(Received for publication, July 8, 1994; and in revised form, November 14, 1994)
From the
Arrestins play an important role in quenching signal
transduction initiated by G protein-coupled receptors. To explore the
specificity of arrestin-receptor interaction, we have characterized the
ability of various wild-type arrestins to bind to rhodopsin, the
-adrenergic receptor (
AR), and the m2
muscarinic cholinergic receptor (m2 mAChR). Visual arrestin was found
to be the most selective arrestin since it discriminated best between
the three different receptors tested (highest binding to rhodopsin) as
well as between the phosphorylation and activation state of the
receptor (>10-fold higher binding to the phosphorylated
light-activated form of rhodopsin compared to any other form of
rhodopsin). While
-arrestin and arrestin 3 were also found to
preferentially bind to the phosphorylated activated form of a given
receptor, they only modestly discriminated among the three receptors
tested. To explore the structural characteristics important in arrestin
function, we constructed a series of truncated and chimeric arrestins.
Analysis of the binding characteristics of the various mutant arrestins
suggests a common molecular mechanism involved in determining receptor
binding selectivity. Structural elements that contribute to arrestin
binding include: 1) a C-terminal acidic region that serves a regulatory
role in controlling arrestin binding selectivity toward the
phosphorylated and activated form of a receptor, without directly
participating in receptor interaction; 2) a basic N-terminal domain
that directly participates in receptor interaction and appears to serve
a regulatory role via intramolecular interaction with the C-terminal
acidic region; and 3) two centrally localized domains that are directly
involved in determining receptor binding specificity and selectivity. A
comparative structure-function model of all arrestins and a kinetic
model of
-arrestin and arrestin 3 interaction with receptors are
proposed.
Many cells have the ability to rapidly regulate their
responsiveness to external stimuli. This phenomenon, often termed
desensitization, has been extensively studied in signaling pathways
initiated by G protein-coupled receptors. Two of the best studied G
protein-coupled receptors are rhodopsin, which mediates
phototransduction in retinal rod cells, and the
-adrenergic receptor
(
AR)
, which stimulates cAMP production in
many tissues. The molecular events underlying desensitization of
rhodopsin and the
AR include a rapid
activation-dependent phosphorylation of the receptor mediated by the
specific G protein-coupled receptor kinases rhodopsin kinase (1) and the
-adrenergic receptor kinase (
ARK) (2) , respectively. Receptor phosphorylation then promotes the
binding of another protein to the receptor. This protein has been
termed S-antigen or arrestin in the phototransduction system while in
the
AR system a related protein termed
-arrestin
has been implicated. The binding of arrestin to the phosphorylated
receptor appears to play the primary role in quenching signal
transduction via its apparent ability to directly decrease receptor/G
protein coupling (3, 4, 5, 6) .
Recent studies suggest a significant diversity in the arrestin gene
family with four mammalian arrestins having been identified to
date(5, 7, 8, 9, 10, 11, 12) .
The mammalian arrestins include visual arrestin, which is predominantly
localized in rods and appears to play a primary role in quenching
phototransduction(13, 14) ; -arrestin, which may
play a role in desensitization of the
AR(5, 6) ; arrestin 3 (also termed
-arrestin 2), a recently identified member of the family that may
interact with the
AR and possibly odorant
receptors(8, 9, 10, 15) ; and a
recently cloned retinal-specific arrestin termed X-arrestin (11) or C-arrestin(12) . Recent studies suggest that at
least three of the mammalian arrestins may also be represented by
multiple polypeptide variants(10, 16, 17) .
These variants include a form of
-arrestin,
arrS, that lacks
8 amino acids found in the originally identified bovine brain
-arrestin (
arr)(5, 10, 16) .
Interestingly, the 8 amino acids that distinguish these two forms of
-arrestin are encoded by exon 13 in the human visual arrestin
gene(18) . While a similar variant may also exist for visual
arrestin(16) , a variant that has an alanine in place of the
C-terminal 35 amino acids has also been identified(17) . In
addition, a form of arrestin 3 (arr3L) that contains an 11-amino acid
insert between residues 361 and 362 has also been
identified(10) . While the predominant form of
arr varies
between tissues and arr3S is the predominant form of arr3 in most
tissues, little is presently known about the functional significance of
these polypeptide variants.
The tremendous diversity of the G
protein-coupled receptors, as compared to the more limited number of G
protein-coupled receptor kinases and arrestins, raises obvious
questions concerning the receptor specificity of the various G
protein-coupled receptor kinases and arrestins. Here we have compared
the ability of visual arrestin, arr,
arrS, arr3L, and arr3S
to bind to four different functional forms (phosphorylated,
phosphorylated and agonist activated, unphosphorylated, and
unphosphorylated and agonist activated) of three functionally distinct
receptors: rhodopsin, the
AR, and the m2 muscarinic
cholinergic receptor (m2 mAChR). We have utilized these receptors since
they each have been demonstrated to undergo an activation-dependent
phosphorylation both in vitro and in intact
cells(2, 19, 20, 21, 22, 23) .
Moreover, more recent studies suggest that each of these receptors is
also able to interact with arrestins in
vitro(4, 5, 6, 9, 24) .
The recent progress in elucidating the molecular mechanisms involved
in visual arrestin binding to phosphorylated light-activated rhodopsin
(P-Rh)(25, 26, 27) , as well as
the cloning of an increasing number of arrestins, raises several
important questions concerning arrestin function. First, do other
members of the arrestin family use the same sequential multisite
binding mechanism as does visual arrestin? In addition, what domains of
the arrestin molecule determine its receptor specificity and what
structural elements of arrestins and receptors are involved? In an
attempt to address these questions we have utilized two strategies of
mutagenesis. First, several truncated forms of
arr and arr3,
analogous to the previously characterized truncated forms of visual
arrestin (arr)(25, 26) , were produced and
functionally characterized. This approach allowed us to establish a
similar localization of the major functional domains in all arrestin
proteins and suggested that the molecular mechanisms involved in their
binding may also be similar. These findings set the stage for the
construction of a series of arr/
arr chimeras. In constructing the
chimeric arrestins we attempted to divide the arrestins into the four
functional domains previously identified in visual
arrestin(25, 26) . These studies have enabled us to
obtain further insight into the mechanism of arrestin-receptor
interaction and the structural basis of arrestin specificity.
Figure 4:
Structure and binding of chimeric
arrestins to human P-mAChR and
P-
AR
. The portion of the chimera derived
from
arr is shaded in the schematic. The four structural
elements of the chimeric arrestins are: 1) N-terminal region: residues
1-47 of arr or 1-43 of
arr (49% identity, 64%
similarity); 2) N-terminal binding domain: residues 48-213 of arr
or 44-207 of
arr (60%, 74%); 3) C-terminal binding domain:
residues 214-345 of arr or 208-340 of
arr (60%, 77%);
4) regulatory C-terminal region: residues 346-404 of arr or
341-418 of
arr (32%, 39%). The average K
and B
from two to three experiments
(± S.D.) is presented. ND, not
determined.
As previously demonstrated, visual arrestin preferentially binds to
P-Rh with a P-Rh
/P-Rh binding ratio of
12 (Fig. 1A). Arr3S, arr3L,
arr, and
arrS
demonstrate a 2-3-fold lower level of binding to P-Rh
compared to arr. In addition,
-arrestin and arrestin 3 also
bind to rhodopsin less selectively compared to arrestin with
P-Rh
/P-Rh binding ratios from 1.3 for arr3L to 2.1 for
arr. The selectivity profiles of
arr and arr3 binding to the
m2 mAChR qualitatively resemble those seen with rhodopsin (Fig. 1B). All of the arrestins bind preferentially to
P-mAChR
with arr3S binding being
1.5-fold higher than
arr3L,
arr, and
arrL, and
19-fold higher than arr. The
P-mAChR*/P-mAChR binding ratio is 1.6-1.9 for all arrestins,
similar to that observed for
arr and arr3 binding to rhodopsin.
Figure 1:
Interaction of in vitro translated arr, -arr,
-arrS, arr3L, and
arr3S with rhodopsin A. 50 fmol of the various in vitro translated arrestins (1068-1205 dpm/fmol) were incubated
with 7.5 pmol of the various functional forms of rhodopsin in 50 mM Tris-HCl, pH 7.5, 100 mM potassium acetate, 0.5 mM MgCl
, 1.5 mM dithiothreitol in 50 µl for
5 min at 37 °C either in the dark or with illumination (room
light). The samples were then cooled on ice before separation of bound
and free arrestins on a 2-ml Sepharose 2B column as described under
``Experimental Procedures.'' B, interaction of in vitro translated arrestins with the human m2 mAChR. 50 fmol
of the various arrestins were incubated with 200 fmol of the different
functional forms of the m2 mAChR and either 100 µM atropine or carbachol in 50 mM Tris-HCl, pH 7.5, 50
mM potassium acetate, 0.5 mM MgCl
, 0.2
mM dithiothreitol in 50 µl at 30 °C for 50 min, cooled
on ice, and chromatographed as described under ``Experimental
Procedures.'' The phosphorylation stoichiometry of the m2 mAChR
used in these studies was 3.7 mol of phosphate/mol of m2 mAChR. C, interaction of in vitro translated arrestins with
the human
AR. 50 fmol of the various arrestins were
incubated with 200 fmol of the different functional forms of the
AR and 100 µM alprenolol
or(-)-isoproterenol in 50 mM Tris-HCl, pH 7.5, 50 mM potassium acetate, 0.5 mM MgCl
in 50 µl
at 30 °C for 35 min, cooled on ice and chromatographed as described
under ``Experimental Procedures.'' The phosphorylation
stoichiometry of the
AR used in these studies was 2.4
mol of phosphate/mol of
AR. The specific binding (mean
± S.D. from two to four independent experiments performed in
duplicate) is shown.
Since no direct binding studies of arrestin interaction with the
AR have been reported, we initially attempted to
optimize the conditions to study
arr and arr3 binding to the
AR. Binding to the
AR was found to
reach an apparent equilibrium following a 30-min incubation at 30
°C, which was maintained for at least another 30 min. The minimum
phosphorylation level necessary for
arr and arr3S to recognize the
phosphorylated form of the
AR was 2-3 mol of
P
/mol of
AR, while additional
phosphorylation up to 10-11 mol/mol did not increase
arr or
arr3S binding (not shown). The selectivity profiles of
arr, arr3,
and arr binding to the
AR are very similar to those
observed with the m2 mAChR (Fig. 1C). Arr3S
demonstrates the highest binding, followed by arr3L,
arr, and
arrS. Arr binding to the
AR was 4-9-fold
lower than the other arrestins. The discrimination of
arr and arr3
between the P-
AR
and P-
AR
is also very similar to that observed for the m2 mAChR
(1.5-1.8-fold).
Previously, we demonstrated that a
conventional Scatchard analysis could be used to study arr and arr
binding to the P-mAChR
(24) . More extensive
analysis demonstrates that arr3S and arr3L (K
= 0.35-0.38 nM), and
arr and
arrS (K
= 0.5-0.6 nM) have
similar affinities for the human P-m2 mAChR
, while arr (K
= 7.2 nM) has a substantially
lower affinity (Table 1). The number of binding sites, determined
in the presence of 50 fmol of P-mAChR
, was also
substantially different among the arrestins with a B
of 15-19 fmol for arr3S, arr3L,
arrS, and
arr and
only
7 fmol for arr. A similar analysis of arrestin binding to the
human P-
AR
demonstrates that
arr and
arrS have the highest affinity (K
=
0.14-0.19 nM), followed by arr3S and arr3L (K
= 0.33-0.36 nM), and arr (K
= 2.1 nM) (Table 1).
Thus, the affinity of
arr and
arrS for the
P-
AR
is 3-4 times higher than for
the P-mAChR
, while the affinity of arr3L and arr3S for both
receptors is comparable. The number of binding sites, determined in the
presence of 50 fmol of P-
AR
, was highest
for arr3S (17 fmol), followed by arr3L (11 fmol),
arrS (7.1 fmol),
arr (5.5 fmol), and
arr (4.1 fmol).
A prominent difference
between the binding of visual arrestin and the non-visual arrestins is
the substantially greater selectivity of visual arrestin for the
phosphorylated and activated form of the receptor. While -arrestin
and arrestin 3 preferentially bind to the phosphorylated form of a
given receptor, they appear to be less dependent on the activation
state of the receptor. To further analyze this phenomenon we studied
the binding of
arr and arr3S to the P-mAChR and
P-
AR in the presence of either agonist or antagonist.
These studies revealed that the agonist promotes a 2-3-fold
increase in B
for
arr and arr3S compared to
the antagonist with no appreciable difference in the K
(Table 1). These results suggest that the agonist
predominantly alters the number of phosphorylated receptors capable of
interacting with a given arrestin, presumably by converting more
receptors to an ``active'' conformation. However, it is clear
that not all of the receptors are active even in the presence of
agonist since the B
is always lower than the
number of receptor molecules in the assay. The differences in B
may reflect the ability of a given arrestin to
promote the formation of and/or stabilize an
agonist-phosphoreceptor-arrestin complex, analogous to the
agonist-receptor-G protein ternary complex(29) .
The
relatively low B observed for arrestin binding (Table 1) prompted us to search for experimental conditions that
favored the formation of a high affinity agonist-receptor-arrestin
complex. To this end we studied the effects of G protein
subunits on
arr and arr3S binding. For these studies the
AR and m2 mAChR were initially phosphorylated to a
stoichiometry of 3-4 mol/mol in the absence of
subunits. When purified bovine brain
subunits (at a 1:1
ratio with receptor) were included in the binding assay they
significantly increased the B
of both
arr
and arr3S binding to the P-mAChR
(31 and 38%,
respectively), without effecting the K
. Similar
experiments with the P-
AR
revealed that
subunits had no effect on either the K
or B
of
arr or arr3S binding. Thus,
it appears that
subunits do not directly interact with
arrestins since the effects observed are similar for both
arr and
arr3S and are strictly receptor-dependent. Conceivably,
subunits are able to bind to the P-m2 mAChR
shifting the
equilibrium toward the high-affinity arrestin-phosphoreceptor complex.
In an attempt to resolve different interactions involved in
arrestin-receptor binding we compared the salt sensitivity of arr
and arr3 binding to P-
AR,
P-
AR
, P-mAChR, and P-mAChR
(Fig. 2, upper panel). The binding of
arr
and arr3S to P-
AR
is modestly stimulated
at physiological salt concentrations followed by slight inhibition at
higher ionic strength. This resembles the effects of ionic strength on
visual arrestin binding to P-Rh
(26) , and suggests
that both ionic (inhibited by salt) and hydrophobic (stimulated by
salt) interactions are involved in
arr and arr3S binding to the
P-
AR
. Physiological salt concentrations
also stimulate
arr and arr3S binding to the P-mAChR
,
while high salt significantly inhibits binding (Fig. 2, upper panel). This again suggests a role for both ionic and
hydrophobic interactions, although both effects are more pronounced
compared to the
AR. Thus, the contribution of ionic
and hydrophobic interactions involved in arrestin binding appears to be
determined in part by the receptor. However, these data demonstrate
that both
arr and arr3 possess the sites necessary for high
affinity interaction with either the P-
AR
or P-mAChR
. Previous studies have demonstrated that
visual arrestin binding to dark P-Rh is predominantly mediated by ionic
interactions since it is very sensitive to salt
inhibition(26) . In contrast, when the effect of receptor
activation was tested on
arr and arr3S binding to the
P-
AR, no appreciable difference in salt sensitivity
was observed between P-
AR and
P-
AR
(Fig. 2, upper
panel). A similar salt sensitivity was also observed for
arr
and arr3S binding to the P-mAChR
and P-mAChR (Fig. 2, upper panel). These results suggest that
similar interactions mediate the binding of both arrestins to the
phosphorylated
AR and m2 mAChR regardless of the
activation state of the receptor. This result is in agreement with the
similar affinities of
arr and arr3 for the phosphorylated and
phosphorylated/activated forms of the
AR and m2 mAChR (Table 1).
Figure 2:
Salt effects on arr, arr3S,
arr(1-206), and arr3(1-183) binding to hamster
P-
AR (circles) and human P-m2 mAChR (triangles) in the
presence of 100 µM of the agonists isoproterenol or
carbachol (solid lines) or the antagonists atropine or
alprenolol (dotted lines). All assay conditions were as in the
legend to Fig. 1except for the potassium acetate concentration
which varied from 50 to 500 mM. The nonspecific binding was
determined at all salt concentrations (it decreased with an increase in
salt) and subtracted. The specific binding (mean ± S.D. from two
to three independent experiments performed in duplicate) is shown as
the percentage of control binding determined at 50 mM potassium acetate.
In order to initially
probe the structural domains involved in arr and arr3 interaction
with G protein-coupled receptors, we utilized truncation mutagenesis,
an approach previously used to study visual arrestin interaction with
rhodopsin(25, 26) . The truncated proteins produced in
this study were
arr(1-367) and arr3S(1-375), which
lack the putative regulatory C-terminal region (
40 amino acids)
identified in arr(24, 25, 26) , and
arr(1-217),
arr(1-206), and arr3(1-183),
which lack the C-terminal half of the molecule. Previously we
demonstrated that similarly truncated arr(1-191) largely retains
its phosphorylation- and activation-recognition sites, two primary
regions involved in arr interaction with
rhodopsin(25, 26, 30) .
Deletion of the C
terminus of arr to produce
arr(1-367) increases binding
to all functional forms of rhodopsin, the m2 mAChR, and the
AR, with the predominant effect observed on the
phosphorylated and the activated forms of the receptor (Fig. 3).
The analogous deletion in arr3S to generate arr3S(1-375) modestly
increases binding to the activated form of the various receptors while
decreasing binding to the phosphorylated/activated receptors. Thus, a
major effect of C-terminal truncation of
arr and arr3S is to
reduce the selectivity of the arrestin for the phosphorylated/activated
form of the receptor. These results are very similar to the effects of
C-terminal truncation of arr (25, 26) , suggesting
that the C terminus of
arr, arr3S, and arr plays a similar
functional role.
Figure 3:
Interaction of full-length and truncated
arrestins with rhodopsin (A), human m2 mAChR (B), and
hamster AR (C). 50 fmol of
arr,
arr(1-367),
arr(1-217),
arr(1-206),
arr3S, arr3(1-375), or arr3(1-183) (1006-1485
dpm/fmol) were incubated with receptor and then treated as described
under ``Experimental Procedures.'' The specific binding (mean
± S.D. from two to four independent experiments performed in
duplicate) is shown.
The deletion of the entire C-terminal half of
arr and arr3S substantially decreases the binding to rhodopsin and
the
AR, while increasing the binding to the m2 mAChR (Fig. 3). In general, the interaction of
arr(1-217),
arr(1-206), and arr3(1-183) with all three receptors
demonstrate a uniformly lower selectivity compared to the wild type
arrestins. However, it is noteworthy that for interaction with
rhodopsin and the m2 mAChR, it is the activation-recognition of the
truncated
arr and arr3 that is predominantly impaired, while for
interaction with the
AR it is the
phosphorylation-recognition that is primarily impaired.
The deletion
of the C-terminal 40 residues (
arr(1-367) and
arr3S(1-375)) appears to decrease the affinity of
arr and
arr3S for binding to human P-mAChR
and hamster
P-
AR
(Table 2). Since this region is
likely not involved directly in receptor interaction (24, 25, 26) , these data underscore the
importance of this domain in keeping the proper functional conformation
of
arr and arr3S. The deletion of the entire C-terminal half
(
arr(1-217),
arr(1-206), and arr3(1-183))
further impairs the affinity of both
arr and arr3 for
P-mAChR
and P-
AR
(Table 2). In contrast, the effect of C-terminal
truncations of
arr and arr3S on the B
for
binding to P-mAChR
and P-
AR
is
dramatically different between the receptors. Truncations of
arr
modestly increase the B
for the
P-
AR
while truncations of arr3S
significantly decrease the B
(Table 2). By
comparison, all of the C-terminal truncations of
arr dramatically
increase the B
for the P-mAChR
while
only arr3(1-183) was affected in a similar manner. It is
noteworthy that the B
for 4 out of the 5
truncated arrestins was increased to 70-90% of the level of
P-mAChR
present in the assay (Table 2). This
demonstrates that most, if not all, of the receptors are accessible for
binding arrestins and suggests that unavailability of receptors is not
the reason for the relatively low B
measured for
full-length
arr and arr3 ( Table 1and Table 2).
In
order to compare the interactions involved in the binding of truncated
arrestins to phosphorylated receptors in the presence of agonists and
antagonists, we studied the salt sensitivity of arr(1-206)
and arr3(1-183) binding to P-
AR and P-mAChR (Fig. 2, lower panel). In contrast to the binding of
the corresponding full-length arrestins, the binding of
arr(1-206) and arr3(1-183) to the
P-
AR
is not stimulated by salt, suggesting
that a substantial portion of the hydrophobic interaction observed with
arr and arr3S is contributed by the C-terminal domain. In
contrast, physiological salt concentrations stimulate the binding of
both
arr(1-206) and arr3(1-183) to the P-mAChR
suggesting that the N-terminal domain significantly contributes
to the hydrophobic interaction. Thus, the contribution of ionic and
hydrophobic interactions involved in full-length and truncated arrestin
binding appears to be determined in part by the receptor.
In
contrast to arr-rhodopsin interactions(26) , the salt
sensitivity for arr, arr3S,
arr(1-206), and
arr3(1-183) binding to the activated or non-activated
P-
AR and P-mAChR were found to be very similar (Fig. 2). These results suggest that similar interactions
mediate the binding of all arrestins to the phosphorylated
AR and m2 mAChR regardless of the activation state of
the receptor.
We initially assessed
chimera binding to rhodopsin. Analysis of wild type arrestin
demonstrates a relatively low level of arr binding to P-Rh
compared to arr.
Arr also does not discriminate between
Rh
and Rh (as compared to a 3-4-fold discrimination
for arr). Moreover,
arr binds with low (
2-fold) selectivity
for P-Rh
versus P-Rh (in contrast to an
14-fold difference for arr). When chimeras that varied in their
C-terminal regulatory region were compared, the only chimera virtually
indistinguishable from arr was AAAB, which has the C-terminal
regulatory region of
arr (Fig. 5A). These data
further corroborate the hypothesis that this region is not directly
involved in rhodopsin interaction (24, 25, 26, 27) and demonstrate
that the
arr C terminus is capable of fulfilling the functions of
its arr counterpart. Chimera BBBA was also found to be very similar to
arr, although its binding to P-Rh and P-Rh
is somewhat
higher. Both chimeras were found to bind to Rh
better than
to Rh, while the P-Rh
/P-Rh binding ratio is
arr-like
(2.3) for BBBA and arr-like (14.4) for AAAB.
Figure 5:
The binding of -arr, arr, and 12
chimeric arrestins to rhodopsin (A), human m2 mAChR (B), and human
AR (C). 50 fmol of
the respective [
H]arrestins (953-1177
dpm/fmol) were incubated with phosphorylated, phosphorylated activated,
unphosphorylated, and unphosphorylated activated forms of the indicated
receptors as described under ``Experimental Procedures.'' The
specific binding (mean ± S.D. from two to four independent
experiments performed in duplicate) is
shown.
The substitution of the
N-terminal regulatory region of arr into arr to generate the
chimera BAAA significantly decreases binding to P-Rh
(compared to arr binding), but does not impair the preference for
Rh
over Rh or the P-Rh
/P-Rh binding ratio
(14.2). The symmetric substitution in
arr to generate the chimera
ABBB substantially improves its binding to P-Rh
(compared
to
arr) while the P-Rh
/P-Rh binding ratio remains
arr-like (1.5). Simultaneous exchange of both the N- and
C-terminal regulatory regions yields the chimeras ABBA and BAAB which
have comparable binding to P-Rh
(
50% of the arr
binding level). The P-Rh
/P-Rh binding ratios of these
chimeras appear to be determined by the origin of the central binding
domains, being
arr-like (2.7) for ABBA and arr-like (15.2) for
BAAB. These data suggest that while the N-terminal region has some
direct participation in rhodopsin binding, the middle portion of the
molecule determines the overall binding characteristics of the chimera.
We also constructed a series of 6 chimeras containing two central
binding domains of different origin. The first pair of these (BBAA and
AABB) demonstrates virtually no preference between Rh and
Rh and have P-Rh
/P-Rh binding ratios of 4.4 and 4.9,
respectively (i.e. intermediate between
arr and arr).
While the binding of AABB to P-Rh
is
70% that of arr,
BBAA binding to P-Rh
is only
25% that of arr and is
even lower than
arr binding. The chimeras ABAA and BABB
demonstrate P-Rh
/P-Rh binding ratios of 5.6 and 5.5;
however, their binding to P-Rh
was reduced compared to the
binding of BBAA and AABB. Chimera AABA has a P-Rh
/P-Rh
binding ratio of 3.7 while the symmetric chimera BBAB binds very poorly
to P-Rh
and demonstrates no preference for P-Rh
over P-Rh. Thus, most of the chimeras that contain one binding
domain from
arr and one from arr demonstrate a binding selectivity
between that of
arr (
2-fold) and arr (
14-fold). The
three chimeras with the structure XBAX (where X can be either A or B) demonstrate an unexpectedly low binding to
P-Rh
(i.e. even lower than the corresponding
arrestins with the structure XBBX) (compare BBAA with
BBBA). In contrast, in all other chimeras a change from XXBX to XXAX enhances binding to
P-Rh
as might be expected (compare AABB with AAAB).
When
chimera binding to the m2 mAChR is compared, the most noticeable
difference between arr and arr is the extremely low level of arr
binding to P-mAChR
(Fig. 5B). This is
reflected in both a higher affinity of
arr for P-mAChR
(K
= 0.48 nM) compared to
arr (K
= 7.2 nM) and a higher B
for
arr (16.3 fmol) compared to arr (7.0
fmol) (Fig. 4). However, while the levels of
arr and arr
binding to the m2 mAChR are very different, the selectivity of the
binding (P-mAChR
/P-mAChR ratio) is similar. Substitution of
the arr C terminus into
arr (BBBA) increases binding to all forms
of the m2 mAChR without significantly affecting the affinity, while
substitution of the N terminus (ABBB) has minimal effect on the binding
but decreases the affinity
2.4-fold ( Fig. 4and Fig. 5B). When both the N- and C-terminal regulatory
domains are changed (ABBA) the binding is decreased
2-fold without
a change in affinity. The symmetric series of substitutions in arr
(AAAB, BAAA, and BAAB) do not significantly improve arr binding to the
m2 mAChR and AAAB actually binds with an affinity
1.7-fold lower
than that of arr. Overall, substitutions of either the C- or N-terminal
regulatory regions lead to modest changes (
2-fold) in affinities
and B
for binding to the P-mAChR
.
Chimeras BBAA and AABB demonstrate comparable binding to
P-mAChR (between that of
arr and arr), with BBAA being
more selective than AABB (Fig. 5B). BBAA also has a
modestly higher affinity for the P-mAChR
compared to AABB (K
= 1.38 versus 2.11 nM,
respectively) (Fig. 4). Chimera BABB is similar to BBAA in terms
of selectivity profile and affinity (K
=
1.25 nM) even though these proteins have the opposite binding
domain combinations ( Fig. 4and Fig. 5B). In
contrast, chimera ABAA, which has the same combination of binding
domains as BBAA, demonstrates very poor selectivity, a low affinity (K
= 3.1 nM) and a low B
(2.3 fmol). Thus, the structure XBAX is as unfavorable for binding to the m2 mAChR as
it is for binding to rhodopsin. The binding of chimera BBAB to the
P-mAChR
most resembles the binding profile of BBAA although
the affinity of BBAB is
2-fold lower. While the chimera AABA most
resembles AABB structurally, the B
of AABA is
significantly increased over that of AABB although there is a modest
reduction in affinity (Fig. 4). Interestingly, AABA demonstrates
almost as poor a discrimination between P-mAChR
and P-mAChR
as did the symmetric chimera BBAB between P-Rh
and P-Rh (Fig. 5, A and B). Thus, both central binding
domains play an important role in arrestin interaction with the m2
mAChR, in agreement with the rhodopsin binding studies.
In general,
the binding of arr and arr to the P-
AR
most resembles the P-mAChR
binding profiles (Fig. 5). This is seen in both the selectivity profile
(P-
AR
/P-
AR binding ratio
of
2) and the
15-fold higher affinity of
arr (K
= 0.14 nM) compared to arr (K
= 2.1 nM) (Fig. 4).
However, in contrast to the m2 AChR binding studies, the B
for arr and
arr binding to the
P-
AR
are similar (Fig. 4). The
chimeric arrestin studies demonstrate that substitution of the C
terminus of
arr (BBBA) again modestly increases the B
without changing the affinity (K
= 0.12 nM) ( Fig. 4and Fig. 5C). Substitution of the arr C terminus (AAAB), N
terminus (BAAA), or both (BAAB) again does not improve the binding to
the
AR (Fig. 5C). However,
substitution of the arr N terminus into
arr (ABBB) dramatically
reduces the affinity (
11-fold) and increases the B
(
8-fold) compared to
arr (Fig. 4). Substitution of both the N and C terminus of arr into
arr (ABBA) has a less dramatic reduction in affinity (
3-fold)
and increase in B
(
3-fold) (Fig. 4). These
results suggest that the N terminus plays a direct role in
arr
interaction with the
AR. An examination of the central
binding domains reveals that, as expected, chimeras containing both
arr-derived binding domains bind better to the
P-
AR
as compared to chimeras containing a
combination of
arr- and arr-derived binding domains (Fig. 5C). The structural pattern XBAX appears to be even less favorable for binding to the
AR than it was for rhodopsin and the m2 mAChR, since
BBAA, ABAA, and BBAB all demonstrate lower binding to the
AR than the corresponding symmetric chimeras AABB,
BABB, and AABA.
In an attempt to more directly assess the role of
the N-terminal half of arr and arr in receptor interaction, we
compared the binding of truncated arr(1-191),
arr(1-217), ABBB(1-221), and BAAA(1-187) (Fig. 6). In contrast to the corresponding full-length proteins (Fig. 5A), there is no dramatic difference between the
ability of arr(1-191),
arr(1-217) and the truncated
chimeras to bind to rhodopsin, although the chimeras appear to have a
reduced preference for the phosphorylation state of the receptor (Fig. 6A). However, the binding levels of all truncated
arrestins are lower than that of the corresponding full-length proteins
(compare Fig. 5A and Fig. 6A),
underscoring the important role of the C-terminal binding domain.
Binding of the truncated arrestins to the m2 mAChR reveals that only
arr(1-217) demonstrates high binding and selectivity, while
ABBB(1-221) and BAAA(1-187) do not significantly differ
from arr(1-191). These data suggest that both the N terminus and
N-terminal binding domain are required for high affinity interaction
with the P-mAChR
. ABBB binding to the P-mAChR
is not significantly different from
arr (Fig. 5B), again underscoring the important role of the
C-terminal binding domain. The binding of all four truncated proteins
to the
AR is also surprisingly similar (Fig. 6C), unlike the binding of the full-length
proteins (Fig. 5C). The selectivity profiles of both
truncated chimeras resemble that of
arr(1-217), while
arr(1-191) is less selective. Interestingly, while the binding of
arr(1-217) and ABBB(1-221) to the
P-
AR
is lower than that of the full-length
proteins, the binding of arr(1-191) and BAAA(1-187) is
comparable to the full-length proteins (compare Fig. 5C and 6C). This suggests that the C-terminal binding
domains of arrestin and BAAA do not significantly interact with the
AR. In general, both truncated chimeras and
arr(1-191) demonstrate a greater specificity for rhodopsin and
the
AR compared to the m2 mAChR. This may correlate
with the localization of the relevant phosphorylation sites in these
receptors and suggests that the specificity of wild type and chimeric
arrestins is determined to a large extent by the C-terminal binding
domain.
Figure 6:
Truncated arr, arr, and chimeric
arrestin interaction with receptors. 50 fmol of the indicated truncated
arrestins were incubated with the indicated functional form of
rhodopsin (A), human m2 mAChR (B), or human
AR (C), as described under
``Experimental Procedures.'' The specific binding (mean
± S.D. from two to three independent experiments performed in
duplicate) is shown.
Rhodopsin, AR, and m2 mAChR represent three
distinct subfamilies of G protein-coupled receptors. Rhodopsin via its
specific light-promoted interaction with the G protein transducin
activates cGMP phosphodiesterase, the
AR via
interaction with G
activates adenylyl cyclase, and the m2
mAChR via its association with G
inhibits adenylyl cyclase
and activates K
channels. There are also distinct
structural differences among these receptors (reviewed in (31) ) that likely play a role in arrestin binding. One
distinction is the localization of relevant phosphorylation sites on
the receptors. Another significant difference is the size and amino
acid sequence of the third cytoplasmic loop and C-terminal domains.
These domains have previously been implicated in receptor interaction
with G proteins (32, 33, 34, 35, 36, 37, 38) ,
G protein-coupled receptor
kinases(39, 40, 41, 42, 43, 44) ,
and more recently in visual arrestin interaction with
rhodopsin(30) . Rhodopsin has a relatively short third
cytoplasmic loop (29 residues) and a C terminus (39 residues) that
contains all of the rhodopsin kinase phosphorylation sites. The third
cytoplasmic loop of the
AR is intermediate in size (54
residues) and most (if not all) of the
ARK phosphorylation sites
are localized in the C terminus (84 residues). In contrast, the third
cytoplasmic loop of the m2 mAChR is much longer (181 residues) and it
is this loop, rather than the short C terminus (23 residues), that
appears to contain all of the
ARK phosphorylation
sites(45) .
The low level of binding of all XBAX chimeras suggests that it is this particular
structure per se, rather than the origin of the binding domains, that
makes these arrestins bind poorly. In fact, all XBAX chimeras have even lower binding than arr(1-217), which
lacks the C-terminal half of the molecule (Fig. 3Fig. 4Fig. 5, Table 2). This
demonstrates that the C-terminal binding domain in XBAX not only does not contribute to the binding, but actually seems to
inhibit receptor interaction. These results suggest that there may be
an obligatory interaction between the N- and C-terminal binding domains
in arrestin, which is dramatically impaired in chimeras with the XBAX structure but not impaired in XABX chimeras. The reason for this apparent lack of symmetry may be
that a portion of one binding domain (the key) must fit into a pocket
in the other binding domain (the keyhole). In this scenario a
``small key/big keyhole'' mismatch is less detrimental for
interaction than a ``big key/small keyhole'' mismatch.
Figure 7:
Molecular architecture of arrestins. The
putative major functional regions in the arrestins are shown with
different fill patterns and are designated as follows: R1,
N-terminal regulatory region (25-29 residues); R2,
C-terminal regulatory region (60-80 residues); A,
activation-recognition region; P, phosphorylation-recognition
region (residues 163-182 in arr, residues 157-177 in
arr and arr3); S, region containing secondary binding
site(s) (equivalent to the hydrophobic booster site in visual
arrestin(26) ). Arrowheadsabove show the
borders between regions used in the chimera constructions (residue
numbers are indicated in the legend to Fig. 4). Arrowheadsbelow show the position where differences between
polypeptide forms of arr (I and II),
arr (I), and arr3 (III) were
found. See text for details.
Figure 8:
a, general model of
arrestin-phosphoreceptor interaction in the presence of agonist (L). Wild type non-visual arrestin (A) binds to
receptor-agonist complex (RL) in which phosphoreceptor (R) is in the basal (RL) or activated (ℝL)
conformation, to form ARL or AℝL. Arrestin can then undergo a
transition into its high-affinity binding conformation (𝔸), giving
rise to 𝔸RL and 𝔸ℝL complexes. While all four
arrestin-phosphoreceptor-agonist complexes are at equilibrium, the
𝔸ℝL complex has considerably higher affinity for arrestin and
therefore represents the experimentally measured B. b, C-terminal truncated arrestin
binding to receptor. C-terminally truncated arrestins such as
arr(1-367) and arr3(1-375) more readily assume the
high-affinity binding conformation (𝔸) effectively eliminating one
of the limiting steps leading to 𝔸ℝL complex formation. c, binding of arrestins lacking their secondary binding sites.
Arrestins lacking the C-terminal half of the molecule such as
arr(1-217),
arr(1-206), and arr3(1-183)
also lack the secondary binding sites mobilized during the A
𝔸 transition. d, arrestin binding to receptor in the
presence of antagonist(N). The same model for full-length arrestin
binding in the presence of an antagonist(N), which does not induce
receptor activation. In this case the 𝔸ℝN complex can be
formed only via 𝔸RN due to the ability of non-visual arrestins to
assume a high-affinity binding conformation (𝔸) while interacting
with non-activated receptors (R). The resulting percentage of receptors
in an 𝔸ℝN complex (B
) would be lower
than an 𝔸ℝL complex. See text for
details.
In the presence of agonist there
is an equilibrium between free receptor (R) (here and below the
receptor is presumed to be phosphorylated since this is a prerequisite
for high affinity arrestin binding), free agonist (L), receptor-agonist
complex (RL), and active receptor-agonist complex (ℝL). While a
high concentration of agonist converts virtually all receptors into RL,
the number of ℝL complexes depends on the equilibrium between RL
and ℝL. Conceivably, arrestin (A) can bind to both forms of the
receptor forming either an ARL or AℝL complex. Bound arrestin then
undergoes a conformational transition from a low to a high affinity
state (A 𝔸) giving rise to 𝔸RL and 𝔸ℝL
complexes, which are again at equilibrium. The linear Scatchard plots
obtained for the binding of all arrestins to the
AR
and m2 mAChR indicate that one of these 4 arrestin-receptor-agonist
complexes has a significantly higher affinity than the others.
Therefore it is the formation of this particular complex, which is most
likely 𝔸ℝL, that we are measuring. By increasing the arrestin
concentration we can drive all of the receptor-agonist complexes to
bind arrestin, however, the actual number of 𝔸ℝL complexes
(the experimental B
) still depends on the
equilibrium between ARL, AℝL, 𝔸RL, and 𝔸ℝL (Fig. 8). The RL to ℝL equilibrium depends on the intrinsic
characteristics of agonist binding to a particular receptor, while the
ARL to AℝL and 𝔸RL to 𝔸ℝL equilibrium may be
shifted compared to RL to ℝL. The magnitude of this shift is
arrestin-dependent and gives rise to the observed differences in B
for different wild type and mutant arrestins.
The deletion of the C terminus of arrestin promotes its transition
into the high-affinity binding conformation
(𝔸)(25, 26) , thus eliminating one of the
limiting steps in the sequence of events leading to 𝔸ℝL and
increasing the observed B (Table 1). While
deletion of the entire C-terminal half of the arrestin molecule
produces an arrestin that cannot form a high affinity binding
conformational state(27) , it still appears capable of forming
an AℝL complex that is stable enough for detection (25-27, Fig. 3and Fig. 6). This truncation also shortens the
chain of events leading to the high-affinity complex and therefore also
increases the B
(Table 1). While
antagonists(N) do not induce receptor activation, arrestins are still
capable of assuming a high-affinity binding conformation (𝔸RN),
albeit with a lower probability, and consequently induce a
conformational change in the receptor to form 𝔸ℝN. Thus,
while a lower percentage of the receptors will be present in the
𝔸ℝN complex at equilibrium (compared to 𝔸ℝL), the
complex itself is essentially the same as in the presence of agonist
(at least as far as arrestin-receptor interaction is concerned). This
would translate into no change in the K
but a
lower B
as was experimentally observed (Table 1). Truncated arrestins would be expected to shift the
equilibrium toward 𝔸ℝN in the presence of an antagonist more
effectively than the full-length arrestins, again in agreement with the
experimental data (Fig. 3).
The kinetic model of
arrestin-receptor interaction suggests that analogous to visual
arrestin, non-visual arrestins are selective toward the
activated/phosphorylated form of receptors. The major difference
between the visual and non-visual arrestins appears to be in their
ability to induce an activation-like conformational change in the
phosphorylated inactive receptor. Thus, arr and arr3 appear to be
able to promote formation of an 𝔸ℝ (L) complex even in the
absence of ligand. The data with truncated arrestins ( Fig. 3and Fig. 6) suggest that both rhodopsin and the
AR
are rather rigid while the m2 mAChR appears to be more flexible, since
even the shortest arrestin species are able to induce an active
conformation in a large proportion of the P-mAChR
(Table 1). Arr3S appears to possess the best ability to
shift the equilibrium toward a high-affinity complex since it has the
highest B
with both P-
AR
and P-mAChR
, and is second only to visual arrestin in
binding to P-Rh
(Fig. 1, Table 1).
The
model of arrestin-receptor interaction is analogous to the current
model of G protein-receptor interaction in several important aspects (48, 49, 50) . Both arrestins and G proteins
preferentially bind to agonist-activated receptors, although both are
capable of interacting with the receptor in its basal
state(49, 50) . In the presence of a saturating
concentration of agonist, only a certain percentage of the receptors
are able to form a high-affinity complex with either an arrestin (Table 1) or G protein(48, 49) . Since the same
functional state of the receptor is capable of interacting with both G
protein and arrestin, this suggests that direct competition between the
two is the most plausible mechanism of arrestin action. Since receptor
associated with G protein has a higher affinity for agonists than the
receptor alone(48, 49) , the 𝔸ℝL complex
might also be expected to have a higher affinity for agonists. In the
visual system, where all-trans-retinal bound to metarhodopsin
II is analogous to the agonist-occupied activated form of the receptor,
transducin interaction stabilizes the metarhodopsin II state. This is
fully analogous to the stabilization of the receptor-agonist complex by
other G proteins. Interestingly, visual arrestin was also shown to
stabilize the metarhodopsin II state of phosphorylated
rhodopsin(47) . The affinity of P-AR and
P-mAChR for agonists in the presence and absence of non-visual
arrestins remains to be determined.
The other implication of these
data and the model of arrestin-phosphoreceptor interaction is a very
slow dissociation of arrestin. While dissociation of visual arrestin
from P-Rh is facilitated by the decay of
phosphometarhodopsin II to phosphoopsin (26) , dissociation of
the agonist from 𝔸ℝL only modestly facilitates arrestin
dissociation (Table 1). Thus, either non-visual arrestin
dissociation is indeed a relatively slow process in the cell or it
might be facilitated by either receptor sequestration or modification (e.g. phosphorylation) of the bound arrestin. In any event the
arrestins are likely to stay bound to the phosphoreceptor at least
until agonist dissociation, thus ensuring high fidelity of the signal
termination.