From the Ralph and Muriel Roberts Laboratory for
Vision Science, Sun Health Research Institute, Sun City, Arizona 85372 and the § Howard Hughes Medical Institute,
Department
of Molecular Biophysics and Biochemistry, Yale University, New
Haven, Connecticut 06510
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ABSTRACT |
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Visual arrestin quenches light-induced
signaling by binding to light-activated, phosphorylated rhodopsin
(P-Rh*). Here we present structure-function data, which in conjunction
with the refined crystal structure of arrestin (Hirsch, J. A.,
Schubert, C., Gurevich, V. V., and Sigler, P. B. (1999)
Cell, in press), support a model for the conversion of a
basal or "inactive" conformation of free arrestin to one that can
bind to and inhibit the light activated receptor. The trigger for this
transition is an interaction of the phosphorylated COOH-terminal
segment of the receptor with arrestin that disrupts intramolecular
interactions, including a hydrogen-bonded network of buried, charged
side chains, referred to as the "polar core." This disruption
permits structural adjustments that allow arrestin to bind to the
receptor. Our mutational survey identifies residues in arrestin
(Arg175, Asp30, Asp296,
Asp303, Arg382), which when altered bypass the
need for the interaction with the receptor's phosphopeptide, enabling
arrestin to bind to activated, nonphosphorylated rhodopsin (Rh*). These
mutational changes disrupt interactions and substructures which the
crystallographic model and previous biochemical studies have shown are
responsible for maintaining the inactive state. The molecular basis for
these disruptions was confirmed by successfully introducing
structure-based second site substitutions that restored the critical
interactions. The nearly absolute conservation of the mutagenically
sensitive residues throughout the arrestin family suggests that this
mechanism is likely to be applicable to arrestin-mediated
desensitization of most G-protein-coupled receptors.
The visual amplification cascade (light-activated rhodopsin
(Rh*)1 Visual signal transduction has a special requirement, since the
lifetime of activated rhodopsin is far longer than the required time
resolution. Hence, a molecular mechanism is necessary to rapidly shut
down the receptor. This is accomplished by the combined effect of
rhodopsin kinase and arrestin on light-activated rhodopsin; the former
quickly phosphorylates the COOH-terminal segment of Rh* and arrestin
then binds to P-Rh*, blocking further transducin activation (2, 3).
Arrestin appears to stay bound until metarhodopsin II decays into
opsin, but ultimately it must dissociate to allow the dephosphorylation
of phosphoopsin (4).
It has been proposed that free arrestin exists in an inactive state and
is activated only by interaction with the phosphorylated form of Rh*
(5, 6). Inactive arrestin has a bipartite molecular architecture with
an N- and C-domain each comprised of a Materials--
[ Site-directed Mutagenesis--
Bovine visual arrestin cDNA
(11) was generously supplied by Dr. T. Shinohara. The plasmid pARR-VSP
was constructed and modified as described earlier (12, 13). This
pGEM2-based plasmid encodes bovine wild type arrestin with an
"idealized" 5'-untranslated region (10) under control of a SP6
promoter. Construct pARR-SC was used for all further mutagenesis. All
mutations were introduced by PCR using an appropriate mutagenizing
oligonucleotide as a forward primer and an oligonucleotide downstream
from the far restriction site to be used for subcloning as a reverse
primer. Resulting fragments of various lengths and an appropriate
primer upstream of the near restriction site were then used as reverse and forward primers, respectively, for the second round of PCR. The
resulting fragments were purified, digested with
EcoRI/BamHI (mutations in positions 2-6 and 30),
XhoI/Nsi I (positions 296 and 303), and
BstBI/HindIII (position 382), and subcloned into appropriately digested pARR-SC (13). Double and triple mutants were
constructed by excising fragments carrying one mutation and subcloning
them into the appropriately digested plasmid carrying another mutation.
The sequence of all PCR-generated portions of all constructs was
confirmed by dideoxy sequencing.
In Vitro Transcription, Translation, and Evaluation of
Mutants' Stability--
Plasmids were linearized with
HindIII before in vitro transcription to produce
mRNAs encoding full-length arrestin proteins. In vitro
transcription and translation were performed as described previously
(6, 10). All arrestin proteins were labeled by incorporation of
[3H]leucine and [14C]leucine with the
specific activity of the mix 1.5-3 Ci/mmol, resulting in the specific
activity of arrestin proteins within the range of 54-90 Ci/mmol
(120-200 dpm/fmol). The translation of each of the arrestin mutants
used in this study produced a single labeled protein band with the
expected mobility on SDS-polyacrylamide gel electrophoresis. Two
parameters were used for the assessment of mutant stability. First,
protein yields in the in vitro translation are known to
correlate with stability, most likely because misfolded or denatured
proteins are rapidly destroyed by proteases present in rabbit
reticulocyte lysate (10). Second, denatured proteins tend to aggregate
and are pelleted by centrifugation at 350,000 × g for
1 h. As an estimate of a mutant's relative stability we used its
yield multiplied by the percentage of protein remaining in the
supernatant after incubation for 10 min at 37 oC followed
by centrifugation. This integral parameter calculated for a mutant was
expressed as a percent of that for wild type arrestin. The relative
stability of all mutants used in this study exceeds 0.5.
Rhodopsin Preparations--
Urea-treated rod outer segment
membranes were prepared, phosphorylated with rhodopsin kinase, and
regenerated with 11-cis-retinal as described (14). The
stoichiometry of phosphorylation for the rhodopsin preparations used in
these studies was 1.2-1.6 mol of phosphate/mol of rhodopsin.
Arrestin Binding to Rhodopsin--
In vitro
translated tritiated arrestins (100 fmol) were incubated in 50 mM Tris-HCl, pH 7.5, 0.5 mM MgCl2,
1.5 mM dithiothreitol, 50 mM potassium acetate
with 7.5 pmol of the various functional forms of rhodopsin in a final
volume of 50 ml for 5 min at 37 °C either in the dark or in room
light (6). The samples were immediately cooled on ice and loaded under
dim red light onto 2-ml Sepharose 2B columns equilibrated with 10 mM Tris-HCl, pH 7.5, 100 mM NaCl. Bound
arrestin eluted with the rod outer segments in the void volume (between
0.5 and 1.1 ml). Nonspecific binding determined in the presence of 0.3 mg of liposomes (<10% of the total binding and <1% of the arrestin
present in the assay) was subtracted.
Perturbing the Polar Core--
Our previous mutagenesis studies
have identified Arg175 as a phosphorylation-responsive
trigger in visual arrestin (12, 14, 15). Every mutation in the 175 position, with the exception of the most conservative R175K, yields
proteins with dramatically enhanced binding to nonphosphorylated Rh*
and even 329G-Rh* (rhodopsin from which the COOH terminus
along with all rhodopsin kinase phosphorylation sites has been
proteolytically removed) (14, 15). These mutations led us to conclude
that Arg175 may interact with negatively charged
neighboring residues and that this type of intramolecular interaction
contributes to the stability of arrestin's inactive basal state. We
hypothesized that the association of the receptor's phosphate with
Arg175 neutralizes its positive charge, disrupting these
interactions, thereby releasing some of the constraints that prevent
arrestin from assuming its high affinity rhodopsin binding state.
Therefore, mutant arrestins with a particular residue's charge
neutralized or reversed by mutagenesis can bypass the need for
phosphorylated receptors, i.e. they bind with high affinity
to any light-activated form of rhodopsin, P-Rh*, Rh*, or
329G-Rh* (12, 14, 15).
Both crystal structures (9, 20) show that Arg175 in the
basal state of visual arrestin has three negatively charged partners: Asp30, Asp296, and Asp303. These
residues are the primary components of a network of interactions, which
we refer to as the polar core (Fig. 1).
We replaced each of these Asp residues with Asn (to test whether the
residue serves as a counter-ion or forms a hydrogen bond), with Ala (to
disrupt any potential interaction the side chain may be engaged in),
and with Arg (to preclude the interaction with Arg175 while
creating a potential for an alternative ion pairing with the remaining
negative charges). All mutants were expressed in the in
vitro translation system (6), and their binding to four functional
forms of rhodopsin (dark P-Rh, P-Rh*, dark Rh, and Rh*) was compared
with that of wild type (WT) arrestin, as well as the most potent
Arg175 mutant, R175E, whose receptor binding is least
dependent on receptor phosphorylation (Fig.
2A). Mutations in position 296 markedly increase binding to Rh* (Asp (WT) = Asn
As shown in Fig. 1, the polar core also includes Arg382 of
the C-tail (20). The C-tail constitutes the last 22 ordered residues of
the crystal structure and loops back into the center of the molecule,
where its interactions appear to help stabilize arrestin's inactive
basal state (20). We changed Arg382 to Ala, Glu, and Asn
(Fig. 2C). Importantly, all these mutations increase the
binding to Rh*, although the effect of these mutations on Rh* binding
is less than half that of R175E (Fig. 2C). The results of
all three 382 mutations are comparable. Apparently, the removal of the
positive charge in either position Arg175 (15) or
Arg382 rather than the exact chemical nature of the
substituting residue effects the altered binding profile. The crystal
structure of Granzin et al. (9) modeled Lys2 as
a part of the polar core in place of Arg382. Various
mutations of Lys2 (K2R, K2A, and even K2E) have no
appreciable effect. Moreover, the deletion of residues 2 through 6 has
no effect (Fig. 2B). These data strongly argue against any
functional role of Lys2 and, by inference, against its
positioning as suggested by Granzin et al. (9), supporting
the conclusion that Arg382 occupies this place in the polar
core (20). In the Arg382 mutations, the binding to dark
P-Rh is also increased, similar to the polar core mutations described above.
To position Arg382 in the polar core, additional
interactions of the C-tail with the NH2-terminal domain are
necessary. Previous studies demonstrated that mutations of important
aromatic residues Phe375, Phe377, and
Phe380 increase Rh* binding in a manner similar to
Arg382 mutations (13). The crystal structure (20) indicates
that these phenylalanines interact with residues 11-13. Deletion of residues 11-16 results in a marked increase of binding to Rh* (Fig.
2B). Thus, any mutation that perturbs the positioning of the
C-tail with concomitant destabilization of the polar core compromises
arrestin's selectivity for P-Rh*.
Reconstructing the Polar Core--
Simultaneous charge reversal of
Arg175 and its negatively charged partner could reestablish
the ion pair with an altered structure so that it would no longer
respond to the phosphorylated COOH-terminal segment of rhodopsin, thus
yielding an arrestin "locked" in its basal state. Alternatively,
this double charge reversal could reconstruct a fully functional polar
core. We constructed mutants that combine the R175E mutation with
charge reversal mutations (Asp to Arg) that change each of its partners
(Asp30, Asp296, Asp303). The
combination of R175E with the D296R mutation, each of which alone
dramatically increases arrestin binding to Rh*, yields an arrestin with
a virtually wild type binding profile (Fig.
3). Less potent by themselves, either
mutation D30R or D303R counteracts the effect of R175E but to a
substantially lesser degree than D296R. In addition, we constructed two
double mutations (D296R/D303R as well as D296R/D30R) and tested their
effects on arrestin's selectivity with and without a concurrent R175E
mutation. These results are presented in Fig. 3. In summary, the more a
given mutation enhances arrestin binding to Rh*, the more effective it
is in suppressing the effect of the R175E mutation (the order of
potency being D296R = D296R + D303R
These results substantiate the assertion that the basal state of
arrestin is restrained by a network of interactions in which the
electrostatic forces of the polar core play an important role. While
the constraints of compensating charge reversal variants (such as
R175E/D296R and R175E/D30R/D303R) in the polar core of arrestin may not
be identical in structural detail to those of wild type arrestin, the
stabilizing principles are the same, and they respond similarly to the
intrusion of the receptor's negatively charged phosphate. This
conclusion is consistent with the fact that the presumed disruptive
intrusion of the phosphopeptide is not structurally explicit, that is,
arrestin responds to a receptor that is phosphorylated at any one or
more of seven positions in the receptor's COOH-terminal segment
in vivo.
Conclusions--
It appears that the mechanism whereby
Arg175 functions as a phosphorylation-sensitive trigger is
more complex than was originally proposed (12). The x-ray crystal
structures (9, 20) provide detailed information on the basal state of
arrestin. The major drawback is that the crystal structures give a
detailed stereochemical framework of the molecule only in the basal
state prior to assuming the conformation necessary to bind P-Rh* (6,
8). Mutagenesis studies, on the other hand, provide a wealth of
phenomenological data on the role of various elements of the molecule,
but cannot unequivocally indicate whether the changes in arrestin
behavior occur because of the destabilization of the basal state,
stabilization of the high-affinity rhodopsin binding state, or some
other primary effect of the mutation. Combining inferences from
stereochemistry and mutagenesis allows one to overcome some of
these limitations and to reconstruct arrestin's behavior in terms of a
consistent model. High-affinity binding to receptor occurs after the
phosphorylated COOH-terminal segment of rhodopsin intrudes upon
arrestin's polar core (Fig. 1) and releases the interactions that
constrain free arrestin in a binding-incompetent form. This model is
analogous to the activation of a proenzyme in which the active site is
formed only after a strong stereochemical constraint is released.
Mutations that neutralize the charge on Arg175 or generate
an opposing charge in its primary partner, Asp296, mimic
the effect of phosphate and allow these arrestin mutants to bypass the
need for receptor phosphorylation. Compensating second site charge
reversal mutations restore the requirement for receptor phosphorylation.
Receptor binding to arrestin involves both the N- and C-domains (6, 16)
as discussed in greater detail by Sigler and co-workers (20). In the
basal state, the position of the two domains relative to each other is
supported by multiple interdomain interactions (20). The disruption of
such interactions occurs as the consequence of the intrusion into the
polar core by the receptor's phosphorylated COOH-terminal segment.
Arrestin's transition into its high-affinity rhodopsin binding state
(6) involves a conformational rearrangement (8). It is tempting to
speculate that this event is, in fact, the reorientation of the two
domains relative to each other, since significant change in the
orientation of the domains would be required to bring all of the
regions of arrestin implicated by chemical protection (16) into
receptor contact. The need for a substantial domain rearrangement
follows from the fact that some of the contact sites are 70 Å apart in arrestin's basal state (16, 20), but the maximum dimension of the
cytosolic surface of Rh* is less than 40 Å (17). Based on the crystal
structure of free arrestin (20), the effects of the 3A (F375A, V376A,
F377A) mutation (13) and short deletions in the NH2
terminus (Ref. 6, Fig. 2B) tempt us to speculate that a
rearrangement of the three-element interaction involving residues
9-14, 103-111, and 373-380 is an integral part of the transition
into the rhodopsin binding competent state. Interestingly, residues
100-120 were recently implicated in rhodopsin binding (18). Further
structure-function studies are necessary to elucidate the molecular
mechanism whereby arrestin recognizes the activated and phosphorylated
state of rhodopsin, ensuring the exquisite selectivity of visual
arrestin for P-Rh*. The elucidation of the three-dimensional structure
of an arrestin/rhodopsin complex would be most informative. Since the
mutagenically sensitive residues are almost absolutely conserved among
all members of the arrestin family, the presented mechanism for
arrestin's activation is most likely to be a general model for
arrestin-mediated desensitization of most G-protein-coupled receptors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
transducin
cGMP phosphodiesterase) is an archetypal signaling pathway initiated by
a G protein-coupled receptor (1). A single Rh* can activate
sequentially hundreds of transducin molecules. Active cGMP
phosphodiesterase hydrolyzes hundreds to thousands of cGMP molecules.
Thus, the potential for signal amplification is enormous, providing for
a very high sensitivity and requiring mechanisms to moderate the
response within a vast dynamic range.
-sandwich (9, 20). In
addition, it has an extended carboxyl-terminal chain called the C-tail
bound to the surface of both N- and C-domains (20). In this study we
used site-directed mutagenesis, which combined with previous
mutagenesis, biochemical studies (5, 6), and the crystal structure (20)
of arrestin, allows us to propose a mechanism for arrestin's response
to the phosphorylated, activated receptor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP,
[14C]leucine, and [3H]leucine were
purchased from NEN Life Science Products. All restriction enzymes were
purchased from New England Biolabs. Sepharose 2B and all other
chemicals were from sources described previously (6). Rabbit
reticulocyte lysate and SP6 RNA polymerase were prepared as
described previously (10). 11-cis-Retinal was generously
supplied by Dr. R. K. Crouch.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Ala
Arg).
This order of potency is the functional expression of the fact that
Asp296 forms a hydrogen-bonded ion pair with
Arg175, as seen in the crystal structures (9, 20).
Asp303 mutations moderately increase the binding to Rh*,
whereas Asp30 mutations have minimal effect. None of these
mutations compromise P-Rh* binding. Several of these polar core
mutations show an increased binding to an inactive phosphorylated
receptor, i.e. the altered protein has partially lost its
ability to discriminate against inactive rhodopsin. The stereochemical
basis of this effect will require more structural information on the
receptor binding form of arrestin.
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Fig. 1.
Essential residues of the polar core.
Rendering of the five charged side chains and their relative
orientation based on the crystal structure is shown.2
Hashed green lines denote hydrogen bonds whose lengths are
3.0 Å or less. Figure was generated with Molscript/Raster3D (7,
19).
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Fig. 2.
Perturbations in the polar core. Effects
of point mutations of negatively charged partners of Arg175
(A), mutations in the NH2 terminus
(B), and of Arg382 (C) on arrestin
binding to four functional forms of rhodopsin. The indicated forms of
rhodopsin (150 nM) were incubated with the indicated
arrestin proteins (2 nM, specific activities 120-200
dpm/fmol) at 37 °C for 5 min in a total volume of 50 µl. Samples
were then cooled on ice, and bound and free arrestin were separated by
Sepharose 2B chromatography as described under "Experimental
Procedures." Means ± S.D. from two to three experiments each
performed in duplicate are shown.
D30R + D303R > D303R > D30R). This correlation suggests that the enhancement of
Rh* binding by the disruptive effect of charge reversal and second site
suppression of the R175E mutation are related stereochemically.
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Fig. 3.
Reconstruction of the polar core.
Effects of the charge reversal mutations of the negatively charged
partners of Arg175 in combination with R175E mutation on
arrestin binding selectivity. The experiments were performed as
described in the legend to Fig. 2. Means ± S.D. from two
experiments each performed in duplicate are shown.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. L. Benovic for purified rhodopsin kinase, Dr. R. K. Crouch for 11-cis-retinal, and Dr. T. Shinohara for arrestin cDNA.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants EY11500 (to V. V. G.) and GM22324 (to P. B. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Fellow of the Deutsche Forschungsgemeinschaft.
** National Eye Institute postdoctoral fellow.
To whom correspondence and reprint requests should be
addressed: Ralph & Muriel Roberts Laboratory for Vision Science, Sun Health Research Institute, 10515 W Santa Fe Dr., Sun City, AZ 85372. Tel.: 602-876-5462; Fax: 602-876-6663; E-mail:
vgurevich{at}sunhealth.org.
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ABBREVIATIONS |
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The abbreviations used are: Rh*, light-activated nonphosphorylated rhodopsin; Rh, dark nonphosphorylated rhodopsin; P-Rh*, light-activated phosphorylated rhodopsin; P-Rh, dark phosphorylated rhodopsin; PCR, polymerase chain reaction; WT, wild type.
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