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J. Biol. Chem., Vol. 277, Issue 46, 43961-43967, November 15, 2002

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Transition of Arrestin into the Active Receptor-binding State Requires an Extended Interdomain Hinge^*

Sergey A. Vishnivetskiy, Joel A. Hirsch, Maria-Gabriela Velez, Yulia V. Gurevich, and Vsevolod V. Gurevich^§

From the Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the  Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel

Received for publication, July 11, 2002, and in revised form, August 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The signaling by a vast family of G protein-coupled receptors is quenched by the binding of arrestin proteins to activated receptors that have been phosphorylated by specific receptor kinases (1). Selective binding to an appropriate functional form of the receptor and timely dissociation are prerequisites for the proper function of arrestin in the cell (1, 2). Phosphorylation and activation recognition sites on arrestin bind simultaneously to the phosphorylated receptor C terminus and regions of receptor that change conformation upon activation, respectively (2). These two interactions activate arrestin, allowing its transition into a high affinity receptor-binding state (2). Several lines of evidence suggest that a major conformational rearrangement of the arrestin molecule is a part of this transition (2-5). First, arrestin binding to the receptor has a very high Arrhenius activation energy (3). Second, the susceptibility of arrestin to proteolysis upon its binding to phosphorylated and light-activated rhodopsin (P-Rh*)¹ changes dramatically (4). Third, an additional hydrophobic binding site, which is not involved in its interaction with light-activated (Rh*) or phosphorylated dark (P-Rh) rhodopsin participates in arrestin binding to P-Rh* (2). This site is localized in the C-terminal half of arrestin molecule (2).

In its inactive basal state, arrestins have an elongated bipartite architecture (5-8). Mutagenesis (2), construction of chimeric arrestins (9), peptide competition (10), and protection from chemical modification by P-Rh* (11) all indicate that rhodopsin-binding residues are present in both domains. The largest distance between implicated residues is approximately 70 Å (5), whereas the whole cytoplasmic surface of rhodopsin is <40 Å (12). Because arrestin binds P-Rh* at a 1:1 ratio (13), all of these data suggest that in the process of arrestin activation and transition to its receptor-binding conformation, its two domains move relative to each other. To test this idea, we manipulated the length of the interdomain hinge (a loop between -strands X and XI, residues 179-190 (5, 6)) (Fig. 1) using deletion and insertion mutagenesis. Our data show that although the lengthening of the hinge region does not appreciably change arrestin binding characteristics, its shortening results in a progressive decrease in P-Rh* binding. These results suggest that there is a minimum length of the hinge necessary for high affinity arrestin binding, consistent with the idea that movement of the two domains relative to each other occurs during the transition of arrestin into its active state.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [-³²P]ATP, [¹⁴C]leucine, and [³H]leucine were purchased from PerkinElmer Life Sciences. All restriction enzymes were purchased from New England Biolabs. Sepharose 2B and all other chemicals were from sources described previously (2, 14). Rabbit reticulocyte lysate and SP6 RNA polymerase were prepared as described previously (15). 11-cis-Retinal was generously supplied by Dr. R. K. Crouch.

Site-directed Mutagenesis-- Bovine visual arrestin cDNA (16) was a gift from Dr. T. Shinohara. The plasmid pARR-VSP was constructed and modified as described earlier (14, 17). This pGEM2-based plasmid encodes bovine wild type arrestin with an "idealized" 5'-untranslated region (15) under the control of an SP6 promoter. Its modification pARR-SC (18) was used for mutagenesis. Mutations were introduced by PCR using an appropriate mutagenizing oligonucleotide as a forward primer and an oligonucleotide downstream from the far restriction site to be used for subcloning as a reverse primer. Resulting fragments of various lengths and an appropriate primer upstream of the near restriction site were then used as reverse and forward primers, respectively, for the second round of PCR. Sites SalI (codons 145-146) plus StuI (codons 191-193) were used for mutations in the hinge region. The resulting fragments were purified, digested with appropriate enzymes, and subcloned into appropriately digested pARR-SC. Combination mutants were constructed by excising fragments carrying one mutation and subcloning them into the appropriately digested plasmid carrying another mutation. The sequences of all constructs were confirmed by dideoxy sequencing.

In Vitro Transcription and Translation-- Plasmids were linearized with HindIII before in vitro transcription to produce mRNAs encoding full-length arrestin proteins. In vitro transcription and translation were performed as described previously (2, 17, 19). Arrestins were labeled by the incorporation of [³H]leucine and [¹⁴C]leucine with the specific activity of the mixture of 1.5-3 Ci/mmol, resulting in the specific activity of proteins within the range of 54-80 Ci/mmol (120-180 dpm/fmol). The translation of each of the arrestin mutants used in this study produced a single labeled protein band with the expected mobility on SDS-PAGE.

Evaluation of Stability and Folding of Mutants-- Two parameters were used for the assessment of mutant stability (14). First, protein yields in the in vitro translation are known to correlate with stability, most probably because misfolded or denatured proteins are rapidly destroyed by proteases present in rabbit reticulocyte lysate (14, 19). Second, denatured proteins tend to aggregate and are pelleted by centrifugation at 350,000 × g for 1 h. As an estimate of the relative stability of a mutant, we used its yield multiplied by the percentage of the protein remaining in the supernatant after incubation for 10 min at 37 °C followed by centrifugation. This integral parameter calculated for a mutant was expressed as a percent of that for wild type arrestin (14). The relative stability of all mutants used in this study exceeds 60%.

Improperly folded or denatured proteins are very sensitive to proteolysis. In contrast, arrestin in its native conformation (similar to most proteins) is relatively insensitive to low trypsin concentrations, whereas in the presence of heparin, its C-tail is clipped off under similar conditions (4) because heparin interacts with arrestin N terminus, thereby displacing the C-tail (20). Therefore, we used limited trypsinolysis to ascertain that hinge deletions do not dramatically change arrestin folding and do not obliterate its proper conformational response to heparin. To this end, 5 µl of in vitro translated (without protease inhibitors) radiolabeled arrestins were treated with 10 µg/ml trypsin in the presence or absence of 0.3 mg/ml heparin. The reaction was allowed to proceed for 30 min at 30 °C in 20 µl of 10 mM Hepes-K, pH 7.3, 100 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA and then was stopped by the addition of 1 µl of 10 mM phenylmethylsulfonyl fluoride dissolved in ethanol. The protein was precipitated by the addition of 9 volumes of methanol supplemented with phenylmethylsulfonyl fluoride and centrifugation. The pellet was washed with 1 ml of 90% methanol, dried, and dissolved in 20 µl of SDS sample buffer. The samples along with undigested controls were subjected to SDS-PAGE. The gels were stained with GelCode Blue reagent (Pierce) soaked in 20% 2,5-diphenyloxazole in glacial acetic acid for 10 min. The fluorochrome was precipitated by washing the gel in three changes of water, and the gels were dried and exposed at 80 °C for 36-72 h.

Rhodopsin Preparations-- Urea-treated rod outer segment membranes were prepared, phosphorylated with rhodopsin kinase, and regenerated with 11-cis-retinal as described previously (2, 21). The stoichiometry of phosphorylation for the rhodopsin preparations used in these studies was 3.8 mol phosphate/mol rhodopsin (for review see Ref. 18 and references therein).

Arrestin binding to rhodopsin was performed as described previously (2, 19). In vitro translated tritiated arrestins (100 fmol) were incubated in 50 mM Tris-HCl, pH 7.5, 0.5 mM MgCl₂, 1.5 mM dithiothreitol, 50 mM potassium acetate with 7.5 pmol (0.3 µg) of the various functional forms of rhodopsin in a final volume of 50 µl for 5 min at 37 °C either in the dark or in room light. The samples were immediately cooled on ice and loaded under dim red light onto 2 ml of Sepharose 2B columns equilibrated with 10 mM Tris-HCl, pH 7.5, 100 mM NaCl. 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 liposomes (<10% of the total binding and <0.5% arrestin present in the assay) was subtracted.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The crystal structure of visual arrestin (5, 6) and arrestin2 (7, 8) as well as previous mutagenesis studies (2, 9) demonstrates that there are three major structural and functional units in the arrestin molecule, the N- and C-domains that are connected by a hinge region and the C-tail (Fig. 1). Both N- and C-domains play a significant role in arrestin binding to P-Rh* (2, 5, 9, 10, 21). Because of the relative size and shape of arrestin (5, 6) and rhodopsin (12), a major conformational rearrangement of the arrestin molecule is necessary to bring all of the residues implicated in binding in contact with the cytoplasmic surface of P-Rh*. We have previously proposed that in the process of arrestin activation, the N- and C-domains move relative to each other (5, 22, 23). Such movement would require a minimal length and flexibility for the interdomain hinge. Hence, a measurable decrease in P-Rh* binding of arrestin mutants with shortened hinge can be expected, whereas an increase in hinge length should not be detrimental for the binding. To test this hypothesis, we manipulated the length of the hinge by introducing deletions and insertions into it. We specifically chose residues that do not participate in intramolecular interactions in the basal state of arrestin (5, 23) and were not directly implicated in receptor binding (17, 22). We also avoided prolines because of their possible importance for the geometry of the hinge (5-8).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Graphical representation of the crystal structure of arrestin. The hinge region is colored in yellow with a thicker worm depiction. Residues 179 and 191 denote the borders of the hinge. The distance indicated by the line is measured from the C of residue 179 to the C of residue 190. The hashed worm section represents a crystallographically disordered part of the polypeptide. The figure was generated with Molscript and Raster3D (28-30).

Hinge Mutations in the Context of Wild Type Arrestin-- To systematically test the effect of hinge length, we introduced deletions of 1-4 residues in two different parts of this region and then constructed 16 possible combinations, producing 24 mutants with deletions ranging from 1 to 8 residues (Fig. 2). Increasing deletions of the hinge result in diminished arrestin binding to P-Rh*. Binding is abolished by the elimination of 7 or 8 residues, which leaves the hinge only of 4-5 residues long. Importantly, the distance between the end of -strand X and the beginning of -strand XI in the basal state of arrestin (5) is ~20 Å, i.e. the length of five residues in a fully extended conformation (Fig. 1). Notably, none of the deletions changes the arrestin binding profile to the various rhodopsin forms (Fig. 2). The analysis of the pooled data for 24 mutants reveals a strong correlation between the number of deleted residues and the decrease in P-Rh* binding (r = 0.91, F(1,23) = 107; p < 0.0001) (Fig. 3). Although P-Rh* binding tends to decrease with hinge length, the binding of different mutants with the same size of deletion varies (Fig. 2). To test whether other factors contribute to observed changes in P-Rh* binding, we analyzed the effect of protein stability. We found no statistically significant correlation between mutant relative stability (determined as described under "Experimental Procedures") and P-Rh* binding (p > 0.05) or between the size of hinge deletion and protein stability (p > 0.05). The effect of no single deletion is smaller than expected based on the slope of the correlation, suggesting that the reduction of hinge length per se is detrimental to P-Rh* binding. The effects of several deletions are stronger than would be expected, suggesting that these deletions have effects in addition to merely shortening the hinge. For example, the deletion of Ala-190 decreases P-Rh* binding by 40%, possibly because it brings Arg-189 and Glu-191 close enough to interact, thereby reducing hinge flexibility. Indeed, an additional deletion of Arg-189 does not suppress the binding further (Fig. 2). Note that all of the deviating mutations yield proteins with lower than expected binding. Thus, they do not contradict the conclusion that progressive shortening of the hinge reduces the ability of arrestin to assume the active conformation necessary for high affinity P-Rh* binding.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Functional effects of the deletions of 1-8 residues in the hinge region. The deletions in regions A (residues 180-184) and B (residues 187-190) are designated as follows (deleted residues in parentheses): A1 (180), A2 (180, 182), A3 (180, 182, 183), A4 (180, 182, 183, 184), B1 (190), B2 (189, 190), B3 (188, 189, 190), and B4 (187, 188, 189, 190). Combination names are used for the mutants with deletions in both regions, e.g. A1B2 has A1 and B2 deletions. The large number over the bar indicates the overall length of the deletion. The binding to four functional forms of rhodopsin was tested in a standard direct binding assay as described under "Experimental Procedures," with 2 nM of each arrestin. Means ± S.D. from two experiments with each performed in duplicate are shown.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   The loss of P-Rh* binding correlates with the extent of the deletion in the hinge region. The P-Rh* binding of 24 mutants with various deletions in the hinge region (the absolute values are shown in Fig. 2) was analyzed as a function of the total number of deleted residues using GraphPad Prism (version 2a, Power MacIntosh). The correlation was found to be statistically significant (r = 0.91, F(1,23) = 107; p < 0.0001).

Because hinge deletions affecting the junction between the two arrestin domains are loss-of-function mutations, it is important to ascertain that these mutations do not cause protein misfolding. To this end, we compared the susceptibility to limited proteolysis of all of the mutants used in this study with insertions of 1-3 residues and deletions ranging from 1 to 8 residues to that of wild type arrestin. We chose trypsin for these experiments for two reasons. First, its effect on WT arrestin in the basal conformation was characterized previously (4). More importantly, it has been convincingly demonstrated that in response to heparin, the conformation of arrestin changes, making its C-tail susceptible to trypsinolysis under the same conditions where arrestin alone is not affected (4). Heparin was later shown to interact with arrestin N terminus, thereby displacing its C-tail (20), which apparently makes the C-tail accessible to trypsin. Thus, trypsinolysis provides a test for the preservation of the protease-resistant native conformation of the mutants and, as an added bonus, an even more rigorous test for the well characterized conformational response of arrestin to heparin (4). We found that all three insertion mutants and 16 of 24 deletion mutants behave exactly like WT arrestin, i.e. they are resistant to trypsinolysis in the absence of heparin and yield a 44-kDa product in the presence of heparin. The remaining eight mutants rapidly yield a 21-kDa product (similar to the product that WT arrestin yields after longer digestion with trypsin (4)). This 21-kDa fragment has the mobility of arrestin-(1-191) truncated mutant, suggesting that trypsin cuts at Arg-189 in the hinge (data not shown). The analysis of the actual hinge sequence of the mutants yielding this fragment shows that all of them have a deletion of two or more residues and an intact Arg-189, suggesting that an increased accessibility to the protease of Arg-189 rather than improper global folding is the reason for their higher sensitivity to trypsin. Nonetheless, we re-analyzed the correlation between the size of the deletion and P-Rh* binding for the group of 16 mutants that behave exactly like WT arrestin (Fig. 4) and found the same strong correlation (r = 0.91; F(1,15) = 64; p < 0.0001) as with the whole set of 24 deletion mutants (Fig. 3). Fig. 4 shows that the mutants with a wide range of deletion lengths (up to seven residues) demonstrate relative resistance to proteolysis by 10 µg/ml trypsin similar to WT arrestin. Moreover, under the same conditions in the presence of 0.3 mg/ml heparin, a fragment of the same size (approximately 4 kDa) is cleaved off WT arrestin and these hinge mutants. These data strongly indicate that neither the native conformation of arrestin nor its conformational response to heparin is affected by hinge deletions. Thus, the progressive decrease in P-Rh* binding that is roughly proportional to the size of hinge deletions cannot be explained by improper folding. We believe that it reflects progressive reduction in the ability of the mutant to assume and maintain the active conformation, which apparently requires relatively long and flexible interdomain hinge.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Mutants with various hinge deletions demonstrate the same high resistance to trypsin and conformational response to heparin as WT arrestin. In vitro translated radiolabeled arrestins (5 µl) were left untreated (lanes 1, 4, and 7) or treated with 10 µg/ml trypsin in the presence (lanes 3, 6, and 9) or absence (lanes 2, 5, and 8) of 0.3 mg/ml heparin for 30 min at 30 °C in 20 µl of 10 mM Hepes-K, pH 7.3, 100 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA. The reaction was stopped by 1 µl of 10 mM phenylmethylsulfonyl fluoride, and the protein was precipitated by methanol. The pellets were dissolved in 20 µl of SDS sample buffer and subjected to SDS-PAGE. Gels were soaked in 20% 2,5-diphenyloxazole in glacial acetic acid. The fluorochrome was then precipitated by water. Gels were dried and exposed at 80 °C for 36-72 h. The mutants that behaved like WT (lanes 1-3 in each panel) in this assay had the following deletions: 180; 190; 180,190; 189,190 (A, lanes 4-6); 180,189,190 (A, lanes 7-9); 188-190; 187-190 (B, lanes 4-6); 180,182,189,190; 180,187-190; 180,182,188-190 (B, lanes 7-9); 180,182,183,189,190; 180,182,183,188-190; 180,182,183,188-190; 180,182-184,189.190 (C, lanes 4-6), 180,182,187-190; and 180, 182-184,188-190 (C, lanes 7-9). Representative results of 2-4 experiments are shown.

We next tested the effects of increasing hinge length by inserting 1-3 alanines after Ala-180 and found that these insertions are not detrimental to P-Rh* binding (Fig. 5). We reasoned that if the hinge length determines the ability of arrestin to undergo transition into its active state, the deletion of three residues will decrease P-Rh* binding. At the same time, the insertion of three residues does not markedly affect binding, because the hinge has more than the requisite length and flexibility to enable activation (Fig. 5). A combination of the three residue deletion with the three residue insertion should then restore the wild type length of the hinge and thereby reconstitute full P-Rh* binding. We constructed such a mutant with both a deletion and insertion of three residues, effectively changing a three-residue sequence in the hinge. Reconstitution of WT activity is exactly what we observed (Fig. 5, 3 + i3). Thus, approximately a 12-amino acid long hinge is required for wild type arrestin transition into its active state in the process of binding to P-Rh*. Additional "slack" in the hinge added by insertions does not affect function, whereas the shortening of the hinge progressively diminishes the ability of arrestin to bind P-Rh*.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   An insertion of the same length fully compensates for a deletion in another part of the hinge. The mutants are designated as follows: i1, i2, i3 (insertions of 1, 2, or 3 extra alanines after Ala-180), respectively; 3 (deletion of residues 182-184); and 3+i3 (the combination of the triple insertion and triple deletion). The binding was performed as in Fig. 2 in the presence of 1.7 nM of each arrestin. Means ± S.D. from three experiments with each performed in duplicate are shown. Phosph., phosphorylation.

Hinge Deletions in Constitutively Active Mutants-- Several structurally distinct constitutively active mutants of various arrestins that bind with high affinity not only to activated phosphoreceptor but also to activated unphosphorylated receptor and inactive phosphoreceptor have been described previously (2, 9, 14, 17, 18, 22-27). The interaction of WT visual arrestin with P-Rh* is resistant to high salt inhibition, whereas its binding to Rh* and dark P-Rh is very sensitive to high ionic strength (2). Based on the reduced salt sensitivity of constitutively active mutant binding to Rh* and dark P-Rh, we hypothesized that the mutants bound to these forms of rhodopsin assume the same or very similar active conformation as wild type arrestin bound to P-Rh* (2, 17, 22). If this proposal is correct, effective binding of any constitutively active mutant to any form of rhodopsin would require the same length of the hinge as wild type arrestin binding to P-Rh*, i.e. the binding of constitutively active mutants to all functional forms of rhodopsin would progressively decrease with the shortening of the hinge. On the other hand, the hinge region is adjacent to -strand X that contains arrestin main phosphate sensor Arg-175 and other phosphate-binding residues (17). Thus, it is conceivable that the shortening of the hinge somehow affects the function of these phosphate-binding elements, resulting in lower P-Rh* binding. If this is the case, we could expect similar progressive inhibition of P-Rh* and dark P-Rh binding, whereas Rh* interaction, which cannot involve arrestin phosphate-binding sites, would not be affected.

Only constitutively active mutants yield sufficient binding to dark P-Rh and Rh* to test these predictions quantitatively and with high precision. Therefore, we combined four activating mutations with hinge deletions of 3, 5, and 8 residues and compared the binding of these proteins to all four forms of rhodopsin with that of the parental constitutively active mutants. For these experiments, we chose activating mutations that do not remove phosphate-binding residues in the -strand X: triple alanine substitution (F375A,V376A,F377A) (14); the deletion of 26 C-terminal residues yielding arrestin (1-378) (14); deletion of residues 11-16 in the N terminus (18); and charge-reversal mutation D296R in the polar core that does not affect Arg-175. As shown in Fig. 6, an incremental decrease in hinge length progressively inhibits arrestin binding to all functional forms of rhodopsin. If anything, Rh* binding is even more sensitive to hinge deletions than P-Rh* and dark P-Rh binding, ruling out direct interference of the hinge deletions with the function of the phosphate sensor. It is noteworthy that the binding to both non-preferred forms (Rh* and dark P-Rh) tends to be more sensitive to hinge deletions than P-Rh* binding (Fig. 6). Apparently, with a shortened hinge, the active conformation of arrestin is more strained. When rhodopsin has fewer elements holding bound arrestin in this state (the active conformation alone in Rh* or the phosphates only in dark P-Rh), the probability of bound mutants relaxing back into the basal state and dissociating grows faster with an increasing strain induced by insufficient hinge length. In short, the mutant binding to Rh* and dark P-Rh requires the same length of the hinge as does wild type arrestin binding to P-Rh*. Apparently, high affinity binding of arrestin mutants to any form of rhodopsin involves the same or very similar movement of the two domains relative to each other, i.e. the transition into the same active conformation.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   The effects of selected hinge deletions on the binding of constitutively active arrestin mutants to P-Rh*, dark P-Rh, and unphosphorylated Rh*. Activating mutations are designated as follows: 3A, triple mutation F375A,V376A,F377A (14); Tr, truncated arrestin-(1-378) (14); DN, arrestin with residues 11-16 in the N terminus deleted (18); and DR, arrestin-(D296R) (23). Deletion designations 3, 5, and 8 indicate the deletion of 3 (positions 188-190), 5 (180, 182, 188-190), and 8 (180, 182-184, 187-190) residues, respectively. The binding was performed as in Fig. 2. Means + S.D. from two experiments with each performed in duplicate are shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The finding by Hofmann and colleagues (3) over a decade ago that the binding of arrestin to rhodopsin has unusually high activation energy led to the conclusion that arrestin undergoes a substantial conformational rearrangement in the process. Consistent with this notion, bound arrestin was shown to be more susceptible to proteolysis (4). Subsequent structure-function studies of visual arrestin and other members of this protein family (2, 9, 14, 17-19, 22-27) lend additional support to this hypothesis. An analysis of the crystal structures of the inactive state of arrestin and rhodopsin (5, 6, 12) in conjunction with the localization of arrestin elements implicated in rhodopsin binding (2, 9-11, 17, 18, 22-27) leads to the conclusion that the shape of arrestin must change dramatically to bring all of these elements in contact with P-Rh* simultaneously. The exact nature of this conformational change still remains to be determined. Elucidation of the three-dimensional structure of the arrestin-receptor complex will provide the most direct information; however, such data are not yet available. Nonetheless, targeted mutagenesis based on the structure of the basal state of the arrestin can provide numerous clues.

Two groups of intramolecular interactions appear to be largely responsible for the stabilization of the basal arrestin conformation (5). One is an unusual network of five solvent-excluded charged residues in the fulcrum of the two-domain molecule, which we termed the "polar core" (5, 23). The other is a three-element interaction of -strand I, -helix I, and -strand XX of the C-tail (5, 18). Both interactions stabilize the relative positions of the two arrestin domains (5). Our recent studies show that the disruption of either group of interactions by mutagenesis yields constitutively active mutants (5, 9, 14, 17, 22-27). Both sets of interactions appear to be disrupted by rhodopsin-attached phosphates in the process of normal arrestin activation for P-Rh* binding (18, 23). The simultaneous disruption of the polar core and the three-element interaction leaves another element to tether the two domains together, the interdomain connector that we have dubbed the hinge region.

In contrast to the polar core and the three-element interaction, the hinge is flexible and its residues do not participate in stabilizing intramolecular interactions. Its inherent flexibility is indicated by significantly higher than average crystallographic temperature factors (also known as thermal mobility factors) for the hinge atoms. The hinge region has an average temperature factor of 77 Å², whereas the immediately preceding strand X has an average temperature factor of 47 Å², the immediately following strand XI has an average temperature factor of 60 Å², and the whole molecule has an average temperature factor of 62 Å² (5).

In addition to the flexibility of the hinge, its sequence is not highly conserved (data not shown) (for review see Ref. 5). In fact, it is more variable than most other parts of the molecule. This relative lack of conservation is consistent with the current study (Fig. 5), which has shown that significant scrambling of the sequence for this region has little effect as long as the requisite length is maintained. The residues that are conserved in the region are primarily prolines (three in the visual arrestin subfamily and four in the -arrestin subfamily). Indeed, proline is an amino acid characterized by its low propensity for secondary structure formation (28), again suggestive that the hinge is designed for tethering and not intramolecular interactions.

Both rearrangements accompanying arrestin activation (18, 23) essentially make the movement of the two domains relative to each other possible. We hypothesized that such a movement is a crucial part of arrestin transition into its high affinity receptor-binding state. If our hypothesis is correct, the length of the interdomain hinge is a factor limiting this movement. Assuming that wild type arrestin has a hinge of sufficient length to allow activation, one would expect its increase to have no effect on arrestin binding. Incremental shortening of the hinge, however, would progressively hinder domain movement, eventually making it impossible. This is exactly what we observed both in case of wild type arrestin binding to P-Rh* (Figs. 2, 3, and 5) and the constitutively active mutant binding to P-Rh*, Rh*, and dark P-Rh (Fig. 6).

These data allow us to refine further and substantiate our model of arrestin activation. Initially, receptor-attached phosphates encounter the N-terminal phosphate-binding element, Lys-14 + Lys-15 (18), which participates in guiding the phosphates to the main phosphate sensor Arg-175 (17, 22-24). The movement of the two lysines distorts -strand I, thereby disrupting the three-element interaction and releasing arrestin C-tail (18), which is one of the elements holding the two domains together. The displacement of the C-tail removes Arg-382 from the polar core. Phosphate binding then neutralizes the other positive charge in the polar core, that of Arg-175. Consequently, the mutual repulsion of the three remaining negative charges in the polar core further destabilizes interdomain interactions, allowing arrestin to relax into its active conformation and bind receptor.

The contention that both arrestin domains, while in the active state, participate in receptor binding is supported by ample experimental evidence (2, 5, 9-11, 17, 18, 22, 23). Hence, simple geometric considerations suggest that a substantial "rigid body"-like movement of the two arrestin domains relative to each other is necessary for receptor association. This movement requires an extended flexible interdomain hinge. This dependence is born out by the effects of hinge perturbations observed here. Finally, the now reconfigured bipartite arrestin will make specific high affinity interactions with the receptor, terminating the signal.

	ACKNOWLEDGEMENTS

We thank Dr. J. L. Benovic for purified rhodopsin kinase, Dr. R. K. Crouch for 11-cis-retinal, Dr. T. Shinohara for arrestin cDNA, and Drs. C. Schubert and M. Han for stimulating discussions.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants EY11500 and GM63097 (to V. V. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-322-7070; E-mail: Vsevolod.Gurevich@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, September 4, 2002, DOI 10.1074/jbc.M206951200

	ABBREVIATIONS

The abbreviations used are: P-Rh*, light-activated phosphorylated rhodopsin; P-Rh, dark phosphorylated rhodopsin; Rh*, light-activated unphosphorylated rhodopsin; Rh, dark unphosphorylated rhodopsin; WT, wild type (Note that here we use the systematic names of non-visual arrestins. The synonyms of arrestin2 are -arrestin and -arrestin1; arrestin3 is also called -arrestin2)..

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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