From the ¶ Ralph & Muriel Roberts Laboratory for Vision
Science, Sun Health Research Institute, Sun City, Arizona 85372 and
the Department of Pharmacology, University of Washington,
Seattle, Washington 98195-7280
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ABSTRACT |
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Arrestin proteins play a key role in the
desensitization of G protein-coupled receptors (GPCRs). Recently we
proposed a molecular mechanism whereby arrestin preferentially binds to
the activated and phosphorylated form of its cognate GPCR. To test the
model, we introduced two different types of mutations into The decrease of a response to a persistent stimulus
(desensitization) is a widespread biological phenomenon. Signaling by diverse G protein-coupled receptors
(GPCRs)1 is believed to be
terminated by a uniform two-step mechanism (1). According to the model,
activated receptor is first phosphorylated by a G protein-coupled
receptor kinase (GRK). An arrestin protein binds to the activated
phosphoreceptor, thereby blocking G protein interaction.
Arrestin-receptor complex is then internalized, whereupon receptor is
either dephosphorylated and recycled back to the plasma membrane
(resensitization) or sorted to lysosomes and destroyed (down-regulation). Thus, the formation of the arrestin-receptor complex
appears to be the final step of desensitization and the first step of
resensitization and/or receptor down-regulation, which puts it at the
crucial cross-roads of the processes regulating cellular
responsiveness. The tremendously diverse superfamily of G
protein-coupled receptors with more than 1000 members is the largest
known group of proteins that translate a wide variety of external
stimuli into intracellular "language." In contrast, the repertoire
of receptor kinases and arrestins involved in the desensitization of
these receptors is rather limited: only six GRKs and four arrestins
have thus far been found in mammals (reviewed in Ref. 1). This suggests
that at least some of the kinases and arrestins regulate numerous
receptors. Thus, these proteins are attractive targets for research
designed to delineate common molecular mechanisms underlying the
regulation of GPCR signaling in cells (and to create fairly universal
tools for the experimental and/or therapeutic intervention in the process).
Mutagenesis and Biochemical Characterization of
Direct Binding Assay--
In vitro translated
tritiated arrestins (50 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 or with
P- Agonist Affinity Shift Assay--
P- Desensitization Studies in Xenopus Oocytes--
Stage IV oocytes
from mature female Xenopus laevis frogs were harvested,
defolliculated, and cultured as described previously (8). cRNA was
prepared for oocyte injection from cDNA template using Ambion
message machine kit (Ambion, TX) according to manufacturer's protocol.
cDNAs (GenBankTM accession numbers in parentheses) for
rat GRK3 (AA144588), human Recently we have proposed a molecular mechanism that explains an
amazing selectivity of arrestins for the activated phosphorylated forms
of GPCRs (2, 3). According to previous in vitro studies (2,
3) arrestins have two primary binding sites: an activation-recognition site that recognizes the agonist-activated state of the receptor and a
phosphorylation-recognition site that interacts with GRK-phosphorylated elements of the receptor. A potent secondary receptor-binding site is
mobilized for the interaction only when both primary sites are
simultaneously engaged, i.e. when an arrestin encounters
activated and phosphorylated receptor (2, 3). In this model, arrestin is kept in its basal conformation by several intramolecular
interactions in which certain residues in the primary binding sites
("trigger" residues) are involved. One of these triggers is pulled
by binding to an activated form of the receptor, the other, by the
interaction of arrestin with phosphate(s) introduced by GRK. Thus,
arrestin works like a coincidence detector, assuming its high-affinity receptor-binding conformation when both triggers are simultaneously pulled. The model of sequential multisite binding (2, 3), and the
recent crystal structure of visual arrestin (4) set the stage for the
targeted construction of arrestin mutants in which one of the triggers
is constitutively pulled by an appropriate mutation.
In order to test the validity of the model we constructed three
First, we tested the ability of these mutants to interact with purified
-arrestin that were expected to disrupt two crucial elements that make
-arrestin binding to receptors phosphorylation-dependent. We
found that two
-arrestin mutants (Arg169
Glu and Asp383
Ter) (Ter, stop codon)
are indeed "constitutively active." In vitro these
mutants bind to the agonist-activated
2-adrenergic receptor (
2AR) regardless of its phosphorylation status.
When expressed in Xenopus oocytes these
-arrestin
mutants effectively desensitize
2AR in a
phosphorylation-independent manner. Constitutively active
-arrestin
mutants also effectively desensitize
opioid receptor (DOR) and
restore the agonist-induced desensitization of a truncated DOR lacking
the critical G protein-coupled receptor kinase (GRK) phosphorylation
sites. The kinetics of the desensitization induced by
phosphorylation-independent mutants in the absence of receptor
phosphorylation appears identical to that induced by wild type
-arrestin + GRK3. Either of the mutations could have occurred
naturally and made receptor kinases redundant, raising the question of
why a more complex two-step mechanism (receptor phosphorylation
followed by arrestin binding) is universally used.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-Arrestins--
Mutations Arg169
Glu (CGG
GAG),
Gln394
Ter (CAA
TAA), and Asp383->Ter (GAT
TAG) were introduced by polymerase chain reaction in
-arrestin
construct pBARR (3), that was used for in vitro transcription and translation, as described (3).
NcoI/HindIII 1404-base pair open reading frame
was then subcloned into appropriately digested Escherichia
coli expression vector pTrcHisB (Invitrogen). All
-arrestin
species were expressed in the in vitro translation system
and tested in the direct binding assay (3), overexpressed in E. coli, purified to apparent homogeneity (16), and characterized in
the agonist affinity shift assay (7), essentially as described.
2AR or
2AR (100 fmol/assay) in a final
volume of 50 µl for 5 min at 37 °C in room light (rhodopsin) or
for 60 min at 30 °C in the presence of 0.1 mM
-agonist isoproterenol. The samples were immediately cooled on ice
and loaded onto 2 ml Sepharose 2B columns equilibrated with 20 mM Tris-HCl, pH 7.5, 2 mM EDTA. Bound arrestin
eluted with receptor-containing membranes in the void volume (between
0.5 and 1.1 ml). Nonspecific binding determined in the presence of 0.3 µg of liposomes was subtracted.
2AR or
2AR (10-15 fmol/assay) was incubated in 0.25 ml of 10 mM Tris-HCl, pH 7.4, 100 mM NaCl (buffer A)
containing 0.1 mg/ml bovine serum albumin in the presence of 65-75
fmol of [125I]iodopindolol (NEN Life Science Products)
and the indicated concentrations of arrestins and agonists for 60 min
at 22 °C. Samples were then cooled on ice and loaded at 4 °C onto
2 ml of Sephadex G-50 columns. Receptor-containing liposomes with bound
radioligand were eluted with buffer A (between 0.6 and 1.5 ml), and
radioactivity was quantitated in a liquid scintillation counter.
Nonspecific binding was determined in the presence of 10 µM alprenolol. All experiments were repeated two to three
times, and data are presented as means ± S.D.
2AR (AI052644), mouse
opioid receptor (L06322), and rat G protein-gated inwardly rectifying
potassium channel subunits Kir3.1 (U01071) and Kir3.4 (X83584) were
amplified and linearized prior to cRNA synthesis. cDNAs for all
forms of
-arrestin were first amplified by polymerase chain reaction
using oligonucleotides designed to add a T7 promoter upstream and a 45-base poly(A) tail downstream of
-arrestin open reading frame. Standard two-electrode voltage clamp recordings were performed to
register Kir3 currents activated by agonist perfusion as described (8).
The expression levels in oocytes of all forms of
-arrestin were
determined by quantitative Western blot with F4C1 anti-arrestin antibody (22), as described (16). Means ± S.D. from four to six
measurements are presented.
RESULTS AND DISCUSSION
-arrestin mutants: 1) Arg169
Glu, that
reverses the charge of the putative phosphorylation-sensitive trigger
(Arg169
Glu is homologous to the Arg175
Glu mutation in visual arrestin, that makes its binding to rhodopsin
phosphorylation-independent (5)); 2) Gln394
Ter;
and 3) Asp383
Ter, that delete a part or all of the
regulatory arrestin COOH terminus, which keeps arrestin in a basal
conformation and suppresses an untimely mobilization of the secondary
binding site (6).
2AR reconstituted into phospholipid vesicles by
performing direct binding studies (3) and agonist affinity shift assays (7) in vitro. Wild type
-arrestin and
-arrestin-(1-393) bind poorly to activated unphosphorylated
receptor (Fig. 1A). In
contrast,
-arrestin-(Arg169
Glu) and
-arrestin-(1-382) demonstrate significantly higher binding to
activated unphosphorylated receptor (Fig. 1A). Wild type
-arrestin and all three mutants readily bind to activated and
phosphorylated
2AR (Fig. 1A). Thus,
-arrestin-(Arg169
Glu) and
-arrestin-(1-382)
bind to activated
2AR in a phosphorylation-independent fashion.
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Fig. 1.
Direct binding assay. A, 100 fmol of ARK-phosphorylated (P-
2AR,
2.7 ± 0.2 mol phosphate/mol receptor) or unphosphorylated
purified
2AR reconstituted into liposomes was incubated
in a 50 µl reaction with 50 fmol of the indicated form of tritiated
arrestin (specific activities: 140-160 dpm/fmol) in the presence of
100 µM agonist isoproterenol in 50 mM
Tris-HCl, pH 7.5, 50 mM potassium acetate, 0.5 mM MgCl2 for 45 min at 30 °C. B,
0.3 µg of rhodopsin kinase-phosphorylated (P-Rh*,
1.6 ± 0.1 mol of phosphate/mol) or unphosphorylated
(Rh*) rhodopsin was incubated with the same set of arrestins
under room light for 5 min at 37 °C. The samples were then cooled on
ice and loaded at 4 °C onto 2-ml Sepharose 2B columns, equilibrated
with 20 mM Tris-HCl, pH 7.5, 2 mM EDTA. Bound
tritiated arrestins were eluted with receptor-containing membranes in
the void volume (between 0.5 and 1.1 ml), and the radioactivity was
quantitated in a liquid scintillation counter. *, p < 0.01, Student's t test, compared with the binding of
corresponding wild type arrestin.
Recently (7) we found that arrestin-receptor complex is similar to G
protein-receptor complex in two respects: agonists have higher affinity
for arrestin-receptor complex than for receptor alone, and only a
fraction of the receptors forms such a high agonist affinity complex
(HAC) even at saturating concentrations of arrestin. The maximum
percentage of the receptor in HAC gives a good estimate of the
propensity of a given arrestin protein to bind tightly to the receptor
(arrestin competency) (7). Consistent with the direct
binding data (Fig. 1A), -arrestin-(Arg169
Glu) and
-arrestin-(1-382) induced the formation of HAC by unphosphorylated
2AR (22 ± 4% in both cases)
(Fig. 2A). In contrast, all
forms of
-arrestin induced the formation of HAC by phosphorylated
2AR (P-
2AR) (Fig. 2B). The
percentage of HAC formed by P-
2AR in the presence of
saturating (1 µM) concentration of
-arrestin,
-arrestin-(Arg169
Glu),
-arrestin-(1-382), and
-arrestin-(1-393) was 31 ± 6, 52 ± 3, 41 ± 3, and
20 ± 4%, respectively (Fig. 2B). In summary, in both
in vitro assays
-arrestin-(Arg169
Glu)
and
-arrestin-(1-382) demonstrate constitutive activity (phosphorylation-independent receptor binding).
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Next we tested whether these -arrestin species can functionally
desensitize unphosphorylated
2AR in living cells. To
this end GPCRs were expressed in Xenopus oocytes and the
activation of coexpressed G protein-gated inwardly rectifying
K+ channel Kir3 was used as a measure of receptor function.
Under these conditions, the application of receptor agonists produced a
large increase in inwardly rectifying potassium conductance (8).
Undetectable levels of endogenous arrestins and GRKs are expressed in
these cells (data not shown). As a result, only a very slow response
desensitization was evident during prolonged agonist treatment when
2AR (or another GPCR) is expressed alone. The rate of
desensitization was not significantly increased when the receptor is
coexpressed with either GRK alone or arrestin alone. However, a
dramatic increase in desensitization rate was observed when both GRK
and arrestin were coexpressed with a receptor (8, 9). To compare the
relative activity of different forms of
-arrestin, we expressed
2AR with or without GRK3 (also called
-adrenergic
receptor kinase 2 or
ARK2) in the presence or absence of different
forms of
-arrestin. As shown on Fig. 3
(A and C), both wild type and
-arrestin-(1-393) facilitated
2AR desensitization only when
ARK2 was present. In contrast,
-arrestin-(Arg169
Glu) and
-arrestin-(1-382) in
the absence of
ARK2 produced high rates of desensitization similar
to that produced by wild type
-arrestin in the presence of
ARK2,
suggesting that these mutants did induce phosphorylation-independent
desensitization of
2AR in the cell.
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In order to test whether the constitutively active forms of
-arrestin retain the characteristic broad receptor specificity of
wild type nonvisual arrestins (9, 10), we performed similar series of
experiments with
opioid receptor (DOR) (Fig. 3, B and
D), which was previously shown to be desensitized following agonist activation in oocytes coexpressing wild type
-arrestin and
ARK2 (8). Again, the constitutively active mutants induced DOR
desensitization, even in the absence of
ARK2, suggesting that these
mutations do not appreciably change receptor specificity of
-arrestin (or, rather, lack thereof). It should be noted that wild
type visual and
-arrestin, visual arrestin mutant
(Arg175
Glu), and
-arrestin mutants
(Arg169
Glu), (1-382), and (1-393), readily bind to
activated phosphorylated forms of both rhodopsin and
2AR
(Fig. 1). Visual arrestin mutant (Arg175
Glu) also
binds to unphosphorylated activated rhodopsin, while
-arrestin
mutants (Arg169
Glu) and (1-382) bind to
unphosphorylated activated
2AR. However, phosphorylation-independent visual arrestin mutant does not bind to
unphosphorylated activated
2AR and
phosphorylation-independent
-arrestin mutants do not bind to
unphosphorylated activated rhodopsin (Fig. 1). Thus, the preference of
-arrestin for
2AR over rhodopsin and that of visual
arrestin for rhodopsin over
2AR (3) appears, if
anything, enhanced by these mutations.
Interestingly, in the presence of ARK2 both of the
phosphorylation-independent
-arrestin mutants induced a more rapid
receptor desensitization than wild type
-arrestin (Fig. 3), although
the expression levels of all forms of
-arrestin in oocytes were
virtually the same (0.72 ± 0.34, 0.90 ± 0.27, 0.85 ± 0.32, and 1.44 ± 0.87 ng/µg of total protein for wild type,
(Arg169
Glu), (1-382), and (1-393) forms,
respectively). Apparently, faster desensitization in the presence of
ARK2 reflects stronger binding of the mutants to phosphorylated
receptor (Figs. 1, 2). Because the peak agonist-induced
2AR and DOR responses were not significantly different
in oocytes expressing constitutively active
-arrestins (compared
with oocytes expressing no
-arrestin or wild type
-arrestin; data
not shown), the mutants do not appear to be prebound to the receptor
before agonist application.
Our previous studies demonstrated that the crucial GRK phosphorylation
sites are localized on the carboxyl-terminal part of DOR, and that the
truncation of the receptor yielding DOR-(1-339) blocked homologous
desensitization mediated by -arrestin +
ARK2 (8). We tested
whether constitutively active
-arrestin mutants can rescue the
desensitization of DOR-(1-339). Both phosphorylation-independent
-arrestin mutants induced the desensitization of truncated DOR with
virtually the same kinetics as evident for the full-length DOR (Fig.
4). These data suggest that
constitutively active
-arrestins are equally capable of tight
binding to (and blocking the signaling of) a receptor without
phosphates on the COOH terminus and without the COOH terminus itself.
An important implication of this finding is that the major role of the
GRK-phosphorylated elements of the receptor is to pull the
phosphorylation-sensitive trigger on the arrestin molecule; they do not
appear to be required for tight arrestin binding to the receptor
per se.
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Thus, the binding of -arrestin-(Arg169
Glu) and
-arrestin-(1-382) to unphosphorylated receptor detectable in both
in vitro assays (Figs. 1 and 2) translates into the ability
of these mutants to induce phosphorylation-independent receptor
desensitization in the living cell (Figs. 3 and 4). Taken together, the
data corroborate the model of sequential multisite arrestin-receptor
interaction (2, 3) and open an enticing prospect of targeted
construction of mutant arrestins with different special functional
characteristics. Recent studies suggest that arrestin binding targets
the receptors for internalization (10, 11), apparently by virtue of the ability of nonvisual arrestins to interact with clathrin (12), which is
unaffected by the mutations introduced in this study (data not shown).
-Arrestin mutants capable of tight phosphorylation-independent binding to the receptor may change the pattern of intracellular receptor trafficking.
Phosphorylation-independent arrestins are likely to prove valuable
tools for the experimental manipulation of the efficiency of signaling
by different GPCRs. Uncontrolled signaling by various naturally
occurring mutant forms of G protein-coupled receptors has been linked
to a wide variety of pathological conditions in humans, from stationary
night blindness and retinitis pigmentosa (Refs. 13 and 14 and
references therein) to Jansen-type metaphyseal chondrodysplasia (15),
autosomal dominant hypocalcemia (17, 18), autosomal dominant
hyperthyroidism (19, 20), and numerous forms of cancer (reviewed in
Ref. 21). Arrestin mutants with an enhanced ability to block this
excessive signaling appear promising tools for the gene therapy of
these disorders.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. L. Benovic for purified
ARK, Dr. J. G. Krupnick for purified rhodopsin kinase, Dr.
J. J. Onorato for purified
2AR, Dr. J. H. Keen
for purified clathrin, and Dr. L. A. Donoso for the arrestin
monoclonal antibody F4C1.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants EY 11500 (to V. V. G.) and DA 04123 (to C. C.).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.
§ These authors contributed equally to this work.
To whom correspondence and requests for materials should be
addressed: Ralph & Muriel Roberts Laboratory for Vision Science, Sun
Health Research Institute, 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:
GPCR, G
protein-coupled receptor;
GRK, G protein-coupled receptor kinase;
ARK2,
-adrenergic receptor kinase 2 (GRK3);
Kir3, G protein-gated
inwardly rectifying K+ channel;
2AR,
2-adrenergic receptor;
P-
2AR, phosphorylated
2AR;
DOR,
opioid receptor;
Rh*, light-activated rhodopsin;
P-Rh*, phosphorylated Rh*;
HAC, high agonist
affinity complex;
Ter, stop codon.
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REFERENCES |
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