(Received for publication, September 27, 1996, and in revised form, December 3, 1996)
From the Department of Molecular Cardiology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195-5069
The binding of heterotrimeric GTP-binding
proteins (G-proteins) to serpentine receptors involves several
independent contacts. We have deduced the points of interaction between
mutant bovine rhodopsins and t-(340-350), a peptide
corresponding to the C terminus of the
subunit (
t)
of bovine retinal G-protein, transducin. Direct binding of
t-(340-350) to rhodopsin stabilizes the activated metarhodopsin II state (M II), consequently uncoupling the
rhodopsin-transducin interaction. This peptide action requires two
segments on the cytoplasmic domain of rhodopsin: the
Tyr136-Val137-Val138-Val139
sequence on the C-D loop and the
Glu247-Lys248-Glu249-Val250-Thr251
sequence on the E-F loop. We propose that a tertiary interaction of
these two loop regions forms a pocket for binding the
t
C terminus of the transducin during light transduction in
vivo. In most G-proteins, the C termini of
subunits are
important for interaction with receptors, and, in several serpentine
receptors, regions similar to those in rhodopsin are essential for
G-protein activation, indicating that the interaction described here
may be a generally applicable mode of G-protein binding in signal transduction.
Activation of heterotrimeric guanine nucleotide-binding proteins
(G-proteins)1 by transmembrane receptors is
a general paradigm for signal transduction by a large variety of
hormones, neurotransmitters, and physical stimuli. The G-protein
coupled receptors (GPCRs) contain an extracellular N-terminal tail,
seven transmembrane helices, three interhelical loops on either side of
the membrane, and a cytoplasmic C-terminal tail. The cytoplasmic domain
of the receptors binds and activates the G-protein (1-5). Visual
transduction in rod cells is a prototypical example of a
G-protein-coupled signaling system. In rod cells, signal transduction
is initiated by photon-induced isomerization of the
11-cis-retinal chromophore, to all-trans-retinal.
As shown in Fig. 1, this generates an inactive intermediate,
metarhodopsin I (M I), and structural changes in the apoprotein leads
to an active intermediate, metarhodopsin II (M II). The M II then binds and activates the retinal G-protein, transducin (Gt).
Evidence from peptide competition (6), mutational (7-9), and
biochemical (10) studies have implicated three cytoplasmic regions of M II as being critical for Gt interaction. Likewise, in
transducin, the subunit residues 340-350 at the C terminus,
311-323 at
4/
6/
5 regions, 8-23 at the N terminus, and the
farnesylated at the C-terminal tail of the
1 subunit
have been shown to be specific contact sites for rhodopsin (11, 12).
Additional contact sites involving the
subunit are anticipated but
have not been mapped. Thus, several distinct contacts are involved in
the signal transfer from rhodopsin to Gt, but which segment
of Gt interacts specifically with a particular region of
rhodopsin is not known.
This paper focuses on the identification of the residues of bovine
rhodopsin that interact with the transducin subunit C-terminal residues 340IKENLKDCGLF350, a region that is
important in rhodopsin-transducin coupling (11, 13-16). The ability of
an 11-amino acid
t-(340-350) peptide to directly
stabilize the M II state of rhodopsin mutants was employed. We report
that the binding site consists of the residues Tyr136
through Val139 in the C-D loop and the residues
Glu247 through Thr251 in the C-terminal portion
of the E-F loop of bovine rhodopsin.
Procedures for the construction of mutants and
expression of opsins have been described earlier (17, 18). Wild-type
and mutant opsin genes (Table I) were expressed in COS1 cells by transient transfection of corresponding gene. The rhodopsin chromophore was generated by adding 11-cis-retinal (40 µM)
to a cell suspension, the cells were solubilized in 1% dodecyl
maltoside, and rhodopsin was purified by immunoaffinity chromatography
(17, 18). The pigment concentration was calculated from its absorbance
at 500 nm based on E500 = 42,700 M1 cm
1. For rhodopsin samples
prepared for M I
M M II equilibrium studies, the
dodecyl maltoside was replaced with 1% digitonin in all washes and
elution.
|
Transducin was isolated from the
bovine rod outer segment as described by Fung et al. (19).
Catalytic activation of transducin by wild-type and mutant rhodopsins
was assayed by a (GTPS) binding assay as described by
Wessling-Resnick and Johnson (20). The assay mixtures consisted of 1-5
nM purified rhodopsin, 2 µM transducin, 20 µM [35S]GTP
S (1130 Ci/mmol) in 10 mM Tris-HCl, pH 7.2, 100 mM NaCl, 5 mM dithiothreitol, and 0.012% dodecyl maltoside. The assay
was initiated by illumination for 2 min at a wavelength greater than 495 nm. The reaction mixture then remained in the dark at 23 °C for
60 min. The number of moles of [35S]GTP
S bound per mol
of rhodopsin in 60 min was estimated from the
[35S]GTP
S retained on the filter after filteration and
washing.
The
t-(340-350) peptide, Ac-IKENLKDCGLF, and seven
analogues (Fig. 2A) were synthesized, purified, and
characterized by the protein chemistry core services of the Research
Institute of the Cleveland Clinic as described earlier (18). These
peptides will be referred to as peptides 1 (the parent peptide) through
8.
The
The principles and procedure for the M I M II
equilibrium assay have been described previously (16, 18). In a typical experiment, ~5 to 8 × 10
8 M rhodopsin
was evaluated with 1 × 10
4, 5 × 10
4, and 1 × 10
3 M
concentrations of each peptide. The
t-(340-350) peptide
or its analogues were mixed with wild-type and mutant rhodopsins in 1%
digitonin in the dark and kept on ice for 20 min. Dark spectra were
recorded at 5 °C. The samples were then exposed to light for 20 s using a 150-watt Fiber-Lite fitted with 490 nm cut-off filter; the
sample was allowed to equilibrate in the dark at 5 °C for 20 min,
and then light spectra were recorded (see Figs. 2B and
3).
The conclusions in this study are based on the analysis of the
ability of the t-(340-350) peptide to directly bind and
stabilize the M II intermediate. Two different assays were employed:
(i)
t-(340-350) inhibition of rhodopsin-stimulated
transducin activation and (ii) formation of M II from the M I
intermediate by
t-(340-350)-dependent stabilization of M II. The relationship between these two assays is
schematically shown in Fig. 1. Previous studies have
found that bleaching rhodopsin in dodecyl maltoside in the absence of Gt yields the active M II state with a
max
~380 nm, an intermediate that stimulates [35S]GTP
S
binding to Gt (steps 2 through 7). In contrast, bleaching rhodopsin in 1% digitonin in the absence of Gt yields
predominantly the inactive M I (
max ~480 nm)
intermediate (step 2) and a small amount of M II (7, 17, 18, 21).
Formation of the M II state in digitonin requires a stoichiometric
mixture of rhodopsin and Gt because instantaneous
stabilization of M II by Gt is necessary (steps 3 and 5).
Adding the non-hydrolyzable GTP analogue, GTP
S, destabilizes the M
II:Gt complex (step 7) and shifts the equilibrium in favor
of the M I state (21). The formation of the M II state in digitonin can
also be induced by
t-(340-350) rather than
Gt (steps 3 and 4). The stabilization of the M II state
depends on the amino acid sequence and concentration of the
t-(340-350) peptide (11, 18). Thus,
t-(340-350), through binding to a site on rhodopsin,
competes with Gt to shift the M I
M II equilibrium and
thus inhibits [35S]GTP
S binding to Gt
(step 6).
To identify the site on rhodopsin required for binding
t-(340-350), wild-type and mutant rhodopsins purified
from transfected COS1 cells were used in both assays. The mutant opsins
(Table I) expressed well (expression was estimated by
Western blot analysis, not shown), bound 11-cis-retinal and
yielded a chromophore within 90% of that yielded by the wild-type,
indicating that the mutant polypeptides folded normally to a native
state. All mutant rhodopsins purified in dodecyl maltoside and
digitonin yielded a chromophore with a
max ~500 nm
(data not shown). Light-activated rhodopsin samples in dodecyl
maltoside were used for measuring [35S]GTP
S binding to
Gt (Fig. 2A and Table I). The
wild-type rhodopsin activated nearly 267 ± 29 mol of
Gt/mol of rhodopsin. Unregenerated opsin and rhodopsin not
exposed to light activated 15 ± 2 mol of Gt/mol of
rhodopsin.
The synthetic 11-amino acid t-(340-350) peptide 1 inhibited (50 ± 10% of maximal) Gt activation by
bleached rhodopsin. The apparent Ki for inhibition
by peptide 1 was 80 µM. In agreement with earlier studies
using urea-washed rod outer segment disc membranes, an inhibition
greater than 50% could not be achieved at a higher peptide 1 concentration (11). As shown in Figs. 2B and 3, bleaching
wild-type rhodopsin in 1% digitonin yielded predominantly the M I
intermediate (
max ~480 nm) and a small amount of the M
II intermediate. The peptide did not affect the spectrum of rhodopsin
in the dark. The presence of 500 µM
t-(340-350) yielded
50% M II at the expense of M I. Comparing the potency of the peptide in the two assays indicated that
an approximately 200-fold molar excess of peptide 1 was required for
half-maximal inhibition of [35S]GTP
S binding to
Gt (Ki = 80 µM), and an
approximately 1000-fold molar excess of peptide was needed for a
half-maximal shift of M I to M II. Thus, the interaction of
t-(340-350) and Gt with the
detergent-solubilized wild-type rhodopsin obtained from COS1 cells had
properties identical to those reported for interaction with bovine
retinal rhodopsin (11).
To determine which amino acid side chains of
t-(340-350) are important for M II stabilization, the
synthetic analogues shown in Fig. 2A were used. The
C-terminal 347CGLF350 sequence was not examined
because this region has already been shown to form a
-turn
structure, and the subtype of the
-turn is speculated to be
important for receptor selectivity (16, 22). Conservative single amino
acid substitution of the remaining seven residues led to varying
phenotypes (Fig. 2). Peptides 2 and 8 competed as effectively against
Gt as the parent peptide 1 in both assays. Peptide 5 was a
slightly (
2-fold) more effective competitor of Gt and
also better shifted the M I
M II equilibrium in favor of M II (Fig.
2, A and B). Peptide 3 had a slightly lower potency (
2.5-fold less) than peptide 1. Peptides 4, 6, and 7 were
very poor competitors in the Gt activation assay and were completely ineffective in shifting the M I
M II equilibrium (Fig.
2B).
Studies using transferred nuclear Overhauser effect spectroscopy
suggested that a salt bridge between Glu342 and
Lys345 exists in t-(340-350) bound to
rhodopsin in the dark, that is broken during M II stabilization and
replaced by a new salt bridge between Lys345 and the free
-COO
group of the peptide (16). The Gln substitution
can provide hydrogen bonding interactions in the place of a salt
bridge. However, the Lys345
Gln change (peptide 7) is
likely to affect both conformations required for binding to rhodopsin,
in the dark, as well as M II. But, the Glu342
Gln
change in peptide 4 is expected to favor the conformation that enables
M II stabilization. The lack of peptide 4 binding suggests that
Glu342 is essential for stabilizing M II state.
Lys341 made a negligible contribution. Leu344
is a critical residue. Substitution with shorter Ala (peptide 6)
produced an inactive peptide indicating that hydrophobicity and the
side chain size of Leu344 side chain are stringent
requirements for interaction with rhodopsin. Consistent with this
observation, Martin et al. (23) discovered that
combinatorial analogues of
t-(340-350) preserve the
shape of the hydrophobic "face" with little variation, whereas
larger changes in the hydrophilic face are tolerated. Mutagenesis
studies suggest a binding preference for hydrophobic amino acids at the Leu344 and Leu349 positions of
t
(13, 14). Thus, the site on rhodopsin that binds
t-(340-350) is expected to consist of charged and
hydrophobic residues. Furthermore, the analogue and mutagenesis studies
combined together indicate that the
t-(340-350) peptide
and residues 340-350 of Gt are similar in their
interactions with M II and therefore very likely bind to a common site
on rhodopsin.
To identify the rhodopsin t-(340-350)
binding site, we created rhodopsin mutants in three distinct
cytoplasmic regions involved in the Gt interaction. Amidst
these, it should be possible to identify mutants in which the
t-(340-350) binding is abolished even though the
interaction with Gt is not completely abolished. The
abolished
t-(340-350) binding should be restored upon
re-introduction of the wild-type amino acid sequence. Five C-D loop
mutants and six E-F loop mutants were chosen for analysis. In these
mutants, formation of rhodopsin and M II-like states determined by
spectral analysis was not altered, but the activation of Gt
by the mutant M II was altered to various degrees (Table I). The
mutants of the remaining cytoplasmic region implicated in
Gt binding (residues 310-322 at the membrane-proximal
carboxyl tail region of rhodopsin) had a low yield of a rhodopsin-like
chromophore and M II-like photo product. Hence, they were not examined
further.
In a previous study we concluded that the residues Glu134
and Arg135 in the C-D loop are not essential for
t-(340-350) binding (18). When the highly conserved
Tyr136 residue was substituted with Gly, the resulting
mutant bound retinal poorly (data not shown). The mutant CD1, in which
the residues 137-140 were replaced by four alanines to preserve the
-helical potential but alter the amino acid sequence, transducin activation was reduced
30%. As shown in Fig. 3, the
M II yield was
20% in the presence of
2000-fold excess of
t-(340-350). To specifically determine the role of the
Tyr136 residue in the context of the CD1 mutant sequence,
the residues 136-140 (YVVVC of the wild-type) were replaced with LAAAA
(mutant CD2). Transducin activation was reduced
80%. A nearly
6000-fold excess of
t-(340-350) did not shift the M II
M I equilibrium (Fig. 3). Thus, Tyr136 is an important
determinant for
t-(340-350) stabilization of the M II
state. Previously, Ridge et al. (24) used cysteine scanning
mutagenesis to demonstrate that individual replacement of
Tyr136, Val137, Val138, and
Val139 led to partial loss of Gt activation.
The Cys140 residue was found to be not essential (17, 24).
Therefore, the VVV sequence following Tyr136 may contribute
stabilizing interactions. In our study, the remaining substitution
mutants (CD3, CD4, and CD5) caused partial loss of Gt
activation but showed normal affinity for the
t-(340-350) (data not shown). Thus, the residues
141-150 of the C-D loop do not participate in
t-(340-350) binding.
Franke et al. (7) found that replacing the E-F loop region
between residues 231 and 252 with an amino acid sequence from the
extracellular loop B-C produced a rhodopsin molecule that was normally
activated by light but stimulated transducin very poorly. We
constructed the same mutant (EF1 in Table I). The mutant exhibited
normal photocycle properties as reported earlier and also activated
transducin at only 9% of the wild-type control. This mutant
produced an M I-like state when bleached in digitonin. The M II
M I
equilibrium of the mutant was not shifted by
t-(340-350), suggesting that this mutant lacks the
binding site for the peptide (Table I and Fig. 3).
We constructed mutants EF2 through EF5 by restoring the wild-type amino
acid sequence in different parts of the E-F loop region. The mutant EF6
was constructed to examine the remaining two residues predicted to be
the part of the E-F loop. As indicated in Fig. 3 and Table I, mutants
EF2, EF3, and EF6 activated transducin at 40-60% of the wild-type.
The mutants EF2 and EF3 bound
t-(340-350) almost as
well as the wild-type. The mutant EF6 exhibited an interesting phenotype. The
t-(340-350) binding was evident because
the M I peak decreased. However, this decrease was not accompanied by a
transition to a distinct M II peak but rather by an increase in light
scattering at the spectral region below 380 nm. The EF6 mutation must
either alter the affinity for
t-(340-350) or decrease the stability of the M II·
t-(340-350) complex.
Therefore, removing the Val250-Thr251 side
chains likely indirectly influences the
t-(340-350)
interaction. The mutant EF4 was essentially inactive in both the
peptide binding and Gt stimulation assays. Examination of
the residues in this mutant indicates that hydrophilic and charged
residues present in the wild-type rhodopsin E-F loop are replaced by
hydrophobic (Leu), shorter (2 Gly residues), and hydrogen-bonding (Asn,
Ser, and Thr) residues.
On the basis of earlier mutagenesis studies, the
Glu247-Lys-Glu-Val-Thr251 sequence is thought
to be essential for efficient activation of transducin and that the
other residues play a relatively minor role (7, 9). The EKE triad
sequence was kept in the mutant EF5. The M II M I mixture generated
by bleaching this mutant was shifted toward M II formation by
t-(340-350), suggesting that the peptide was now able
to bind and stabilize the M II intermediate. Transducin activation was
partially restored (
30%). Therefore, it seems reasonable that the
charged triad Glu247-Lys248-Glu249
is necessary for the M II
M I equilibrium shift promoted by
t-(340-350) binding. We conclude that
t-(340-350)-mediated stabilization requires the
hydrophobic residues Tyr136-Val-Val-Val139 on
the C-D loop and the hydrophilic charged residues
Glu247-Lys-Glu-Val-Thr251 on the E-F loop of
rhodopsin. The type of analysis used here is not sensitive enough to
determine which side chains of the
t-(340-350) interact
with each of the regions.
Fig. 4 depicts the location of the two sites required
for t-(340-350) binding in the cytoplasmic extensions
of the C and F helices. In conventional models, the transmembrane
helices of rhodopsin are terminated at the membrane-aqueous interface.
However, recent site-directed spin-labeling studies suggest that
1
to 3 turns of the helices extend into the cytoplasm, with helix C having a close tertiary interaction with helices B, D, E, and F. In a
revised model, the Tyr136 and Val139 of helix C
faces helix F, and Lys247 of helix F faces helix C (24,
25). This observation supports our hypothesis that the tertiary
interaction of Tyr136-Cys140 and
Glu247-Thr251 regions forms a subsite that is
stabilized by
t-(340-350). The spin-labeling studies
suggest that both these segments are rigid relative to the helix E
extension, which is more dynamic and not essential for binding the
t-(340-350). Based on these observations, some
qualitative conclusions can be drawn regarding M II stabilization by
t-(340-350) and holotransducin in vivo.
Perhaps the rigidity of the cytoplasmic helix C and helix F extensions
is required to provide an optimal surface for binding. The M II
stabilization may occur because entropy is lost after
t-(340-350) has bound to the rigid cytoplasmic
extensions of helices C and F. This loss explains M II·Gt
complex stabilization by the Gt-
residues 340-350,
which are currently believed to be disordered in the heterotrimer (26).
It is noteworthy that these two rhodopsin helices contact the ionone
ring of the 11-cis-retinal chromophore (27, 28).
It is now generally assumed that the G-protein binding site of all
GPCRs comprises regions from the C-D loop, E-F loop, and the
membrane-proximal segment of the cytoplasmic tail (1-9). Various types
of studies indicate that the E-F loop is preeminent in the G-protein
activation process in GPCRs (3-5). A hydrophobic site near the
N-terminal region of the E-F loop that is important for G-protein
coupling in several GPCRs (4, 29) appears not to be crucial for
t-(340-350) interaction with rhodopsin. Instead, our
results indicate that the
t-(340-350) binding involves
a hydrophobic region of the C-D loop. The hydrophilic and charged portion of this pocket near the C terminus of the E-F loop corresponds to a site that is important for G-protein activation in several GPCRs
(4). E-F loop regions of different GPCRs were found to cross-link to
specific
-subunits (30), as well as to
-subunits of G-protein
heterotrimers (31). All these evidences suggest that the E-F loop may
wrap around the G-protein heterotrimer, establishing contacts with
critical regions of the
-subunit, as well as with the
complex. Our results are the first description of an interaction
between a defined region on transducin and a specific site on the
receptor. Our approach could be used to explore the three other
subsites on rhodopsin for Gt regions,
t-(311-323),
t-(8-23), and the
farnesylated
t-(60-71) residues.
We are indebted to Dr. Kunio Misono for assistance in synthesis and characterization of peptides; Dr. R. Crouch, Medical School of South Carolina, Charlotte, SC for supplying 11-cis-retinal, Dr. Ramaswamy Ramachandran for critical reading of the manuscript, and Robin Lewis and Cassandra Talerico for manuscript preparation.
During the review of this manuscript two papers were published (Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L., and Khorana, H. G. (1996) Science 274, 768-770; Sheikh, S. P., Zvyaga, T. A., Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350). Using two different experimental systems the authors have reached the same conclusion which indicates that movement of transmembrane helices C and F is required for light activation of rhodopsin. Furthermore these studies demonstrated the proximity of cytoplasmic extensions of transmembrane helices C and F which are identical to the segments identified in our study.