©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Visual Arrestin Binding to Rhodopsin
DIVERSE FUNCTIONAL ROLES OF POSITIVELY CHARGED RESIDUES WITHIN THE PHOSPHORYLATION-RECOGNITION REGION OF ARRESTIN (*)

(Received for publication, November 18, 1994; and in revised form, January 5, 1995)

Vsevolod V. Gurevich Jeffrey L. Benovic (§)

From the Department of Pharmacology, Jefferson Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

G protein(^1)-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 alphabulletGTP and beta subunits. alphabulletGTP 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 beta-arrestin (bovbeta-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.




EXPERIMENTAL PROCEDURES

Materials

[-P]ATP, [S]dATP, and [^3H]leucine were purchased from DuPont NEN. All restriction enzymes were purchased from Boehringer Mannheim, Promega, or New England Biolabs. Sepharose 2B, Sephadex G-25, and all other chemicals were from Sigma. Rabbit reticulocyte lysate and SP6 RNA polymerase were prepared as described previously(5) . 11-cis-Retinal was generously supplied by Dr. R. K. Crouch, National Institutes of Health. Other reagents were from sources described previously(5, 6, 7, 8) .

Plasmid Construction and Site-directed Mutagenesis

A bovine visual arrestin cDNA was generously supplied by Dr. T. Shinohara(9) . An arrestin construct containing the wild-type N terminus was excised with NcoI and HindIII and subcloned into NcoI-HindIII-digested pG2S6-I yielding the plasmid pARR(7) . Plasmid pVP was constructed by subcloning a 320-base pair SalI-XhoI fragment of the arrestin cDNA (encompassing codons 146-251) into SalI-XhoI-digested pBluescript II KS from which the SacI, SacII, and BstXI restriction sites had been initially removed by sequential digestion with BstXI, SacI, and mung bean nuclease followed by religation. In order to generate the point mutants K163S, K166S, and K167S, three pairs of oligonucleotides each corresponding to the AatII-SacI region (codons 159-169) of a particular mutant were synthesized, annealed, phosphorylated, and subcloned into AatII-SacI-digested pVP. A pair of oligonucleotides corresponding to the SacI-PpuMI region (codons 169-184) of pVP, which encoded the triple mutation R171Q/R175N/K176S and also contained silent mutations creating the unique restriction sites BclI (codons 173-175), SphI (codons 178-180), and SacII (codons 181-182), were annealed, phosphorylated, and subcloned into SacI-PpuMI-digested pVP, yielding the plasmid p3PS. In order to create the point mutations R171Q, R175N, and K176S, pairs of oligonucleotides corresponding to the SacI-BclI and BclI-SphI regions encoding either the respective mutations or silent mutations creating the same restriction sites were synthesized, annealed, and phosphorylated. The appropriate pair of oligonucleotide duplexes was then subcloned into SacI-SphI-digested p3PS. This yielded the three single mutants as well as the plasmid pVSP, which contained the wild-type amino acid sequence with silent mutations creating the same set of restriction sites as in p3PS. In order to generate R189Q, a pair of oligonucleotides corresponding to the PpuMI-StuI region (codons 185-191) were annealed, phosphorylated, and subcloned into PpuMI-StuI-digested pVSP. For the R182Q mutation, a pair of oligonucleotides corresponding to the SphI-PpuMI region (codons 179-184) were annealed, phosphorylated, and subcloned into SphI-PpuMI-digested pVSP. For the H179Q mutation, a pair of oligonucleotides corresponding to the BclI-PpuMI region (codons 174-184) were annealed, phosphorylated, and subcloned into BclI-PpuMI-digested pVSP. The sequences of all constructs were confirmed by dideoxy sequencing. Subsequently, the 320-base pair SalI-XhoI fragments for each mutation were subcloned into SalI-XhoI-digested pARR and resequenced.

In Vitro Transcription and Translation

Plasmids were linearized with HindIII before in vitro transcription to obtain full-length mRNAs. To obtain truncated mRNAs, plasmids were linearized with StyI or StuI to obtain mRNAs encoding ARR(1-365) or ARR(1-191), respectively(5, 6) . In vitro transcription and translation were carried out as described(5, 6, 7, 8) .

Rhodopsin Preparations

Urea-treated rod outer segment membranes were prepared, phosphorylated with beta-adrenergic receptor kinase, and regenerated with 11-cis-retinal as described (6) . The stoichiometries of phosphorylation for the preparations used in these studies were 0, 2.2 and 5.2 mol of phosphate/mol of rhodopsin.

Arrestin Binding to Rhodopsin

The basis of the arrestin/receptor binding assay involves assessing a change in the mobility of arrestin on a gel filtration column upon receptor binding. To study binding to rhodopsin, the in vitro translated tritiated arrestins were incubated in 50 mM Tris-HCl, pH 7.5, 0.5 mM MgCl(2), 1.5 mM dithiothreitol, and 50 mM potassium acetate with 7.5 pmol of the various functional forms of rhodopsin in a volume of 50-100 µl for 5 min at 37 °C either in the dark or in room light. The samples were then cooled on ice, and under dim red light they were loaded onto a 2-ml Sepharose 2B column equilibrated with 20 mM Tris-HCl, pH 7.5, 2 mM EDTA. Bound arrestin eluted with the rod outer segments in the void volume (between 0.5 and 1.1 ml). Nonspecific binding, determined in the presence of 0.3 µg of control liposomes, did not exceed 25% of the total binding and was subtracted.


RESULTS AND DISCUSSION

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) .

Mutagenesis of the Phosphorylation-Recognition Region of Visual Arrestin

The ionic nature of the interaction involved in phosphorylation-recognition suggests that the negatively charged phosphorylated C terminus of rhodopsin interacts with positively charged residues in arrestin(6) . Previous deletion mutagenesis studies suggest that residues 163-191 in arrestin play a significant role in phosphorylation-recognition(6) . In visual arrestin, this region contains 9 positively charged residues, 4 of which are conserved among all arrestin homologs(9, 13, 14, 15, 16, 17, 18, 19, 20) (Fig. 1). In order to probe the functional role of these 9 charged residues, we created point mutations by substituting uncharged residues that were capable of hydrogen bonding (i.e. lysine was replaced with serine, while arginine and histidine were replaced with glutamine or asparagine). In addition, one triple mutant (R171Q/R175N/K176S) was also created. These mutants were then expressed by in vitro translation, and their ability to bind to various functional forms of rhodopsin (P-Rh, P-Rh*, Rh, and Rh*) were compared with that of wild-type arrestin (Fig. 2, Table 1).


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.

The Effects of Mutations on Truncated Arrestin Binding

The binding of full-length arrestin depends not only on its ability to interact with the phosphorylated C terminus of rhodopsin but also on its ability to mobilize secondary binding sites(6, 7, 11) . Apparently, either or both of these functions may be impaired and/or enhanced by a given mutation. Thus, in order to more directly study the effect of each mutation on interaction with the phosphorylated C terminus of rhodopsin, we compared the binding of wild-type and mutant arrestins that were truncated at residue 191 (Fig. 3, Table 2). Truncated ARR(1-191) has previously been shown to retain its phosphorylation-recognition domain while lacking the secondary binding site(6) .


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 (bullet), 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.



Role of Rhodopsin Phosphorylation Level in Phosphorylation-Recognition

Previously, we found that two phosphates/rhodopsin are required to induce high affinity arrestin binding, while additional phosphorylation does not seem to increase binding(6) . Similarly, the m2 muscarinic cholinergic and beta(2)-adrenergic receptors require 2 mol of phosphate/mol of receptor for high affinity binding of beta-arrestin and arrestin 3(10, 11) . Recently 2 residues on the C terminus of arrestin (Ser-338 and Ser-343) were identified as the initial targets for rhodopsin kinase phosphorylation, while additional phosphorylation was shown to occur on 4 adjacent threonine residues (Thr-335, -336, -340, and -342)(21, 22) . In order to address whether any of the positively charged residues within the phosphorylation-recognition region of arrestin are specifically positioned to interact with these additional phosphates, we can compare the interaction of the mutants with P-Rh* and P-Rh phosphorylated to stoichiometries of 2 or 5 mol of phosphate/mol of rhodopsin ( Fig. 2and Fig. 3, Table 1and Table 2).

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).

Role of Individual Arrestin Residues in Phosphorylation-Recognition

Overall, these data allow us to ascribe functional roles to at least 7 positively charged residues within arrestin. Mutation K163S has no detectable effect on truncated arrestin interaction with any functional form of rhodopsin, nor does it affect full-length arrestin interaction with Rh*. However, this mutation decreases the ability of full-length arrestin to bind to both dark and light-activated P-Rh by 15-20%. This pattern suggests that Lys-163 does not directly participate in the interaction with the phosphorylated C terminus of rhodopsin, but its presence appears to be important for the mobilization of secondary binding sites. Mutation K166S decreases full-length arrestin binding to all functional forms of rhodopsin (P-Rh > P-Rh*Rh*). K166S also has a modest inhibitory effect on truncated arrestin binding to P-Rh*, while it enhances interaction with Rh*. These data suggest a minor role of Lys-166 in phosphate binding. Mutation K167S decreases both full-length and truncated arrestin binding to P-Rh-2.2 and P-Rh*-2.2 by 20%, while it has a minimal effect on binding to P-Rh*-5.2. This suggests a role of Lys-167 in phosphate interaction that does not appear crucial for the mobilization of secondary binding sites. The inhibitory effects of mutation R171Q on both full-length and truncated arrestin binding to dark and light-activated P-Rh are comparable and profound, suggesting a major role of Arg-171 in phosphate interaction.

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).

Phosphorylation-Recognition and Its Role in the Sequential Multisite Binding of Arrestin

At least 5 positively charged residues appear to be directly involved in phosphate binding. Arg-171, Arg-175, and Lys-176 play a major role, and Lys-166 and Lys-167 play a minor role in phosphate interaction. The substitution of Lys-163 or Lys-166 appears to be substantially more detrimental for full-length than for truncated ARR(1-191) binding, suggesting a potential role of these residues in facilitating the transition of arrestin into a high affinity binding conformation. In contrast, the substitution of Arg-175 and His-179 enhance full-length arrestin binding suggesting that these 2 residues hinder such a transition.

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.


FOOTNOTES

*
This research was supported in part by Grants GM44944 and GM47417 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
An established investigator of the American Heart Association. To whom correspondence should be addressed: Thomas Jefferson University, 233 South 10th St., Philadelphia, PA 19107. Tel.: 215-955-4607; Fax: 215-923-1098.

(^1)
The abbreviations used are: G protein, guanine nucleotide binding protein; Rh, dark rhodopsin; Rh*, light-activated rhodopsin; P-Rh, phosphorylated rhodopsin; P-Rh*, phosphorylated light-activated rhodopsin; ARR(1-365), arrestin truncated after residue 365.


ACKNOWLEDGEMENTS

We thank Dr. T. Shinohara for the visual arrestin cDNA, Dr. C. M. Kim for the beta-adrenergic receptor kinase preparation, and R. Bodine for technical assistance.


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