(Received for publication, November 18, 1994; and in revised form, January 5, 1995)
From the
Arrestin plays a critical role in quenching phototransduction via its ability to specifically interact with the phosphorylated light-activated form of the visual receptor rhodopsin. In an effort to identify the residues involved in interaction with the phosphorylated C terminus of rhodopsin, we introduced point mutations into a basic region in visual arrestin previously implicated in phosphorylation-recognition (residues 163-189). A total of nine point mutations were made, each substituting a neutral hydrophilic residue for a positively charged Lys, Arg, or His. The functional consequences of these mutations were then analyzed by comparing the binding of full-length and truncated wild-type and mutant arrestin to various functional forms of rhodopsin. These studies demonstrate that Arg-171, Arg-175, and Lys-176 in bovine arrestin play a primary role in phosphate interaction, while Lys-166 and Lys-167 likely play a minor role in phosphate binding. In contrast, Lys-163 and His-179 appear to play a regulatory role, while Arg-182 and Arg-189 are not directly involved in arrestin binding to rhodopsin. Arg-175 also appears to function as a phosphorylation-sensitive trigger since charge neutralization by mutagenesis enables arrestin-R175N to bind to light-activated rhodopsin as well as wild-type arrestin binds to phosphorylated light-activated rhodopsin. The implications of these findings for the sequential multisite binding of arrestin to rhodopsin are discussed.
G protein()-coupled receptors enable eukaryotic cells
to respond to a wide variety of stimuli including hormones,
neurotransmitters, odors, and light. The visual amplification cascade
is perhaps the best studied G protein-coupled receptor-initiated
signaling system(1) . Signal transduction in retinal rod cells
is initiated by photoisomerization of 11-cis-retinal
covalently attached to a lysine residue within the seventh
transmembrane domain of rhodopsin. Rhodopsin, via a series of transient
intermediates, is thus converted into metarhodopsin II, the form of
rhodopsin capable of binding the visual G protein transducin. The
metarhodopsin II-bound transducin then exchanges GTP for GDP, promoting
dissociation of the transducin
GTP and
subunits.
GTP then binds to the inhibitory
-subunit of cGMP
phosphodiesterase, leading to enzyme activation, decreased cGMP levels,
the closing of cGMP-gated sodium channels, and hyperpolarization of the
rod cell.
Quenching of the visual transduction cascade involves a rapid activation-dependent phosphorylation of rhodopsin by the enzyme rhodopsin kinase(2) . This is followed by the highly selective binding of arrestin to activated phosphorylated rhodopsin, a process that appears to attenuate the activation of transducin(3, 4) . Recent mutagenesis studies of visual arrestin have enabled the identification of several important functional regions within arrestin (see Fig. 1) (5, 6, 7, 8) . In particular, a relatively short positively charged region encompassing residues 163-191 emerged as a likely domain involved in interaction with the phosphorylated C terminus of rhodopsin. The moderate size of this domain as well as the presence of a number of potentially important basic residues makes this region a suitable target for systematic site-directed mutagenesis.
Figure 1:
Molecular architecture of arrestin and
sequence homology of putative phosphorylation recognition region. Upper panel, the major functional regions are designated as
follows: R1, basic N-terminal regulatory region; R2,
acidic C-terminal regulatory region; A, activation-recognition
region; P, phosphorylation recognition region; S,
secondary binding site region(6, 11) . The approximate
number of residues corresponding to the borders between the functional
regions are shown below the schematic. Lower panel, sequences of putative phosphorylation-recognition regions of
bovine arrestin (bovarr)(9) , bovine
-arrestin (bov
-arr)(13) , bovine
arrestin 3 (bovarr3)(14) , human X-arrestin (humx-arr)(15) , Drosophila arrestin 2 (droarr2)(16) , Calliphora arrestin (calarr)(17) , Locusta arrestin (locarr)(18) , Limulus arrestin (limarr)(19) , and Drosophila arrestin 1 (droarr1) (20) are shown. Positively charged residues are shown in boldface, while prolines and glycines are underlined.
The number of the first residue is indicated in parentheses.
See text for details.
Previous mutagenesis studies have led to the identification of several key functional regions within the arrestin molecule(5, 6, 7, 8, 10, 11) . The N-terminal half of arrestin (residues 1-191) was found to contain domains involved in both activation-recognition (interacts with the portions of rhodopsin that change conformation upon light-activation, denoted as A in Fig. 1) and phosphorylation-recognition (interacts with the phosphorylated C terminus of rhodopsin, denoted as P in Fig. 1)(6, 11) . When these primary binding sites are simultaneously engaged by binding to phosphorylated light-activated rhodopsin (P-Rh*), arrestin undergoes a rearrangement into a high affinity binding conformation(6, 7, 11, 12) . This conformational change results in the mobilization of a hydrophobic secondary binding site (denoted as S in Fig. 1). The conformational rearrangement necessary for the mobilization of this secondary binding site appears to be partially controlled by the intramolecular interaction of the regulatory N- and C-terminal regions (R1 and R2, respectively, in Fig. 1)(6, 7, 11) . Thus, the deletion of the R1 or R2 region yields a mutant arrestin that more readily assumes a high affinity binding conformation upon binding not only to P-Rh*, but also to phosphorylated rhodopsin (P-Rh) and light-activated rhodopsin (Rh*)(6, 7) . The contributions of the primary and secondary binding sites to arrestin-receptor interaction appear to be comparable(6, 11) . Deletion of the entire C-terminal domain yields an arrestin, ARR(1-191), that is still capable of recognition of the activation and phosphorylation status of rhodopsin via its primary binding sites. However, ARR(1-191) displays a significantly lower selectivity toward P-Rh* compared with full-length arrestin since it lacks a secondary binding site(5, 6, 7) .
Figure 2: Effect of arrestin mutations on full-length arrestin binding to light-activated (shaded bars) or dark (hatched bars) P-Rh-2.2 (A), P-Rh-5.2 (B), and Rh (C). Rhodopsin (150 nM) was incubated with the various arrestin mutants (1 nM, specific activities from 902 to 1271 dpm/fmol) at 37 °C for 5 min in a 100- (dark Rh) or 50-µl assay volume. Samples were then cooled on ice, and bound and free arrestin were separated by Sepharose 2B chromatography as described under ``Experimental Procedures.'' The means ± S.D. from three experiments, each performed in duplicate, are shown.
The ability of several of the point mutants to bind to phosphorylated rhodopsin containing 2.2 mol of phosphate/mol of rhodopsin (P-Rh-2.2) was reduced compared with wild-type arrestin binding. Mutations K163S, K166S, K167S, R171Q, and K176S decrease arrestin binding to P-Rh*-2.2 by 15-28% (Fig. 2A, Table 1). Arrestin binding to dark P-Rh-2.2 was even more affected by these mutations (20-30% decrease), as well as by mutation R182Q. When binding to rhodopsin containing a higher level of phosphorylation (P-Rh-5.2) was assessed, only the effect of the K166S mutant was fully retained (Fig. 2B, Table 1). Similarly, arrestin binding to nonphosphorylated Rh* was significantly reduced only for K166S (Fig. 2C, Table 1). The most dramatic reduction in binding was observed with the triple mutant R171Q/R175N/K176S. While this mutant has reduced binding to all forms of rhodopsin, the biggest reduction is observed in the binding to P-Rh*-2.2 (Fig. 2, Table 1). Overall, these results suggest that K163, K167, R171, and K176 are likely involved in phosphate recognition.
Several of
the point mutants also appeared to increase arrestin binding to various
forms of rhodopsin. Mutation R189Q modestly increased arrestin binding
to P-Rh* without a detectable effect on binding to either P-Rh or Rh* (Fig. 2, Table 1). Mutation H179Q increased arrestin
binding to P-Rh* by 20-25%, while binding to Rh* and P-Rh were
increased to an even greater extent (50 and 75%, respectively).
The binding of arrestin-R175N to P-Rh* was increased 40-90%,
while its binding to P-Rh was increased 140-200%. The R175N
mutation dramatically increased arrestin binding to Rh* such that the
binding of arrestin-R175N to Rh* was comparable with the binding of
wild-type arrestin to P-Rh* (Fig. 2, Table 1). These
results suggest that Arg-175 may play a critical role in sensing the
phosphorylation state of the receptor.
Figure 3:
Effect of mutations on truncated
ARR(1-191) binding to P-Rh-2.2 (A),
P-Rh*-5.2 (B), and Rh* (C). Rhodopsin (150
nM) was incubated with the various ARR(1-191) mutants (1
nM, specific activities from 485 to 491 dpm/fmol) at 37 °C
for 5 min in a 100-µl assay volume. Samples were then cooled on
ice, and bound and free arrestin were separated by Sepharose 2B
chromatography as described under ``Experimental
Procedures.'' The means ± S.D. from two experiments, each
performed in duplicate, are shown.
In these studies, the point mutations R171Q,
R175N, and K176S were found to result in the greatest reduction
(30-60%) in binding to P-Rh* (Fig. 3, A and B, Table 2). This suggests that these 3 residues play a
direct role in phosphate interaction. This is further substantiated by
the triple mutant R171Q/R175N/K176S where the binding to P-Rh* is
reduced 85% (Fig. 3, Table 2). Moreover, the
specificity of these mutants for phosphate-recognition is demonstrated
by the finding that the truncated R171Q, R175N, K176S, and triple
mutants bind to Rh* as well as wild-type ARR(1-191) (Fig. 3C). Mutations K166S and K167S were found to
modestly inhibit ARR(1-191) binding to P-Rh*-2.2, suggesting a
minor role for these residues in phosphate interaction (Fig. 3A, Table 2). Mutation K166S also
significantly stimulates ARR(1-191) binding to Rh*, while
mutations H179Q and R182Q slightly increase ARR(1-191) binding to
both P-Rh* and Rh* (Fig. 3, Table 2). Mutations K163S and
R189Q had no appreciable effect on binding.
A comparison of the effects of the various mutations on full-length and truncated arrestin binding reveals a number of interesting differences (compare Fig. 2and Fig. 3and Table 1and Table 2). For example, the binding of full-length K163S to P-Rh* is significantly reduced while the binding of truncated K163S to P-Rh* is not affected. In contrast, the decrease in binding of R171Q and K176S to P-Rh* is greatest when the truncated arrestins are studied compared with the full-length arrestins.
The effects of mutation R175N are the most divergent since the binding of truncated R175N to P-Rh* is substantially impaired while the binding of full-length R175N to all forms of rhodopsin is increased. The extent of the increase appears to be maximal for Rh*, although significantly increased binding is also observed for P-Rh*, P-Rh, and Rh ( Fig. 2and 4A, Table 1). Mutation H179Q produces similar effects on full-length arrestin binding to different functional forms of rhodopsin, although to a significantly lower extent ( Fig. 2and 4A, Table 1). Interestingly, a similar apparent loss of selectivity for binding to P-Rh* was previously observed for N- and C-terminal deletion mutants in arrestin(6, 7, 8) . The underlying mechanism of these effects was attributed to a loss in the control of the transition of arrestin into a high affinity binding conformation. This results in the mobilization of the secondary binding sites not only in binding to P-Rh* but also in the binding to P-Rh and Rh*(6, 7) . Experimentally, this also manifests itself as a significant reduction in the salt sensitivity of arrestin interaction with P-Rh and Rh*(6) . In order to further probe the mechanism by which R175N and H179Q mutations increase arrestin binding to Rh*, we compared the salt sensitivity of wild-type and mutant arrestin binding to Rh*. Both mutations were found to comparably reduce the salt sensitivity of arrestin interaction with Rh* (Fig. 5A), suggesting that mobilization of the hydrophobic secondary binding sites are indeed involved.
Figure 5:
Salt
sensitivity of full-length (A) and truncated ARR(1-365) (B) binding to Rh*. Wild-type (), R175N mutant
(
), or H179Q mutant (
) arrestins (1 nM, specific
activities from 870 to 1141 dpm/fmol) were incubated with 150 nM of Rh* in a 50-µl assay volume for 5 min at 37 °C. Samples
were then cooled on ice, and bound and free arrestin were separated by
Sepharose 2B chromatography as described under ``Experimental
Procedures.'' Average control binding was 4.2, 20.1, 6.5, 8.8,
10.3, and 12.4 fmol for arrestin, arrestin-R175N, arrestin-H179Q,
ARR(1-365), ARR(1-365)-R175N, and ARR(1-365)-H179Q,
respectively. The means ± S.D. from two experiments, each
performed in duplicate, are shown.
These data indicate that Arg-175 and to a lesser extent His-179 are involved in the control of the transition of arrestin into a high affinity binding conformation. Previous studies have demonstrated that interaction between the basic N-terminus (R1 in Fig. 1) and the acidic C terminus (R2 in Fig. 1) of arrestin participate in this function(6, 7) . This finding raises the question of whether Arg-175 and His-179 also interact with the acidic C terminus of arrestin or with another portion of the C-terminal domain. To address this question, we studied the binding of ARR(1-365) containing the wild-type or mutant sequences. Surprisingly, ARR(1-365)-R175N binds with a selectivity better than the full-length R175N mutant (Fig. 4B). In fact, ARR(1-365)-R175N binding looks strikingly similar to the binding of wild-type ARR(1-365). In contrast, the H179Q mutation enhances the binding of ARR(1-365) to all functional forms of rhodopsin (Fig. 4B). Similarly, the ability of H179Q to reduce the salt sensitivity of ARR(1-365) to Rh* is completely retained, while the R175N mutant has a salt sensitivity between that of the wild type and H179Q mutant arrestins (Fig. 5B). Thus, these results suggest that while both R175N and H179Q disrupt an interaction that helps control mobilization of hydrophobic binding sites, the interaction of these residues may be within distinct regions of the arrestin molecule with Arg-175 possibly involved in an interaction within the acidic C-terminal domain of arrestin.
Figure 4: Effect of mutations R175N and H179Q on selectivity of full-length (A) and truncated ARR(1-365) (B) binding. The indicated functional form of rhodopsin (150 nM) was incubated with the respective arrestin (1 nM, specific activities from 870 to 1141 dpm/fmol) at 37 °C for 5 min in a 50-µl assay volume. Samples were then cooled on ice, and bound and free arrestin were separated by Sepharose 2B chromatography as described under ``Experimental Procedures.'' The means ± S.D. from two experiments, each performed in duplicate, are shown.
Mutations K163S, K166S, K167S, R171Q, K176S, and R182Q inhibit arrestin binding to highly phosphorylated rhodopsin to various extents, mutations R175N and H179Q enhance binding, and R189Q has a minimal effect (Fig. 2B, Table 1). In most cases, the effect on P-Rh-5.2 binding is greater than on P-Rh*-5.2 binding, similar to what was found with P-Rh-2.2. However, in all cases, the inhibitory effects on binding to P-Rh-5.2 are less profound than those with P-Rh-2.2 (Table 1). Thus, it appears that no particular residue within the phosphorylation-recognition region of arrestin is specifically involved in interaction with the additional phosphates on the rhodopsin C terminus. Nevertheless, arrestin interaction with these additional phosphates appears to partially compensate for the loss of any particular positive charge in the phosphorylation-recognition region, thus decreasing the inhibitory effect of any individual point mutation. When binding of the truncated wild-type and mutant arrestins are compared, the effects of the mutations on P-Rh*-5.2 and P-Rh*-2.2 binding appear similar with one exception. The inhibitory effect of mutation R175N on P-Rh*-5.2 binding is even stronger than on P-Rh*-2.2 (Fig. 3, A and B, Table 2). Interestingly, the effect of the R175N mutation on full-length arrestin binding, although stimulatory, is also stronger with P-Rh*-5.2 (Fig. 2, A and B, Table 1).
Mutation R175N also potently inhibits truncated arrestin binding to P-Rh*. However, this mutation also profoundly increases full-length arrestin binding to all functional forms of rhodopsin (Rh* > P-Rh > P-Rh*). Such a pattern suggests that Arg-175 plays a crucial role in both phosphate interaction and mobilization of secondary binding sites. Conceivably, Arg-175 interaction with yet unidentified residue(s) within the C-terminal domain helps to maintain arrestin in a low affinity binding conformation. Upon binding to a phosphate on the C terminus of rhodopsin, the positive charge of Arg-175 becomes neutralized, disrupting the aforementioned interaction and enabling arrestin to assume a high affinity binding conformation provided that it interacts with light-activated rhodopsin. Neutralization of the charge on Arg-175 by mutagenesis substitutes for phosphate interaction, and, as a result, arrestin assumes a high affinity binding conformation upon binding to Rh*. On the other hand, the primary interaction itself (between phosphates on the rhodopsin C terminus and the arrestin phosphorylation-recognition region) is weakened by the R175N mutation, thus inhibiting truncated arrestin binding to P-Rh*.
The function of Lys-176 appears to be very similar to that of Arg-171. The K176S mutation has a moderate inhibitory effect on full-length arrestin binding, while potently inhibiting truncated arrestin binding to P-Rh* but not to Rh*. Thus, Lys-176 appears to be one of the 3 major residues involved in phosphate binding. Indeed, simultaneous substitution of these 3 residues (Arg-171, Arg-175, and Lys-176) virtually obliterates truncated arrestin binding to P-Rh* without affecting Rh* interaction. It should be noted that our data ( (6, 7, 8) and this study) do not unambiguously exclude the role of residues outside the 163-191 region as being involved in phosphorylation-recognition. Nevertheless, the magnitude of the effects of the triple mutation suggests that Arg-171, Arg-175, and Lys-176 play the major role in phosphorylation recognition.
The effects of the H179Q mutation on full-length arrestin binding are qualitatively similar to those of R175N although less profound. However, this mutation has no significant effect on truncated arrestin binding. The binding of arrestin-H179Q to Rh* appears to involve secondary binding sites similar to arrestin-R175N interaction with Rh* (Fig. 5). Thus, while His-179 does not play a direct role in phosphate binding, it may participate in preventing the transition of arrestin into a high affinity binding conformation upon binding to Rh* (and dark P-Rh). Thus, its substitution by mutagenesis may facilitate this transition. Most likely His-179 interacts with a yet unidentified residue within the C-terminal domain of arrestin, although, in contrast to Arg-175, phosphate binding may only indirectly disrupt this interaction.
Mutation R182Q has a weak stimulatory effect on truncated arrestin binding to all functional forms of rhodopsin. With the full-length protein, this mutation has a modest inhibitory effect on P-Rh binding, without appreciably affecting arrestin binding to any other form. Mutation R189Q brings about even less functional consequences. Thus, Arg-182 and Arg-189 do not appear to play any significant role in either phosphate binding or mobilization of secondary binding sites. Interestingly, these two charged residues are the least conserved among the arrestins, and both are localized outside the densely packed cluster of positive charges (residues 163-176, Fig. 1).
The function of Arg-175 is of special interest for several reasons. First, our sequential multisite binding model predicts that intramolecular interactions block the transition of arrestin into a high affinity binding conformation(6, 7) . Some of these interactions should be disrupted upon binding to a phosphorylated form of rhodopsin, while others would be disrupted upon binding to an activated form of rhodopsin. Thus, the transition involving the mobilization of secondary binding sites occurs only upon arrestin binding to P-Rh* (``double-trigger'' mechanism, (6) and (7) ). Our data suggest that Arg-175 participates in one of these constraining intramolecular interactions that appears to be disrupted upon the neutralization of its charge by phosphate binding. As a result, arrestin-R175N becomes largely phosphorylation-insensitive in binding to Rh* with an affinity comparable with that of wild-type arrestin binding to P-Rh*. These data further corroborate the sequential multisite binding model and suggest that Arg-175 (and possibly His-179) function as a crucial part of the phosphorylation-sensitive trigger. These data thus provide the first insight into the molecular mechanism of the conditional constraint release involved in arrestin binding to P-Rh*. A similar simple and elegant mechanism may also be involved in the activation-recognition dependent constraint release of arrestin.
A positively charged residue in the position corresponding to Arg-175 in visual arrestin is present in all arrestins sequenced thus far (Fig. 1). This suggests that the substitution of a neutral residue for this positively charged residue may serve as a universal way to produce a phosphorylation-independent ``constitutively active'' arrestin. This hypothesis can also be tested experimentally, and if proven correct, the use of constitutively active arrestins may substantially facilitate elucidation of receptor specificity of different arrestins in intact cells, since such a mutant would not be dependent on the simultaneous presence of the appropriate G protein-coupled receptor kinase. The use of constitutively active arrestins might also serve a useful purpose to turn-off constitutively active G protein-coupled receptors.
The 5 positively charged residues demonstrated here to be directly involved in phosphate binding are localized within an 11-amino acid portion of the phosphorylation-recognition region (Fig. 1). This virtually matches the close clustering of the rhodopsin kinase sites of phosphorylation within a 9-amino acid stretch (residues 335-343) on the C terminus of rhodopsin(21, 22) . The flanking charged residues (e.g. Lys-163, His-179) appear to play a different regulatory role while the major (Arg-175) and auxiliary (His-179) phosphorylation-sensitive triggers are localized within the C-terminal portion of the phosphorylation-recognition region. Interestingly, all arrestins have a potentially flexible proline- and glycine-rich sequence (e.g. Pro-181, Gly-185, Pro-186, and Pro-188 in visual arrestin, Fig. 1) C-terminal to the phosphorylation-recognition domain. The role of these residues in conformational rearrangements of arrestin can also be tested by mutagenesis.
In this study, we have identified a number of residues that are directly involved in phosphate binding and 2 residues that appear to function as a phosphorylation-sensitive trigger to modulate the transition of arrestin into a high affinity binding state. Similar mutagenesis studies of other members of the arrestin family will be required to test whether the mechanisms of phosphorylation recognition and the subsequent mobilization of secondary binding sites are universal among arrestins.