
Assay of R*-dependent GTP
A fluorescence assay was employed to measure the
ability of each pigment to catalyze GTPS Uptake by
Transducin
S uptake by transducin.
Each of the mutants prepared was reported previously to be
significantly impaired in light-dependent activation of transducin. The
mutants had been studied in a GTPase assay
(5, 6, 8) or in a GTP
S uptake assay using nitrocellulose filters
(12) . The ability of each mutant to catalyze the uptake of
GTP
S by transducin in response to light was evaluated here using a
more sensitive fluorescence assay
(18, 24) . In this
assay, the accumulation of G
GTP
S as
depicted in the scheme in Fig. 2is monitored as an increase in
intrinsic fluorescence. As shown in Fig. 3 A, the
addition of GTP
S to a mixture of transducin and R* results in an
increase of relative fluorescence as a function of time. The signal
reaches a maximum when GTP
S uptake, in a 1:1 stoichiometry with
G
, is complete.
Figure 3:
Measurement of R*-catalyzed
GGTP
S formation and R*-dependent GDP
release from G
GDP. A, a fluorescence
assay was employed to measure the rate of R*-catalyzed
G
GTP
S formation. The increase in relative
fluorescence is recorded as a function of time and is directly related
to the increase in the concentration of
G
GTP
S. GTP
S was injected
( arrow) into a cuvette containing known concentrations of R*
and G
to start the reaction. The assay was
carried out as described under ``Experimental Procedures.''
Assay conditions are: 2 nM pigment, 200 nM
transducin, 10 mM BTP, pH 7.0, 130 mM NaCl, 1
mM MgCl
, 1 mM DTT, 0.01% (w/v) DM,
GTP
S 5 µM at 10 °C. Under these conditions, R*
catalyzes the uptake of 48 pmol GTP
S/min (1.5-ml reaction volume).
B, a nucleotide-release assay was employed to measure the
R*-catalyzed GDP release by G
GDP. The intrinsic
GTPase activity of transducin was used to prepare
G
[
-
P]GDP from
transducin pre-loaded with [
-
P]GTP. The
assay was carried out as described under ``Experimental
Procedures.'' Briefly, [
-
P]GDP-loaded
transducin was incubated with rhodopsin for 5 min in the dark or in the
light. The mixture was filtered through a nitrocellulose membrane, and
the filter-bound radioactivity was quantitated. The amount of bound
G
[
-
P]GDP for the
dark sample was defined as 100%. In the light, R* binds transducin and
catalyzes GDP release from G
GDP. The decrease in
G
[
-
P]GDP bound to
the filter is directly proportional to the R*-dependent release of
[
-
P]GDP from its nucleotide-binding pocket.
The values plotted represent the means of five individual experiments.
An error bar (S.E., n = 5) is shown for the
light experiments. Assay conditions are: 400 nM pigment, 200
nM transducin, 10 mM BTP, pH 7.0, 130 mM
NaCl, 1 mM MgCl
, 0.2 mM DTT, 0.016% (w/v)
DM, at 20 °C.
The activation rate for recombinant
rhodopsin was determined by linear regression analysis of a 35-50
s time interval starting 6 s after GTPS addition (Fig. 4).
Under the conditions of the assay, 2 nM recombinant rhodopsin
(
3 pmol of R*) catalyzed the uptake of 48 pmol of GTP
S/min.
Each of the mutant pigments was tested for its ability to activate
transducin in the fluorescence assay (Fig. 4). The activation
rates were measured as follows: transducin without pigment, 0.4%; AB
70-71, 6.7%; E134R/R135E, 0.6%; CD
143-150, 0.3%;
CD r140-152, 0.8%; EF
237-249, 1.1%. Only mutant AB
70-71 showed a significant transducin activation rate (6.7%
of the rate for rhodopsin). Rates of G
GTP
S
formation slower than about 1% of that of native rhodopsin are within
the noise of the assay and the corresponding mutants are considered to
be unable to induce significant G
GTP
S
formation under conditions of the assay.
Figure 4:
Mutant rhodopsins are deficient in
catalyzing GTPS uptake by transducin. A fluorescence assay was
employed to measure the rate of pigment-catalyzed
G
GTP
S formation. The increase in relative
fluorescence is recorded as a function of time and is directly related
to the increase in the concentration of
G
GTP
S. GTP
S was injected
( arrow) into a cuvette containing known concentrations of
light activated pigment and G
to start the
reaction. Purified recombinant rhodopsin was compared with five mutant
pigments as described in Fig. 1. In the control measurement no pigment
was present. For rhodopsin the slope of the curve in the linear phase
starting 6 s after GTP
S addition was calculated from a linear
regression analysis over a time interval of 35-50 s. For each of
the mutant pigments the slope of the curve starting 60 s after
GTP
S addition was calculated from a linear regression analysis
over a time interval of 500 s. The rates of fluorescence increase
expressed as a percentage of the rhodopsin rate for the experiments
shown were: transducin without pigment, 0.4%; AB
70-71,
6.7%; E134R/R135E, 0.6%; CD
143-150, 0.3%; CD
r140-152, 0.8%; EF
237-249, 1.1%. The rates determined
from independent preparations were reproducible to within
±1%.
Assay of R*
A light-scattering assay was developed to measure
directly the ability of each pigment to bind transducin in response to
light and to release bound transducin in the presence of GTP. Binding
and dissociation signals for recombinant rhodopsin reconstituted into
phosphatidylcholine vesicles are shown in Fig. 5. A flash-induced
scattering increase over time is noted in the presence (binding
signal), but not in the absence of transducin. This signal reflects
binding of the soluble fraction of the protein to the vesicles after
photolysis as observed with reconstituted systems of disk membranes and
transducin
(19, 20) or rhodopsin kinase
(21) .
In presence of transducin and GTP, a flash produces a decrease of
scattering intensity. This is due to the R*-catalyzed activation of the
transducin fraction bound to the vesicle at the time of the flash. GTP
binds to the activated R*G
Complex Formation and
Dissociation
G
(empty)
complex and then G
GTP and G
dissociate from the R* in the vesicle, resulting in a loss of
scattering mass and a decrease in scattering intensity (dissociation
signal). The mutant EF
237-249 is able to bind transducin as
shown by the positive scattering change. However, the dissociation
signal, which is normally observed in the presence of GTP, is absent
with this mutant. Photolysis of mutants E134R/R135E and CD
143-150 does not cause scattering changes in presence of
transducin alone, nor in the presence of transducin and GTP.
Figure 5:
Binding and dissociation signals for
rhodopsin and mutant pigments. Flash-induced near-infrared
light-scattering changes were measured on recombinant pigments
reconstituted into phosphatidylcholine vesicles. Samples containing 700
nM pigment and 4 µM transducin were bleached with
a green flash as indicated by arrows. The mole fraction of
flash-excited rhodopsin (R*/R) was 55% for signals in the a and b traces, or 0.2% for signals in the c
traces. Light-scattering changes as a function of time in the
presence ( a) or absence ( b) of transducin are shown.
The dissociation signal caused by the presence of 1 mM GTP in
addition to transducin is shown ( c). After GTP uptake,
vesicle-bound GGTP and G
dissociate from R
and become soluble, resulting in a
decrease in light-scattering intensity. The very small scattering
decrease in the control without transducin ( b) is due to the
N-signal (21, 41), a rhodopsin-related signal, which is superimposed on
all signals caused by transducin. The N-signal in vesicles is much
smaller than in disk membranes. As shown, rhodopsin binds transducin in
response to light and releases bound transducin in the presence of GTP.
The photoactivated mutant EF
237-249 binds transducin
somewhat less well than rhodopsin, but does not release bound
transducin in the presence of GTP. Mutants E134R/R135E and CD
143-150 fail to bind transducin.
I/I is the normalized
scattering change where
I represents the flash induced scattering
change and I represents the scattering intensity of the sample before
flash minus the scattering intensity of the
buffer.
Assay of R*-dependent GDP Release from
G
A nucleotide-release assay was
developed to measure the ability of each pigment to catalyzed the
release of GDP from GGDP
GDP
(Fig. 3 B). Photoactivated rhodopsin induces almost
complete release of GDP from the nucleotide-binding pocket of
transducin after 5 min of continuous illumination. Only 3.4% of the
dark reference sample was not released. This small residual GDP binding
can be attributed to incomplete hydrolysis of
[
-
P]GTP in the preloading step (see
``Experimental Procedures'') or to transducin which is not
able to bind to R*. The results of this assay for the recombinant
pigments are shown in Fig. 6and . Mutants
E134R/R135E, CD
143-150, and CD r140-152 did not show
a significant release of GDP. The difference in the mean amount of
G
GDP complex between dark and light experiments
was less than 10%. Mutant pigments AB
70-71 and EF
237-249 catalyzed significant GDP-nucleotide releases of
about 84 and 34%, respectively.
Figure 6:
Light-dependent GDP release from
transducin in the presence of recombinant pigments. The assay was
carried out to determine the effect of various photoactivated rhodopsin
mutants on the nucleotide occupancy of transducin. The fluorescence
assay in Fig. 4 shows that mutants CD r140-152 and EF
237-249 do not induce GTP
S uptake by transducin. The
GDP-release assay addresses the question of whether the transducin that
binds to these mutants contains GDP or an empty nucleotide-binding
site. The results are consistent with the failure of transducin to
release GDP upon binding to mutant pigments CD r140-152 and EF
237-249. Mutant pigments E134R/R135E and CD
143-150 were shown not to bind transducin (Fig. 5) (6). GDP
release is not detected from transducin exposed to these
light-activated mutant pigments. Mutant AB
70-71 shows GDP
release consistent with its small transducin-activating ability (Fig.
4). The assay was performed as described for rhodopsin in Fig. 3. The
values plotted represent the means of three to five individual
experiments. The means of the dark values are normalized to 100%. An
error bar (S.E., n = 3-5) is shown for
the light experiments. Numerical values are presented in Table
II.
70-71 showed a very low
but measurable ability to activate transducin in a filter-binding assay
(12) . Mutant CD
143-150 was unable to activate
transducin in a GTPase assay
(6) . Mutant E134R/R135E was also
unable to activate transducin in a GTPase assay
(8) , possibly
because of alteration of the conserved Glu
residue, which
has been shown to regulate light-dependent transducin binding and
activation
(18, 25) . Mutants CD r140-152 and EF
237-249 were also shown to be inactive in a GTPase assay,
but had the additional very interesting property that they bound
transducin in response to light but failed to release the bound
transducin in the presence of GTP
(6) .
237-249, and E134R/R135E were previously
studied by an assay which measured the ability of transducin to shift
the equilibrium between MI- and MII-like forms of the pigment
photoproduct toward MII (extra-MII assay)
(6, 26) .
Mutant E134R/R135E did not bind transducin in the extra-MII assay
(6) . Mutants CD r140-152 and EF
237-249 formed
extra-MII upon photolysis in the presence of transducin in both the
presence and absence of GTP. It was postulated that photoactivated
mutants CD r140-152 and EF
237-249 formed stable
complexes with transducin, and that the bound transducin had an empty
nucleotide-binding pocket that was unable to bind GTP
(6) .
However, the combination of GTPase and extra-MII assays could not
distinguish among three possibilities for defects in mutants CD
r140-152 and EF
237-249.
GTP formation could result
from a defect at any one of three steps: 1) GDP dissociation from the
R*
G
GDP complex, 2) GTP uptake by
the R*
G
(empty) complex, or 3)
dissociation of G
GTP and G
from R*.
GTP
S over time. As shown
in Fig. 3 A, the addition of GTP
S to a mixture of R*
and G
causes an increase in fluorescence
resulting from the formation of G
GTP
S
(27) . Each CD-loop mutant (CD r140-152, CD
143-150, and E134R/R135E) and mutant EF
237-249
was unable to catalyze a significant GTP
S uptake under conditions
of the assay. This result supports the GTPase and filter-binding assay
results of earlier studies
(6, 7, 8) . Mutant AB
70-71 displayed about 7% of the activity of rhodopsin. This
activation rate is somewhat higher than that reported from a
filter-binding assay
(12) . The different rates reported are
most likely due to a higher pH (0.5 unit) in the filter-binding assay.
This pH increase is known to cause a lower activation rate for
rhodopsin
(18) .
value of 474 nm has been shown to activate
transducin
(28, 29, 30) . Such mutants pigments
can be studied by the light-scattering assay but not by the extra-MII
assay.
237-249,
E134R/R135E, and CD
143-150 were studied by the
light-scattering assay (Fig. 5). Mutant EF
237-249
bound but failed to activate transducin in response to light. The
magnitude of binding of transducin by the mutant pigment was about 40%
of that of rhodopsin. Mutants E134R/R135E and CD
143-150 did
not bind transducin. Mutants EF
237-249 and E134R/R135E were
studied previously by the extra-MII assay
(6) . The results of
the light-scattering assay for these mutants are consistent with the
results from the earlier extra-MII assay study
(6) .
GDP (Fig. 6). Photoactivated rhodopsin
induced almost complete release of GDP from the nucleotide-binding
pocket of transducin. The nucleotide release assay is a rigorous test
as to whether a rhodopsin mutant is competent to induce the release of
GDP from bound transducin. Even very low catalytic activity would be
detected in this assay since it extends over minutes and measures the
accumulated GDP release over the entire period of the assay. For
rhodopsin, as calculated from the G
GTP
S
formation rate, total GDP release is reached after only a few seconds.
This is consistent with the time of formation of the
R*
G
GDP complex, for which the
light scattering 1/e time provides an upper limit
(20) .
Therefore, defects detected by this assay must be severe. For example,
in mutant EF
237-249, only 34% of the GDP in the transducin
pool was released after 5 min, while at least 40% of the mutant pigment
preserved the ability to form a complex with transducin based on the
magnitude of the light-scattering binding signal. It can be concluded
that the rate of catalyzed GDP release from transducin was specifically
slowed. A more direct comparison between the results of the
nucleotide-release assay and the light-scattering assay is not possible
and an intrinsic relationship does not necessarily exist between the
values for GDP release and complex formation.
143-150 do not bind transducin as
shown by the light-scattering assay (Fig. 5). Therefore, they are
not expected to induce GDP release ( Fig. 6and ).
Mutant AB
70-71 had a small signal in the fluorescence
GTP
S-uptake assay (Fig. 4), and the significant GDP release
in the nucleotide release assay of about 84% ( Fig. 6and
) is consistent with that activity.
143-150 are
profoundly defective in binding to
G
GDP. Mutant AB
70-71 may
have an incomplete defect in binding
G
GDP or in release of GDP from the
R*
G
GDP complex. Consequently,
GDP release and GTP
S uptake are diminished but detectable. These
three mutants are defective to varying degrees in each of the three
assays carried out. Mutants EF
237-249 and CD r140-152
are able to bind G
GDP, but appear
defective in catalyzing GDP dissociation from the mutant
R*
G
GDP complex. A mutant
defective in dissociation from G
GTP
S and
G
was not found among the five tested. It is
likely that such a mutant phenotype will be rare based upon current
understanding of the nucleotide-dependent switch regions of
G
from the x-ray crystal structure
(31, 32, 33) , and from energetic considerations
where the binding of GTP to the
R*
G
(empty) complex has been shown to
occur in a thermally activated state
(34) .
143-150 and CD
r140-152 is particularly interesting. The 8-amino acid deletion
from loop CD in mutant CD
143-150 causes an inability of the
pigment to interact with transducin in all assays. No transducin
activation occurs and no binding is detected in the light-scattering
assay. However, replacement of the deleted amino acids by a sequence
derived from the intradiscal domain in mutant CD r140-152
restores binding activity as previously reported
(6) . It has
been noted as shown in that the amino acid replacement
retained the same amino acid residues at positions Asn
and Phe
(6) . Thus, the structure of loop CD
appears to be important, although specific interactions between the
central portion of loop CD, including positions 145 and 146, and
transducin require further study. Only one point mutation in this loop
has been reported, R147Q, which had normal ability to activate
transducin in a GTPase assay
(7) . Interestingly, a hydrophobic
amino acid residue in the CD loop in the muscarinic cholinergic
receptor was identified by alanine scanning mutagenesis to play a key
role in G protein coupling
(35) .
at
the cytoplasmic border of helix C
(6, 8, 18, 25, 36) . Glu
in the protonated form regulates a transducin binding and
activation domain that must be made up of additional titratable surface
groups
(18, 25) . The present data provide direct
support for the G protein activation model depicted in Fig. 2.
The specific impairment of catalyzed GDP release in certain mutants
confirms the existence of this discrete step in the activation pathway.
This conclusion is supported by previous indirect evidence: 1) that
binding of R* and nucleotide to transducin are antagonistic
(37) , 2) that GDP dissociates the
R*
G
(empty) complex
(38, 39) , and 3) that R*-bound transducin cannot be
solubilized in the absence of nucleotide
(40) . The present
findings provide a framework for further study to determine the extent
of involvement of particular regions of the cytoplasmic surface of
rhodopsin in mediating discrete steps in the transducin activation
pathway.
Table:
Amino
acid sequence replacement in rhodopsin mutant CD r140-152
Table:
Results of assay of
light-dependent GDP release from G[GDP]
-D-maltoside; DTT, dithiothreitol;
GTP
S, guanosine 5`- O-(3-thiotriphosphate); OG,
n-octyl-
-D-glucoside; MI, metarhodopsin I; MII,
metarhodopsin II; MOPS, 3-( N-morpholino)propanesulfonic acid;
R*, photoactivated rhodopsin; G
or
G
, transducin holoenzyme;
G
,
-subunit of transducin;
G
(empty), transducin with empty
nucleotide-binding pocket.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.