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Volume 272, Number 46, Issue of November 14, 1997
pp. 28849-28852
(Received for publication, July 14, 1997, and in revised form, September 4, 1997)
From the ABSTRACT The rapid decrease of a response to a persistent
stimulus, often termed desensitization, is a widespread biological
phenomenon. Signal transduction by numerous G protein-coupled receptors
appears to be terminated by a strikingly uniform two-step mechanism,
most extensively characterized for the
INTRODUCTION Agonist binding activates G
protein1-coupled receptors
and initiates two intimately intertwined cascades of events, resulting in signal transduction and signal termination (desensitization). The
receptor-agonist complex initially interacts with G protein(s) to form
a transient agonist-receptor-G protein ternary complex that is the
first intermediate in transmembrane signaling (1, 2). This ternary
complex has a higher affinity for agonists than receptor alone (1, 2).
Formation of this complex promotes GDP release from the G protein,
which is followed by rapid GTP binding and dissociation of the active
G EXPERIMENTAL PROCEDURES Bovine arrestin cDNAs were subcloned using the
NcoI and HindIII sites of pTrcB (Invitrogen).
BL-21 cells transformed with the pTrcB-arrestin constructs were grown
at 30 °C in LB containing 0.1 mg/ml ampicillin to an
A600 of 0.2-0.4, induced with 30 µM isopropyl- Hamster P- RESULTS AND DISCUSSION Our initial studies examined ligand binding characteristics of
phosphorylated
[View Larger Version of this Image (20K GIF file)]
Similar curve shifts were observed for the Arrestins bind preferentially to phosphorylated receptors (9, 11). To
further explore this relationship, we tested the phosphorylation
dependence of the high affinity agonist binding to ascertain whether
the increased affinity directly reflects arrestin-receptor interaction.
Additional analysis of the agonist-receptor-arrestin ternary complex
reveals that it is insensitive to physiological concentrations of
MgCl2 (1 mM), CaCl2 (up to 11.6 µM free ± 1 µM calmodulin), ATP, or
GTP (0.1 mM ± 1 mM MgCl2), or 100 nM G protein Various arrestin mutations were previously shown to change the
propensity of arrestins to form a high affinity complex with receptors
(9, 11) and to affect the selectivity of the arrestins (8, 13, 14). For
example, replacing the N-terminal ~45 amino acids of
[View Larger Version of this Image (23K GIF file)]
All non-visual arrestins tested exhibited a profound effect on agonist
affinity and no effect on antagonist binding, suggesting that the
effect may correlate with the intrinsic activity of the ligand. To test
this hypothesis we used two partial agonists: salbutamol, with
relatively high (0.65) intrinsic activity, and dobutamine, with
relatively low (0.25) intrinsic activity. In conjunction with these
ligands we used three arrestin proteins with relatively low, medium,
and high propensity to induce the high agonist affinity state of
P-
[View Larger Version of this Image (16K GIF file)]
To test whether the high agonist affinity of the arrestin-receptor
complexes is observed with other G protein-coupled receptors, we
performed similar experiments with purified, reconstituted m2 mAChR.
Both wild type
[View Larger Version of this Image (18K GIF file)]
In many respects the receptor-arrestin complex behaves similarly to the
previously identified receptor-G protein complex (1, 2, 15). Both
complexes promote high affinity agonist binding to receptors. The
percentage of receptor in the high affinity state appears to be
proportional to the intrinsic activity of the ligand in both cases. The
increase in agonist affinity due to the formation of either of these
complexes is 10-100-fold. A major difference between receptor-arrestin
complex and receptor-G protein complex lies in the sensitivity of the
agonist-receptor-G protein ternary complex to GTP, which promotes G
protein dissociation, whereas the agonist-receptor-arrestin complex is
relatively stable and insensitive to nucleotides, ions, and even whole
cell lysate (not shown). Since the agonist affinity of two structurally
and functionally diverse receptors, the The relative ability of a given arrestin to form arrestin-receptor
complexes with high agonist affinity appears to be its intrinsic
characteristic, which we termed "competency." Conceivably, arrestin
competency may reflect its affinity for a given receptor, the
propensity of an arrestin to undergo a transition into the active
receptor-binding state, or both. We found no apparent relationship between the EC50 values of different arrestins and their
competency with P- FOOTNOTES ACKNOWLEDGEMENTS We thank Dr. L. A. Donoso for the
arrestin monoclonal antibody F4C1, Dr. O. B. Goodman and Dr.
J. H. Keen for purified clathrin, Dr. S. Kennedy for purified G
protein REFERENCES
COMMUNICATION:
Agonist-Receptor-Arrestin, an Alternative Ternary Complex with
High Agonist Affinity*
§,
,,
Sun Health Research Institute, Sun City,
Arizona 85372, the ¶ Department of Molecular Pharmacology and
Biological Chemistry, Northwestern University Medical School, Chicago,
Illinois 60611, the ** Department of Microbiology and Immunology, Kimmel
Cancer Institute, Thomas Jefferson University, Philadelphia,
Pennsylvania 19107, and the ¶¶ Department of Medicine,
University of Wisconsin, Madison, Wisconsin 53706
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
2-adrenergic receptor (2AR), m2
muscarinic cholinergic receptor (m2 mAChR), and rhodopsin. The model
predicts that activated receptor is initially phosphorylated and then
tightly binds an arrestin protein that effectively blocks further G
protein interaction. Here we report that complexes of 2AR-arrestin and m2 mAChR-arrestin have a higher
affinity for agonists (but not antagonists) than do receptors not
complexed with arrestin. The percentage of phosphorylated
2AR in this high affinity state in the presence of full
agonists varied with different arrestins and was enhanced by selective
mutations in arrestins. The percentage of high affinity sites also was
proportional to the intrinsic activity of an agonist, and the
coefficient of proportionality varies for different arrestin proteins.
Certain mutant arrestins can form these high affinity complexes with
unphosphorylated receptors. Mutations that enhance formation of the
agonist-receptor-arrestin complexes should provide useful tools for
manipulating both the efficiency of signaling and rate and specificity
of receptor internalization.
·GTP and G
subunits. The agonist-occupied receptors are
then phosphorylated by G protein-coupled receptor kinases, resulting in
arrestin binding and consequent disruption of receptor-G protein
interaction (3). Recent studies suggest that arrestin binding also
targets the receptors for internalization (4, 5), apparently by virtue
of the ability of non-visual arrestins to interact with clathrin (6), a
process that appears to be a prerequisite for resensitization (3).
Thus, the formation of the arrestin-receptor complex is not only the
final step of signal termination but also an initial step of subsequent
resensitization, representing a critical juncture in the signaling
process. Because of this the arrestin-receptor complex appears to be a
tempting target for a more detailed characterization.
-D-thiogalactopyranoside, and
grown for an additional 4-6 h. Cells were harvested by centrifugation
and lysed, and arrestins were purified by sequential heparin-Sepharose
(6) and Q-Sepharose chromatography as described (6-8), adjusting salt
gradients for elution of different arrestin proteins.
2-adrenergic receptor
(2AR) and human m2 muscarinic cholinergic receptor (m2
mAChR) were expressed in Sf9 cells and purified by affinity
chromatography as described (9). Purified 2AR (90-120
pmol) was mixed with 0.6-0.8 mg of sonicated soybean phosphatidylcholine in 0.4 ml of 10 mM Tris-HCl, pH 7.4, 100 mM NaCl (buffer A) containing 3 mg/ml BSA and incubated
on ice for 7 min. Samples were loaded onto Extracti-Gel columns
(Pierce) at 4 °C, previously equilibrated with 3 ml of buffer A
containing 2 mg/ml BSA and 2 ml of buffer A containing 20 mM MgCl2, and then eluted with 1.7 ml of the
latter buffer. 50% polyethylene glycol 8000 (0.6 ml) was added to the
eluant, mixed, and incubated for 7 min at 22 °C. Samples were
diluted with 30 ml of ice-cold buffer A, and liposomes with
reconstituted receptor were pelleted by centrifugation at 35,000 × g for 90 min. Pellets were resuspended in 0.4 ml of 20 mM Tris-HCl, pH 7.4, 2 mM EDTA. The m2 mAChR
was reconstituted and phosphorylated, as described (10). Receptors were
phosphorylated in the presence of respective agonists by purified
-adrenergic receptor kinase to a stoichiometry of 2.7 ± 0.2 mol/mol (P-2AR) or 8 ± 0.2 mol/mol (P-m2 mAChR),
as described (7, 9, 10). To remove the agonist, P-2AR
was washed with 20 mM Tris-HCl, pH 7.4, 2 mM
EDTA three times by centrifugation as above, whereas P-m2 mAChR was
gel-filtered on a 2-ml Sephadex G-50 column. Control receptors were
similarly prepared but in the absence of kinase.
2AR or
2AR (10-15 fmol/assay) were incubated in 0.25 ml of
buffer A containing 0.1 mg/ml BSA 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 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. P-m2
mAChR and m2 mAChR (10) (50 fmol/assay) were incubated in 0.5 ml of the same buffer A/BSA in the presence of 250 fmol of
[3H]quinuclidinyl benzilate (Amersham Corp.) with the
indicated concentrations of arrestins and agonists for 60 min at
22 °C. 150 µl of ice-cold 30% (w/v) polyethylene glycol were
subsequently added to each assay tube, and the samples were incubated
on ice for 10 min and then filtered through GF/F filters (presoaked for 1 h in 1% polyethyleneimine to reduce nonspecific binding). The radioactivity retained on filters was then quantitated in a liquid scintillation counter. Nonspecific binding was determined in the presence of 10 µM alprenolol (2AR) or
atropine (m2 mAChR). All binding experiments were repeated 2-4 times,
and data are presented as means ± S.D.
2AR in the absence or presence of
arrestins (Fig. 1). Strikingly, both
-arrestin and arrestin3, but not visual arrestin, induced a
pronounced leftward shift of the isoproterenol (ISO) competition curve
(Fig. 1A). We next tested the effects of varying the
arrestin concentration on ligand binding to P-2AR in the
presence of either 30 or 100 nM isoproterenol. This yielded EC50 values of 19.4 ± 7.4 and 5.0 ± 1.8 nM for -arrestin and arrestin3, respectively, values
well within the physiologically relevant range (6, 11).
Fig. 1.
Effect of wild type arrestins on
P-2AR (A-C) or 2AR
(D) affinity for agonists. Receptor was incubated with
280 pM [125I]iodopindolol (IPIN)
in the absence (
) or presence of 1 µM visual arrestin
(
), 1 µM -arrestin (
), or 300 nM
arrestin3 (
) and the indicated concentration of isoproterenol
(A and D), epinephrine (B,
EPI), or norepinephrine (C, NE).
The parameters of the competition curves (two-state model) were as
follows: A, Kl = 660 ± 30 nM (), Kl = 650 ± 70 nM (), Kl = 850 ± 40 nM, Kh = 34 ± 5 nM,
H = 31 ± 6% (), and Kl = 860 ± 120 nM, Kh = 11 ± 3 nM, H = 58 ± 8% (); B,
Kl = 3.3 ± 0.2 µM (),
Kl = 3.8 ± 1.2 µM,
Kh = 110 ± 10 nM,
H = 30 ± 6% (), and Kl = 4.5 ± 0.6 µM, Kh = 65 ± 8 nM, H = 57 ± 3% (); C,
Kl = 26 ± 1.3 µM (),
Kl = 32 ± 4.2 µM,
Kh = 1.6 ± 0.5 µM,
H = 33 ± 5% (), and Kl = 39 ± 11 µM, Kh = 0.7 ± 0.2 µM, H = 56 ± 5% ();
D, Kl = 430 ± 130 nM (), 450 ± 60 nM (), 570 ± 90 nM (), and 140 ± 10 nM (), where Kl is an apparent Kd of low
affinity sites, Kh is an apparent
Kd of high affinity sites, and H is the
percentage of high affinity sites.
-agonists epinephrine and
norepinephrine (Fig. 1, B and C), whereas
affinities for the antagonists alprenolol
(IC50,~3.47 ± 0.14 nM) and propranolol (IC50,~1.19 ± 0.06 nM) were unchanged
by arrestins. As shown in Fig. 1, the agonist competition curves in the
presence of -arrestin or arrestin3 are shallower than control curves
performed in the absence of arrestins. Analysis of these curves reveals
the presence of two distinct sites that differ in their agonist
affinity (Fig. 1). -Arrestin and arrestin3 induce very similar high
and low affinity sites for isoproterenol with IC50 values
of 26.7 ± 9.1 and 871 ± 71 nM, respectively,
the latter value being very similar to the affinity for the receptor in
the absence of arrestin (IC50 of 656 ± 127 nM). -Arrestin and arrestin3 also promote similar high
and low affinity binding sites for epinephrine and norepinephrine with
IC50 values of 90.3 ± 21.8 nM and
3.84 ± 0.69 µM for epinephrine and 1.20 ± 0.40 and 43.1 ± 13.4 µM for norepinephrine,
respectively (Fig. 1). However, the percentage of high affinity sites
is clearly dependent upon the particular arrestin present: 31.5 ± 1.4% with -arrestin and 56.9 ± 1.0% with arrestin3. Thus, it
appears that arrestin3 more efficiently shifts the equilibrium of the
receptor between the two distinct functional states than does
-arrestin. Although neither arrestin drives 100% of the receptors
into a high affinity state, these results are in agreement with direct binding studies where arrestin3 was found to bind better than -arrestin to the P-2AR (9).
-Arrestin did not increase the affinity of unphosphorylated
2AR for ISO, whereas arrestin3 evoked a marginal shift
of the curve (Fig. 1D), apparently reflecting the stronger
propensity of arrestin3 to interact with unphosphorylated receptor (9).
A more systematic study using 2AR phosphorylated by the
-adrenergic receptor kinase to various stoichiometries (0.6-3.4
mol/mol) revealed no effect of -arrestin on the ISO competition
curve at stoichiometries of 0.6 or 1.4 mol/mol, a marginal effect at
1.9 mol/mol, and significant and virtually indistinguishable effects at
2.5-3.4 mol/mol. These data are consistent with our earlier
observation that two phosphates/receptor are necessary and sufficient
for high affinity arrestin interaction (9) and provide additional
evidence that it is the arrestin-receptor complex that demonstrates
higher affinity for agonists. Collectively, all these data strongly
suggest that the receptor complex with high agonist affinity is the
same arrestin-receptor complex previously characterized by direct
binding studies (9).
subunits. Although both -arrestin
and arrestin3 bind with high affinity to clathrin (6) without
influencing clathrin lattice formation (12), the addition of 50 nM purified clathrin had no appreciable effect on the ISO
competition curve shift induced by either 10 or 50 nM
of the non-visual arrestins (not shown). Thus, clathrin interaction
with arrestins does not interfere with arrestin binding to
P-2AR, suggesting that different regions of the arrestin
are involved in these two interactions.
-arrestin
with the corresponding region of visual arrestin (yielding the chimera
ABBB) was found to dramatically increase binding to
P-2AR (9). When this chimera was purified and tested on
the ISO competition curves for P-2AR and
2AR, it induced a more significant change in the curve
than did wild type -arrestin, converting 45.4 ± 6.6% of
P-2AR into a high affinity state (Fig.
2A), with no appreciable
effect on 2AR binding (Fig. 2B). We
previously demonstrated that a single point mutation within the
phosphorylation-recognition region of visual arrestin (R175E) yields a
phosphorylation-independent arrestin (i.e. an arrestin
capable of high affinity binding to both phosphorylated and
unphosphorylated light-activated rhodopsin) (8, 14). Therefore, a
similar form of -arrestin (-arrestin-R169E) was constructed and
assessed for its ability to induce high affinity agonist binding to
P-2AR and 2AR. Interestingly, this mutant not only promotes high affinity agonist binding to 2AR
(21.9 ± 3.9% high affinity sites) but also induces a more
significant shift in the ISO competition curve of P-2AR
(52.1 ± 2.3% high affinity sites) than does -arrestin
(31.4 ± 5.6%) (Fig. 2). Since previous studies demonstrated that
C-terminal arrestin truncation also enhances
phosphorylation-independent receptor binding (9, 11), we tested a
C-terminal truncation of arrestin3. Purified arrestin3-(1-393)
promotes significant high affinity agonist binding to both
P-2AR (82.5 ± 4.6%) and 2AR
(65.6 ± 4.7%) (Fig. 2). Notably, the EC50 values of
both arrestin3-(1-393) (2.98 ± 0.27 nM) and ABBB
(1.91 ± 0.75 nM) for inducing high affinity agonist binding are comparable with the Kd values previously determined in direct binding assays (9), whereas the EC50
values for -arrestin and arrestin3 are substantially higher. These
differences may be attributed to the kinetically complex multistep
nature of arrestin binding to receptor (9, 11), which appears to be
simplified for conformationally unrestrained arrestin mutants (9).
Fig. 2.
Effects of mutant and chimeric arrestins on
P-2AR (A) or 2AR
(B) affinity for isoproterenol. Isoproterenol
competition curves were obtained in the absence () or presence of 1 µM of the chimera ABBB (
), 1 µM
-arrestin-R169E (
), or 300 nM truncated arrestin3-(1-393) (
). The parameters of the competition curves (two-state model) were as follows: A, Kl = 660 ± 30 nM (), Kl = 920 ± 150 nM, Kh = 19 ± 8 nM, H = 45 ± 7% (),
Kl = 830 ± 110 nM,
Kh = 26 ± 10 nM, H = 52 ± 3% (), and Kl = 850 ± 110 nM, Kh = 26 ± 5 nM,
H = 83 ± 5% (); B,
Kl = 430 ± 130 nM (), Kl = 450 ± 30 nM (),
Kl = 760 ± 100 nM,
Kh = 65 ± 18 nM, H = 22 ± 4% (), and Kl = 760 ± 100 nM, Kh = 17 ± 4 nM,
H = 66 ± 5% (), where Kl
is an apparent Kd of low affinity sites,
Kh is an apparent Kd of high
affinity sites, and H is the percentage of high affinity
sites. IPIN, iodopindolol.
2AR, i.e. -arrestin, arrestin3, and
arrestin3-(1-393). These arrestins induced leftward shifts of the
competition curves of both partial agonists, with the effect on
salbutamol being less profound than that on full agonists, although
stronger than that on dobutamine (not shown). The competition curves in
the presence of arrestins were shallow, and analysis of these curves
again revealed the presence of high and low affinity sites. The values
observed for the high affinity sites for salbutamol and dobutamine were
53 ± 9 and 640 ± 240 nM, respectively, in the
presence of all three arrestins tested. The affinities of low affinity
sites were 3.7 ± 1.1 and 15 ± 3 µM for
salbutamol and dobutamine, respectively, i.e. remarkably
close to the affinities observed in the absence of arrestins (3.1 ± 0.2 and 14.5 ± 3.1 µM, respectively). The
percentage of high affinity sites was clearly dependent on the nature
of both the arrestin and the ligand. In the presence of -arrestin,
arrestin3, and arrestin3-(1-393) the percentage of high affinity sites
for salbutamol was 20 ± 2, 39 ± 2, and 42 ± 2%,
respectively, whereas in the case of dobutamine, the percentage was
9 ± 1, 29 ± 6, and 32 ± 3%. As shown in Fig. 3A, the percentage of high
affinity sites in the presence of a given arrestin appears to be
roughly proportional to the intrinsic activity of the ligand. Most
interestingly, the coefficient varies with different arrestin proteins
from 0.32 for -arrestin through 0.59 for arrestin3 to 0.76 for
arrestin3-(1-393). These results suggest that various arrestins each
possess a characteristic propensity to form an arrestin-receptor
complex with high agonist affinity. We propose to term this propensity
of an arrestin its competency. Practically speaking, the
percentage of high affinity sites formed in the presence of a full
agonist appears to give a fairly accurate estimate of the
competency of a given arrestin (compare Figs. 1, 2, and
3).
Fig. 3.
Structure and functional characteristics of
arrestin proteins. A, dependence of the percentage of high
affinity sites on the intrinsic activity of ligands in the presence of
1 µM -arrestin (), 300 nM arrestin3
(), or 300 nM truncated arrestin3-(1-393) ().
Ligands with intrinsic activities of 0 (propranolol, alprenolol), 0.25 (dobutamine), 0.65 (salbutamol), and 1 (isoproterenol, epinephrine, and
norepinephrine) were used. The equation H = aI, where H is the percentage of high affinity
sites and I is an intrinsic activity, yields the best fit.
Coefficients "a" (competency) for -arrestin (), arrestin3
(), and truncated arrestin3-(1-393) () were 0.32, 0.59, and
0.76, respectively. B, structure, phosphorylation
dependence, and competency of arrestin proteins used in this study. The
origin of the sequence of corresponding arrestins is coded by a
filled pattern; the check mark denotes the
position of a point mutation, whereas relative length shows the extent
of truncation. Competency values for -arrestin, arrestin3, and
arrestin3-(1-393) are from the graph on panel A, whereas
the competency of the other three arrestins was estimated by the
percentage of high affinity sites with full agonists.
-arrestin and arrestin3 promote a >10-fold increase
in agonist affinity for phosphorylated m2 mAChR (Fig. 4A), whereas there was no
significant shift in carbachol affinity for the unphosphorylated m2
mAChR (Fig. 4B). In addition, in contrast to the
2AR, there was no significant change in the slope of the curve, suggesting that most of the phosphorylated m2 mAChR forms solely
a high agonist affinity complex with either arrestin. This may be
attributable to the high stoichiometry of phosphorylation of the
P-m2 mAChR preparation (8 mol/mol) or to inherent differences between the two receptors. Interestingly, -arrestin(R169E) had virtually the same effect as wild type -arrestin on the P-m2 mAChR,
whereas arrestin3-(1-393) induced larger shifts on P-m2 mAChR and
exhibited significant phosphorylation-independent interaction with
mAChR (Fig. 4, C and D).
Fig. 4.
Effect of arrestins on P-m2 mAChR (A
and C) and unphosphorylated m2 mAChR (B
and D) affinity for agonists. Receptor was incubated
with 0.5 nM [3H]quinuclidinyl benzilate
(QNB) in the absence () or presence of 1 µM
-arrestin (), or 300 nM arrestin3 (), 1 µM -arrestin-R169E (), or 300 nM
truncated arrestin3-(1-393) (), and the indicated concentration of
carbachol (CCh). IC50 values of carbachol for unphosphorylated m2 mAChR were: 2.78 ± 0.23 (), 1.54 ± 0.16 (), 1.47 ± 0.20 (), 0.90 ± 0.09 (), and
0.33 ± 0.04 () µM; IC50 values on
P-m2 mAChR were: 7.87 ± 1.12 (), 0.38 ± 0.05 (),
0.26 ± 0.02 (), 0.44 ± 0.05 (), and 0.075 ± 0.009 () µM.
2AR and m2
mAChR, increases upon arrestin binding, it is tempting to speculate
that this is a universal phenomenon among G protein-coupled receptors.
The ability of visual arrestin to stabilize the active conformation of
rhodopsin (16) supports this hypothesis. The formation of the
P-2AR--arrestin complex promotes internalization via
clathrin-coated pits (7), suggesting that the bound agonist may also be
internalized, although the biological significance of this is unclear.
Whereas -arrestin and arrestin3 appear to be functionally similar in a number of assays in vitro and in transfected cells (4, 5, 7, 9, 17, 18), we observed a significant difference in their
competency, i.e. the ability to form a high affinity agonist-receptor-arrestin complex with 2AR.
Interestingly, arrestin3 also has a 6-fold higher affinity for binding
to clathrin cages (7). Thus, whereas -arrestin and arrestin3 may
similarly desensitize the 2AR they may differ in their
ability to promote receptor internalization and subsequent
resensitization. The diverse characteristics of the various arrestin
mutants (the ability to form tight complexes with phosphorylated and
unphosphorylated receptors more effectively, the relative specificity
of -arrestin(R169E) toward P-2AR and 2AR, and more promiscuous nature of truncated arrestin3)
makes these proteins useful tools for experimental manipulation of the duration and extent of receptor signaling and internalization in
cells.
2AR. However, mutations that increased
competency appear to reduce arrestin selectivity (Figs. 2 and 4),
suggesting that competency predominantly reflects the ease with which
an arrestin undergoes a transition into its active state. The apparent
similarity of the rank order of competency of different arrestin
proteins with the P-2AR and P-m2 mAChR (Figs. 1, 2, and
4) supports this hypothesis. Competency has no analogous concept for G
proteins, perhaps because the high affinity state of any receptor has
not been systematically studied in the presence of more than one type of G protein. Intuitively the most related concept to arrestin competency in receptor-G protein interactions appears to be coupling efficiency. Our data suggest that certain mutations can substantially increase arrestin competency, and we believe that additional mutants with even higher competency can be engineered. Arrestin competency in vitro may correlate with its ability to desensitize
and/or promote internalization of a given receptor in vivo,
but this hypothesis needs to be experimentally tested.
§
To whom correspondence should be addressed: Sun Health Research
Inst., 10515 W. Santa Fe Drive, Sun City, AZ 85372. Tel.: 602-876-5462;
Fax: 602-876-5695; E-mail: vgurevich{at}sunhealth.org.
Predoctoral fellow of the Howard Hughes Medical Institute.
Established investigator of the American Heart
Association.
1
The abbreviations used are: G protein, guanyl
nucleotide binding protein; 2AR, unphosphorylated
2-adrenergic receptor; P-2AR, phosphorylated 2-adrenergic receptor; m2 mAChR,
unphosphorylated m2 muscarinic cholinergic receptor; P-m2 mAChR,
phosphorylated m2 muscarinic cholinergic receptor; BSA, bovine serum
albumin; ISO, isoproterenol.
subunits, Dr. J. Hirsch for help in protein
characterization by mass spectrometry and N-terminal sequencing, J. Ptasienski for the purification of m2 mAChR, and Dr. R. B. Penn
for critical reading of the manuscript.
Volume 272, Number 46,
Issue of November 14, 1997
pp. 28849-28852
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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 S. Maudsley, B. Martin, and L. M. Luttrell The Origins of Diversity and Specificity in G Protein-Coupled Receptor Signaling J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 485 - 494. [Abstract] [Full Text] [PDF]
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 R. J. Lefkowitz and S. K. Shenoy Transduction of Receptor Signals by {beta}-Arrestins Science, April 22, 2005; 308(5721): 512 - 517. [Abstract] [Full Text] [PDF]
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C. Krasel, M. Bunemann, K. Lorenz, and M. J. Lohse {beta}-Arrestin Binding to the {beta}2-Adrenergic Receptor Requires Both Receptor Phosphorylation and Receptor Activation J. Biol. Chem., March 11, 2005; 280(10): 9528 - 9535. [Abstract] [Full Text] [PDF]
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 R. Jorgensen, L. Martini, T. W. Schwartz, and C. E. Elling Characterization of Glucagon-Like Peptide-1 Receptor {beta}-Arrestin 2 Interaction: A High-Affinity Receptor Phenotype Mol. Endocrinol., March 1, 2005; 19(3): 812 - 823. [Abstract] [Full Text] [PDF]
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M. E. Sommer, W. C. Smith, and D. L. Farrens Dynamics of Arrestin-Rhodopsin Interactions: ARRESTIN AND RETINAL RELEASE ARE DIRECTLY LINKED EVENTS J. Biol. Chem., February 25, 2005; 280(8): 6861 - 6871. [Abstract] [Full Text] [PDF]
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 T. Yoshino, M. Takahashi, H. Takeyama, Y. Okamura, F. Kato, and T. Matsunaga Assembly of G Protein-Coupled Receptors onto Nanosized Bacterial Magnetic Particles Using Mms16 as an Anchor Molecule Appl. Envir. Microbiol., May 1, 2004; 70(5): 2880 - 2885. [Abstract] [Full Text] [PDF]
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 M. Azzi, P. G. Charest, S. Angers, G. Rousseau, T. Kohout, M. Bouvier, and G. Pineyro {beta}-Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors PNAS, September 30, 2003; 100(20): 11406 - 11411. [Abstract] [Full Text] [PDF]
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J. A. Gray, A. Bhatnagar, V. V. Gurevich, and B. L. Roth The Interaction of a Constitutively Active Arrestin with the Arrestin-Insensitive 5-HT2A Receptor Induces Agonist-Independent Internalization Mol. Pharmacol., May 1, 2003; 63(5): 961 - 972. [Abstract] [Full Text] [PDF]
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L. Pan, E. V. Gurevich, and V. V. Gurevich The Nature of the Arrestin{middle dot}Receptor Complex Determines the Ultimate Fate of the Internalized Receptor J. Biol. Chem., March 21, 2003; 278(13): 11623 - 11632. [Abstract] [Full Text] [PDF]
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T. A. Key, T. D. Foutz, V. V. Gurevich, L. A. Sklar, and E. R. Prossnitz N-Formyl Peptide Receptor Phosphorylation Domains Differentially Regulate Arrestin and Agonist Affinity J. Biol. Chem., January 31, 2003; 278(6): 4041 - 4047. [Abstract] [Full Text] [PDF]
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S. A. Vishnivetskiy, J. A. Hirsch, M.-G. Velez, Y. V. Gurevich, and V. V. Gurevich Transition of Arrestin into the Active Receptor-binding State Requires an Extended Interdomain Hinge J. Biol. Chem., November 8, 2002; 277(46): 43961 - 43967. [Abstract] [Full Text] [PDF]
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L. Martini, H. Hastrup, B. Holst, A. Fraile-Ramos, M. Marsh, and T. W. Schwartz NK1 Receptor Fused to beta -Arrestin Displays a Single-Component, High-Affinity Molecular Phenotype Mol. Pharmacol., July 1, 2002; 62(1): 30 - 37. [Abstract] [Full Text] [PDF]
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S. Mukherjee, V. V. Gurevich, A. Preninger, H. E. Hamm, M.-F. Bader, A. T. Fazleabas, L. Birnbaumer, and M. Hunzicker-Dunn Aspartic Acid 564 in the Third Cytoplasmic Loop of the Luteinizing Hormone/Choriogonadotropin Receptor Is Crucial for Phosphorylation-independent Interaction with Arrestin2 J. Biol. Chem., May 10, 2002; 277(20): 17916 - 17927. [Abstract] [Full Text] [PDF]
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R. M. Potter, T. A. Key, V. V. Gurevich, L. A. Sklar, and E. R. Prossnitz Arrestin Variants Display Differential Binding Characteristics for the Phosphorylated N-Formyl Peptide Receptor Carboxyl Terminus J. Biol. Chem., March 8, 2002; 277(11): 8970 - 8978. [Abstract] [Full Text] [PDF]
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B. Cen, Y. Xiong, L. Ma, and G. Pei Direct and Differential Interaction of {beta}-Arrestins with the Intracellular Domains of Different Opioid Receptors Mol. Pharmacol., April 1, 2001; 59(4): 758 - 764. [Abstract] [Full Text]
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G. Wu, G. S. Bogatkevich, Y. V. Mukhin, J. L. Benovic, J. D. Hildebrandt, and S. M. Lanier Identification of Gbeta gamma Binding Sites in the Third Intracellular Loop of the M3-muscarinic Receptor and Their Role in Receptor Regulation J. Biol. Chem., March 17, 2000; 275(12): 9026 - 9034. [Abstract] [Full Text] [PDF]
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