©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Arrestin Interactions with G Protein-coupled Receptors
DIRECT BINDING STUDIES OF WILD TYPE AND MUTANT ARRESTINS WITH RHODOPSIN, beta(2)-ADRENERGIC, AND m2 MUSCARINIC CHOLINERGIC RECEPTORS (*)

(Received for publication, July 8, 1994; and in revised form, November 14, 1994)

Vsevolod V. Gurevich (3) Stephane B. Dion (3) James J. Onorato (2) Judith Ptasienski (1) Chong M. Kim (3) Rachel Sterne-Marr (3) M. Marlene Hosey (1) Jeffrey L. Benovic (3)(§)

From the  (1)From theDepartment of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611, the (2)Department of Medicine, University of Wisconsin, Madison, Wisconsin 53792, and the (3)Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Arrestins play an important role in quenching signal transduction initiated by G protein-coupled receptors. To explore the specificity of arrestin-receptor interaction, we have characterized the ability of various wild-type arrestins to bind to rhodopsin, the beta(2)-adrenergic receptor (beta(2)AR), and the m2 muscarinic cholinergic receptor (m2 mAChR). Visual arrestin was found to be the most selective arrestin since it discriminated best between the three different receptors tested (highest binding to rhodopsin) as well as between the phosphorylation and activation state of the receptor (>10-fold higher binding to the phosphorylated light-activated form of rhodopsin compared to any other form of rhodopsin). While beta-arrestin and arrestin 3 were also found to preferentially bind to the phosphorylated activated form of a given receptor, they only modestly discriminated among the three receptors tested. To explore the structural characteristics important in arrestin function, we constructed a series of truncated and chimeric arrestins. Analysis of the binding characteristics of the various mutant arrestins suggests a common molecular mechanism involved in determining receptor binding selectivity. Structural elements that contribute to arrestin binding include: 1) a C-terminal acidic region that serves a regulatory role in controlling arrestin binding selectivity toward the phosphorylated and activated form of a receptor, without directly participating in receptor interaction; 2) a basic N-terminal domain that directly participates in receptor interaction and appears to serve a regulatory role via intramolecular interaction with the C-terminal acidic region; and 3) two centrally localized domains that are directly involved in determining receptor binding specificity and selectivity. A comparative structure-function model of all arrestins and a kinetic model of beta-arrestin and arrestin 3 interaction with receptors are proposed.


INTRODUCTION

Many cells have the ability to rapidly regulate their responsiveness to external stimuli. This phenomenon, often termed desensitization, has been extensively studied in signaling pathways initiated by G protein-coupled receptors. Two of the best studied G protein-coupled receptors are rhodopsin, which mediates phototransduction in retinal rod cells, and the beta(2)-adrenergic receptor (beta(2)AR)^1, which stimulates cAMP production in many tissues. The molecular events underlying desensitization of rhodopsin and the beta(2)AR include a rapid activation-dependent phosphorylation of the receptor mediated by the specific G protein-coupled receptor kinases rhodopsin kinase (1) and the beta-adrenergic receptor kinase (betaARK) (2) , respectively. Receptor phosphorylation then promotes the binding of another protein to the receptor. This protein has been termed S-antigen or arrestin in the phototransduction system while in the beta(2)AR system a related protein termed beta-arrestin has been implicated. The binding of arrestin to the phosphorylated receptor appears to play the primary role in quenching signal transduction via its apparent ability to directly decrease receptor/G protein coupling (3, 4, 5, 6) .

Recent studies suggest a significant diversity in the arrestin gene family with four mammalian arrestins having been identified to date(5, 7, 8, 9, 10, 11, 12) . The mammalian arrestins include visual arrestin, which is predominantly localized in rods and appears to play a primary role in quenching phototransduction(13, 14) ; beta-arrestin, which may play a role in desensitization of the beta(2)AR(5, 6) ; arrestin 3 (also termed beta-arrestin 2), a recently identified member of the family that may interact with the beta(2)AR and possibly odorant receptors(8, 9, 10, 15) ; and a recently cloned retinal-specific arrestin termed X-arrestin (11) or C-arrestin(12) . Recent studies suggest that at least three of the mammalian arrestins may also be represented by multiple polypeptide variants(10, 16, 17) . These variants include a form of beta-arrestin, betaarrS, that lacks 8 amino acids found in the originally identified bovine brain beta-arrestin (betaarr)(5, 10, 16) . Interestingly, the 8 amino acids that distinguish these two forms of beta-arrestin are encoded by exon 13 in the human visual arrestin gene(18) . While a similar variant may also exist for visual arrestin(16) , a variant that has an alanine in place of the C-terminal 35 amino acids has also been identified(17) . In addition, a form of arrestin 3 (arr3L) that contains an 11-amino acid insert between residues 361 and 362 has also been identified(10) . While the predominant form of betaarr varies between tissues and arr3S is the predominant form of arr3 in most tissues, little is presently known about the functional significance of these polypeptide variants.

The tremendous diversity of the G protein-coupled receptors, as compared to the more limited number of G protein-coupled receptor kinases and arrestins, raises obvious questions concerning the receptor specificity of the various G protein-coupled receptor kinases and arrestins. Here we have compared the ability of visual arrestin, betaarr, betaarrS, arr3L, and arr3S to bind to four different functional forms (phosphorylated, phosphorylated and agonist activated, unphosphorylated, and unphosphorylated and agonist activated) of three functionally distinct receptors: rhodopsin, the beta(2)AR, and the m2 muscarinic cholinergic receptor (m2 mAChR). We have utilized these receptors since they each have been demonstrated to undergo an activation-dependent phosphorylation both in vitro and in intact cells(2, 19, 20, 21, 22, 23) . Moreover, more recent studies suggest that each of these receptors is also able to interact with arrestins in vitro(4, 5, 6, 9, 24) .

The recent progress in elucidating the molecular mechanisms involved in visual arrestin binding to phosphorylated light-activated rhodopsin (P-Rh)(25, 26, 27) , as well as the cloning of an increasing number of arrestins, raises several important questions concerning arrestin function. First, do other members of the arrestin family use the same sequential multisite binding mechanism as does visual arrestin? In addition, what domains of the arrestin molecule determine its receptor specificity and what structural elements of arrestins and receptors are involved? In an attempt to address these questions we have utilized two strategies of mutagenesis. First, several truncated forms of betaarr and arr3, analogous to the previously characterized truncated forms of visual arrestin (arr)(25, 26) , were produced and functionally characterized. This approach allowed us to establish a similar localization of the major functional domains in all arrestin proteins and suggested that the molecular mechanisms involved in their binding may also be similar. These findings set the stage for the construction of a series of arr/betaarr chimeras. In constructing the chimeric arrestins we attempted to divide the arrestins into the four functional domains previously identified in visual arrestin(25, 26) . These studies have enabled us to obtain further insight into the mechanism of arrestin-receptor interaction and the structural basis of arrestin specificity.


EXPERIMENTAL PROCEDURES

Materials

[-P]ATP, [S]dATP, and [^3H]leucine were purchased from DuPont NEN. All restriction enzymes were purchased from either Boehringer Mannheim or Promega. Sepharose 2B, Sephadex G-25, and all other chemicals were from Sigma. Rabbit reticulocyte lysate and SP6 RNA polymerase were prepared as described previously(25, 26) . 11-cis-Retinal was generously supplied by Dr. R. K. Crouch, National Institutes of Health. Other reagents were from sources previously described(25, 26) .

Plasmid Constructions

A bovine visual arrestin cDNA was generously supplied by Dr. T. Shinohara(7) . An arrestin construct containing the wild type N terminus of arrestin (pARR) was subcloned into the vector pG2S6-I as described previously(24) . The bovine beta-arrestin, beta-arrestin S, arrestin 3L, and arrestin 3S cDNAs (10) were excised with NcoI and HindIII and subcloned into NcoI/HindIII digested pG2S6-I (resulting plasmids were termed pBARR, pBARRS, pARR3L, and pARR3S, respectively). Plasmids were linearized with HindIII before in vitro transcription to obtain full-length mRNAs. Plasmids encoding chimeric arrestins were constructed as follows. pBBBA and pAAAB were obtained by exchange of 350- and 377-bp BstXI-HindIII fragments of pARR and pBARR, respectively. pBAAA and pABBB were constructed by exchange of 1225- and 1274-bp BamHI/HindIII fragments of pARR and pBARR, respectively. pBAAB and pABBA were constructed by subcloning 1252- and 1247-bp BamHI/HindIII fragments of pAAAB and pBBBA, respectively, into BamHI/HindIII digested pBARR and pARR, respectively. pBBAA and pAABB were constructed by exchange of 730-and 786-bp BglII/HindIII fragments of pARR and pBARR, respectively. pABAA and pBABB were obtained by subcloning 1218- and 1300-bp BamHI/HindIII fragments of BBAA and AABB, respectively, into BamHI/HindIII-digested pARR and pBARR, respectively. pBBAB and pAABA were constructed by subcloning 377- and 350-bp BstXI/HindIII fragments of pBARR and pARR, respectively, into BstXI/HindIII-digested pBBAA and pAABB, respectively. The sequences of all constructs were confirmed by DNA sequencing (the structures of the resulting proteins are schematically shown in Fig. 4). Plasmids were linearized with HindIII before in vitro transcription to obtain full-length mRNAs. To obtain truncated mRNAs, pARR and pBAAA were linearized with StuI to obtain mRNAs encoding arr(1-191) and BAAA(1-187); pBARR and pABBB were linearized with HindII to obtain mRNAs encoding betaarr(1-217) and ABBB(1-221); pBARR was linearized with either BglII or AspHI to obtain mRNAs encoding betaarr(1-206) and betaarr(1-367), respectively; while pARR3S was linearized with either PvuII or SfuI to obtain mRNAs encoding arr3(1-183) and arr3S(1-375), respectively. In vitro transcription and translation was carried out as described previously(25, 26) .


Figure 4: Structure and binding of chimeric arrestins to human P-mAChR and P-beta(2)AR. The portion of the chimera derived from betaarr is shaded in the schematic. The four structural elements of the chimeric arrestins are: 1) N-terminal region: residues 1-47 of arr or 1-43 of betaarr (49% identity, 64% similarity); 2) N-terminal binding domain: residues 48-213 of arr or 44-207 of betaarr (60%, 74%); 3) C-terminal binding domain: residues 214-345 of arr or 208-340 of betaarr (60%, 77%); 4) regulatory C-terminal region: residues 346-404 of arr or 341-418 of betaarr (32%, 39%). The average K and B(max) from two to three experiments (± S.D.) is presented. ND, not determined.



Receptor Preparations

Urea-treated rod outer segment membranes were prepared, phosphorylated with betaARK, and regenerated with 11-cis-retinal as described(26) . The stoichiometry of phosphorylation for the rhodopsin preparation used in these studies was 2.4 mol of phosphate/mol of rhodopsin. The human m2 mAChR was expressed in Sf9 cells, purified by affinity chromatography, and reconstituted into chick heart phospholipid vesicles as described previously(23) . The reconstituted m2 mAChRs were phosphorylated with betaARK to a stoichiometry of 2.8-3.7 mol of phosphate/mol of m2 mAChR as described previously(24) . Both human and hamster beta(2)AR were expressed in Sf9 cells, purified by affinity chromatography, reconstituted into soybean phosphatidylcholine vesicles, and phosphorylated by betaARK to stoichiometries of 2.4-4.5 mol of phosphate/mol of beta(2)AR as described previously(28) . Activation of the receptors was achieved with room light for rhodopsin, 100 µM(-)-isoproterenol for the beta(2)AR, and 100 µM carbachol for the m2 mAChR.

Arrestin Binding to Receptors

The basis of the arrestin/receptor binding assay was to assess a change in the mobility of an arrestin on a gel filtration column upon binding to the receptor. To study binding to rhodopsin, the in vitro translated [^3H]arrestins were incubated in 50 mM Tris-HCl, pH 7.5, 0.5 mM MgCl(2), 100 mM potassium acetate with 7.5 pmol of the various functional forms of rhodopsin in a volume of 50 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 were loaded onto a 2-ml Sepharose 2B column equilibrated with 20 mM Tris-HCl, pH 7.5, 2 mM EDTA (buffer A). Bound arrestin eluted with the rod outer segments in the void volume (between 0.5 and 1.1 ml). To study binding of the arrestins to the m2 mAChR or beta(2)AR, 200 fmol of the receptor (phosphorylated or nonphosphorylated) were incubated for 50 min at 30 °C in a 50-µl reaction with 50 fmol of the respective [^3H]arrestin and 100 µM of either the respective antagonist (atropine or alprenolol) or agonist (carbachol or isoproterenol) in 50 mM Tris-HCl, pH 7.5, 50 mM potassium acetate, 0.5 mM MgCl(2) (and 0.2 mM dithiothreitol for the mAChR). The samples were then cooled on ice and loaded onto a 2-ml Sepharose 2B column equilibrated with buffer A. The columns were washed with 0.5 ml of buffer A and the phospholipid vesicles containing the receptor and bound arrestin were then eluted with 0.6 ml of buffer A. When saturation isotherms were prepared, samples contained 50 fmol of phosphorylated m2 mAChR or beta(2)AR, 100 µM of the appropriate ligand, and various concentrations of the arrestins. Nonspecific binding was determined in the presence of 0.3-1 µg of control liposomes and did not exceed 15% of the total binding. For all of the arrestin binding experiments the nonspecific binding was subtracted.


RESULTS

Interaction of Arrestins with Rhodopsin, m2 Muscarinic Cholinergic, and beta(2)-Adrenergic Receptors

In an effort to further characterize the interaction of arrestins with G protein-coupled receptors, we have studied the binding of in vitro translated arrestin, beta-arrestin (short and long forms), and arrestin 3 (short and long forms) to various functional forms of rhodopsin (Rh), the beta(2)AR, and the m2 mAChR. These functional forms include the phosphorylated receptors (P-Rh, P-beta(2)AR, P-mAChR), phosphorylated and activated (^2)receptors (P-Rh, P-beta(2)AR, P-mAChR), non-activated receptors (Rh, beta(2)AR, mAChR), and activated receptors (Rh, beta(2)AR, mAChR).

As previously demonstrated, visual arrestin preferentially binds to P-Rh with a P-Rh/P-Rh binding ratio of 12 (Fig. 1A). Arr3S, arr3L, betaarr, and betaarrS demonstrate a 2-3-fold lower level of binding to P-Rh compared to arr. In addition, beta-arrestin and arrestin 3 also bind to rhodopsin less selectively compared to arrestin with P-Rh/P-Rh binding ratios from 1.3 for arr3L to 2.1 for betaarr. The selectivity profiles of betaarr and arr3 binding to the m2 mAChR qualitatively resemble those seen with rhodopsin (Fig. 1B). All of the arrestins bind preferentially to P-mAChR with arr3S binding being 1.5-fold higher than arr3L, betaarr, and betaarrL, and 19-fold higher than arr. The P-mAChR*/P-mAChR binding ratio is 1.6-1.9 for all arrestins, similar to that observed for betaarr and arr3 binding to rhodopsin.


Figure 1: Interaction of in vitro translated arr, beta-arr, beta-arrS, arr3L, and arr3S with rhodopsin A. 50 fmol of the various in vitro translated arrestins (1068-1205 dpm/fmol) were incubated with 7.5 pmol of the various functional forms of rhodopsin in 50 mM Tris-HCl, pH 7.5, 100 mM potassium acetate, 0.5 mM MgCl(2), 1.5 mM dithiothreitol in 50 µl for 5 min at 37 °C either in the dark or with illumination (room light). The samples were then cooled on ice before separation of bound and free arrestins on a 2-ml Sepharose 2B column as described under ``Experimental Procedures.'' B, interaction of in vitro translated arrestins with the human m2 mAChR. 50 fmol of the various arrestins were incubated with 200 fmol of the different functional forms of the m2 mAChR and either 100 µM atropine or carbachol in 50 mM Tris-HCl, pH 7.5, 50 mM potassium acetate, 0.5 mM MgCl(2), 0.2 mM dithiothreitol in 50 µl at 30 °C for 50 min, cooled on ice, and chromatographed as described under ``Experimental Procedures.'' The phosphorylation stoichiometry of the m2 mAChR used in these studies was 3.7 mol of phosphate/mol of m2 mAChR. C, interaction of in vitro translated arrestins with the human beta(2)AR. 50 fmol of the various arrestins were incubated with 200 fmol of the different functional forms of the beta(2)AR and 100 µM alprenolol or(-)-isoproterenol in 50 mM Tris-HCl, pH 7.5, 50 mM potassium acetate, 0.5 mM MgCl(2) in 50 µl at 30 °C for 35 min, cooled on ice and chromatographed as described under ``Experimental Procedures.'' The phosphorylation stoichiometry of the beta(2)AR used in these studies was 2.4 mol of phosphate/mol of beta(2)AR. The specific binding (mean ± S.D. from two to four independent experiments performed in duplicate) is shown.



Since no direct binding studies of arrestin interaction with the beta(2)AR have been reported, we initially attempted to optimize the conditions to study betaarr and arr3 binding to the beta(2)AR. Binding to the beta(2)AR was found to reach an apparent equilibrium following a 30-min incubation at 30 °C, which was maintained for at least another 30 min. The minimum phosphorylation level necessary for betaarr and arr3S to recognize the phosphorylated form of the beta(2)AR was 2-3 mol of P(i)/mol of beta(2)AR, while additional phosphorylation up to 10-11 mol/mol did not increase betaarr or arr3S binding (not shown). The selectivity profiles of betaarr, arr3, and arr binding to the beta(2)AR are very similar to those observed with the m2 mAChR (Fig. 1C). Arr3S demonstrates the highest binding, followed by arr3L, betaarr, and betaarrS. Arr binding to the beta(2)AR was 4-9-fold lower than the other arrestins. The discrimination of betaarr and arr3 between the P-beta(2)AR and P-beta(2)AR is also very similar to that observed for the m2 mAChR (1.5-1.8-fold).

Previously, we demonstrated that a conventional Scatchard analysis could be used to study betaarr and arr binding to the P-mAChR(24) . More extensive analysis demonstrates that arr3S and arr3L (K(d) = 0.35-0.38 nM), and betaarr and betaarrS (K(d) = 0.5-0.6 nM) have similar affinities for the human P-m2 mAChR, while arr (K(d) = 7.2 nM) has a substantially lower affinity (Table 1). The number of binding sites, determined in the presence of 50 fmol of P-mAChR, was also substantially different among the arrestins with a B(max) of 15-19 fmol for arr3S, arr3L, betaarrS, and betaarr and only 7 fmol for arr. A similar analysis of arrestin binding to the human P-beta(2)AR demonstrates that betaarr and betaarrS have the highest affinity (K(d) = 0.14-0.19 nM), followed by arr3S and arr3L (K(d) = 0.33-0.36 nM), and arr (K(d) = 2.1 nM) (Table 1). Thus, the affinity of betaarr and betaarrS for the P-beta(2)AR is 3-4 times higher than for the P-mAChR, while the affinity of arr3L and arr3S for both receptors is comparable. The number of binding sites, determined in the presence of 50 fmol of P-beta(2)AR, was highest for arr3S (17 fmol), followed by arr3L (11 fmol), betaarrS (7.1 fmol), arr (5.5 fmol), and betaarr (4.1 fmol).



A prominent difference between the binding of visual arrestin and the non-visual arrestins is the substantially greater selectivity of visual arrestin for the phosphorylated and activated form of the receptor. While beta-arrestin and arrestin 3 preferentially bind to the phosphorylated form of a given receptor, they appear to be less dependent on the activation state of the receptor. To further analyze this phenomenon we studied the binding of betaarr and arr3S to the P-mAChR and P-beta(2)AR in the presence of either agonist or antagonist. These studies revealed that the agonist promotes a 2-3-fold increase in B(max) for betaarr and arr3S compared to the antagonist with no appreciable difference in the K(d) (Table 1). These results suggest that the agonist predominantly alters the number of phosphorylated receptors capable of interacting with a given arrestin, presumably by converting more receptors to an ``active'' conformation. However, it is clear that not all of the receptors are active even in the presence of agonist since the B(max) is always lower than the number of receptor molecules in the assay. The differences in B(max) may reflect the ability of a given arrestin to promote the formation of and/or stabilize an agonist-phosphoreceptor-arrestin complex, analogous to the agonist-receptor-G protein ternary complex(29) .

The relatively low B(max) observed for arrestin binding (Table 1) prompted us to search for experimental conditions that favored the formation of a high affinity agonist-receptor-arrestin complex. To this end we studied the effects of G protein beta subunits on betaarr and arr3S binding. For these studies the beta(2)AR and m2 mAChR were initially phosphorylated to a stoichiometry of 3-4 mol/mol in the absence of beta subunits. When purified bovine brain beta subunits (at a 1:1 ratio with receptor) were included in the binding assay they significantly increased the B(max) of both betaarr and arr3S binding to the P-mAChR (31 and 38%, respectively), without effecting the K(d). Similar experiments with the P-beta(2)AR revealed that beta subunits had no effect on either the K(d) or B(max) of betaarr or arr3S binding. Thus, it appears that beta subunits do not directly interact with arrestins since the effects observed are similar for both betaarr and arr3S and are strictly receptor-dependent. Conceivably, beta subunits are able to bind to the P-m2 mAChR shifting the equilibrium toward the high-affinity arrestin-phosphoreceptor complex.

In an attempt to resolve different interactions involved in arrestin-receptor binding we compared the salt sensitivity of betaarr and arr3 binding to P-beta(2)AR, P-beta(2)AR, P-mAChR, and P-mAChR (Fig. 2, upper panel). The binding of betaarr and arr3S to P-beta(2)AR is modestly stimulated at physiological salt concentrations followed by slight inhibition at higher ionic strength. This resembles the effects of ionic strength on visual arrestin binding to P-Rh(26) , and suggests that both ionic (inhibited by salt) and hydrophobic (stimulated by salt) interactions are involved in betaarr and arr3S binding to the P-beta(2)AR. Physiological salt concentrations also stimulate betaarr and arr3S binding to the P-mAChR, while high salt significantly inhibits binding (Fig. 2, upper panel). This again suggests a role for both ionic and hydrophobic interactions, although both effects are more pronounced compared to the beta(2)AR. Thus, the contribution of ionic and hydrophobic interactions involved in arrestin binding appears to be determined in part by the receptor. However, these data demonstrate that both betaarr and arr3 possess the sites necessary for high affinity interaction with either the P-beta(2)AR or P-mAChR. Previous studies have demonstrated that visual arrestin binding to dark P-Rh is predominantly mediated by ionic interactions since it is very sensitive to salt inhibition(26) . In contrast, when the effect of receptor activation was tested on betaarr and arr3S binding to the P-beta(2)AR, no appreciable difference in salt sensitivity was observed between P-beta(2)AR and P-beta(2)AR (Fig. 2, upper panel). A similar salt sensitivity was also observed for betaarr and arr3S binding to the P-mAChR and P-mAChR (Fig. 2, upper panel). These results suggest that similar interactions mediate the binding of both arrestins to the phosphorylated beta(2)AR and m2 mAChR regardless of the activation state of the receptor. This result is in agreement with the similar affinities of betaarr and arr3 for the phosphorylated and phosphorylated/activated forms of the beta(2)AR and m2 mAChR (Table 1).


Figure 2: Salt effects on betaarr, arr3S, betaarr(1-206), and arr3(1-183) binding to hamster P-beta(2)AR (circles) and human P-m2 mAChR (triangles) in the presence of 100 µM of the agonists isoproterenol or carbachol (solid lines) or the antagonists atropine or alprenolol (dotted lines). All assay conditions were as in the legend to Fig. 1except for the potassium acetate concentration which varied from 50 to 500 mM. The nonspecific binding was determined at all salt concentrations (it decreased with an increase in salt) and subtracted. The specific binding (mean ± S.D. from two to three independent experiments performed in duplicate) is shown as the percentage of control binding determined at 50 mM potassium acetate.



Truncation Mutagenesis of beta-Arrestin and Arrestin 3

Previous structure-function studies have suggested that there are at least four functional domains within the visual arrestin molecule(25, 26) . These four domains include an N-terminal regulatory region; an N-terminal binding domain containing activation- and phosphorylation-recognition sites (residues 1-191); a C-terminal binding domain that interacts only when the activation- and phosphorylation-recognition sites are engaged (residues 191-365); and a C-terminal regulatory region that maintains the overall conformation of arrestin, possibly via direct interaction with the N-terminal regulatory region(27) .

In order to initially probe the structural domains involved in betaarr and arr3 interaction with G protein-coupled receptors, we utilized truncation mutagenesis, an approach previously used to study visual arrestin interaction with rhodopsin(25, 26) . The truncated proteins produced in this study were betaarr(1-367) and arr3S(1-375), which lack the putative regulatory C-terminal region (40 amino acids) identified in arr(24, 25, 26) , and betaarr(1-217), betaarr(1-206), and arr3(1-183), which lack the C-terminal half of the molecule. Previously we demonstrated that similarly truncated arr(1-191) largely retains its phosphorylation- and activation-recognition sites, two primary regions involved in arr interaction with rhodopsin(25, 26, 30) .

Deletion of the C terminus of betaarr to produce betaarr(1-367) increases binding to all functional forms of rhodopsin, the m2 mAChR, and the beta(2)AR, with the predominant effect observed on the phosphorylated and the activated forms of the receptor (Fig. 3). The analogous deletion in arr3S to generate arr3S(1-375) modestly increases binding to the activated form of the various receptors while decreasing binding to the phosphorylated/activated receptors. Thus, a major effect of C-terminal truncation of betaarr and arr3S is to reduce the selectivity of the arrestin for the phosphorylated/activated form of the receptor. These results are very similar to the effects of C-terminal truncation of arr (25, 26) , suggesting that the C terminus of betaarr, arr3S, and arr plays a similar functional role.


Figure 3: Interaction of full-length and truncated arrestins with rhodopsin (A), human m2 mAChR (B), and hamster beta(2)AR (C). 50 fmol of betaarr, betaarr(1-367), betaarr(1-217), betaarr(1-206), arr3S, arr3(1-375), or arr3(1-183) (1006-1485 dpm/fmol) were incubated with receptor and then treated as described under ``Experimental Procedures.'' The specific binding (mean ± S.D. from two to four independent experiments performed in duplicate) is shown.



The deletion of the entire C-terminal half of betaarr and arr3S substantially decreases the binding to rhodopsin and the beta(2)AR, while increasing the binding to the m2 mAChR (Fig. 3). In general, the interaction of betaarr(1-217), betaarr(1-206), and arr3(1-183) with all three receptors demonstrate a uniformly lower selectivity compared to the wild type arrestins. However, it is noteworthy that for interaction with rhodopsin and the m2 mAChR, it is the activation-recognition of the truncated betaarr and arr3 that is predominantly impaired, while for interaction with the beta(2)AR it is the phosphorylation-recognition that is primarily impaired.

The deletion of the C-terminal 40 residues (betaarr(1-367) and arr3S(1-375)) appears to decrease the affinity of betaarr and arr3S for binding to human P-mAChR and hamster P-beta(2)AR (Table 2). Since this region is likely not involved directly in receptor interaction (24, 25, 26) , these data underscore the importance of this domain in keeping the proper functional conformation of betaarr and arr3S. The deletion of the entire C-terminal half (betaarr(1-217), betaarr(1-206), and arr3(1-183)) further impairs the affinity of both betaarr and arr3 for P-mAChR and P-beta(2)AR (Table 2). In contrast, the effect of C-terminal truncations of betaarr and arr3S on the B(max) for binding to P-mAChR and P-beta(2)AR is dramatically different between the receptors. Truncations of betaarr modestly increase the B(max) for the P-beta(2)AR while truncations of arr3S significantly decrease the B(max) (Table 2). By comparison, all of the C-terminal truncations of betaarr dramatically increase the B(max) for the P-mAChR while only arr3(1-183) was affected in a similar manner. It is noteworthy that the B(max) for 4 out of the 5 truncated arrestins was increased to 70-90% of the level of P-mAChR present in the assay (Table 2). This demonstrates that most, if not all, of the receptors are accessible for binding arrestins and suggests that unavailability of receptors is not the reason for the relatively low B(max) measured for full-length betaarr and arr3 ( Table 1and Table 2).



In order to compare the interactions involved in the binding of truncated arrestins to phosphorylated receptors in the presence of agonists and antagonists, we studied the salt sensitivity of betaarr(1-206) and arr3(1-183) binding to P-beta(2)AR and P-mAChR (Fig. 2, lower panel). In contrast to the binding of the corresponding full-length arrestins, the binding of betaarr(1-206) and arr3(1-183) to the P-beta(2)AR is not stimulated by salt, suggesting that a substantial portion of the hydrophobic interaction observed with betaarr and arr3S is contributed by the C-terminal domain. In contrast, physiological salt concentrations stimulate the binding of both betaarr(1-206) and arr3(1-183) to the P-mAChR suggesting that the N-terminal domain significantly contributes to the hydrophobic interaction. Thus, the contribution of ionic and hydrophobic interactions involved in full-length and truncated arrestin binding appears to be determined in part by the receptor.

In contrast to arr-rhodopsin interactions(26) , the salt sensitivity for betaarr, arr3S, betaarr(1-206), and arr3(1-183) binding to the activated or non-activated P-beta(2)AR and P-mAChR were found to be very similar (Fig. 2). These results suggest that similar interactions mediate the binding of all arrestins to the phosphorylated beta(2)AR and m2 mAChR regardless of the activation state of the receptor.

Structure-Function Analysis Using Arr/betaArr Chimeras

Truncation mutagenesis of arr, betaarr, and arr3 suggests that the localization of the major functional domains in all arrestins is similar. In an attempt to further elucidate the role of these various domains in arrestin function, we generated a series of 12 chimeric arrestins that were roughly divided into the four functional regions of arr and betaarr (Fig. 4, left panel). The chimeras are designated using a four-letter description of their structure, where each letter describes the origin of the four respective regions in the chimeric molecule (A stands for arr, B for betaarr). For example, the chimera ABBB contains the N-terminal regulatory region of arr and the N- and C-terminal binding and C-terminal regulatory regions of betaarr.

We initially assessed chimera binding to rhodopsin. Analysis of wild type arrestin demonstrates a relatively low level of betaarr binding to P-Rh compared to arr. betaArr also does not discriminate between Rh and Rh (as compared to a 3-4-fold discrimination for arr). Moreover, betaarr binds with low (2-fold) selectivity for P-Rhversus P-Rh (in contrast to an 14-fold difference for arr). When chimeras that varied in their C-terminal regulatory region were compared, the only chimera virtually indistinguishable from arr was AAAB, which has the C-terminal regulatory region of betaarr (Fig. 5A). These data further corroborate the hypothesis that this region is not directly involved in rhodopsin interaction (24, 25, 26, 27) and demonstrate that the betaarr C terminus is capable of fulfilling the functions of its arr counterpart. Chimera BBBA was also found to be very similar to betaarr, although its binding to P-Rh and P-Rh is somewhat higher. Both chimeras were found to bind to Rh better than to Rh, while the P-Rh/P-Rh binding ratio is betaarr-like (2.3) for BBBA and arr-like (14.4) for AAAB.


Figure 5: The binding of beta-arr, arr, and 12 chimeric arrestins to rhodopsin (A), human m2 mAChR (B), and human beta(2)AR (C). 50 fmol of the respective [^3H]arrestins (953-1177 dpm/fmol) were incubated with phosphorylated, phosphorylated activated, unphosphorylated, and unphosphorylated activated forms of the indicated receptors as described under ``Experimental Procedures.'' The specific binding (mean ± S.D. from two to four independent experiments performed in duplicate) is shown.



The substitution of the N-terminal regulatory region of betaarr into arr to generate the chimera BAAA significantly decreases binding to P-Rh (compared to arr binding), but does not impair the preference for Rh over Rh or the P-Rh/P-Rh binding ratio (14.2). The symmetric substitution in betaarr to generate the chimera ABBB substantially improves its binding to P-Rh (compared to betaarr) while the P-Rh/P-Rh binding ratio remains betaarr-like (1.5). Simultaneous exchange of both the N- and C-terminal regulatory regions yields the chimeras ABBA and BAAB which have comparable binding to P-Rh (50% of the arr binding level). The P-Rh/P-Rh binding ratios of these chimeras appear to be determined by the origin of the central binding domains, being betaarr-like (2.7) for ABBA and arr-like (15.2) for BAAB. These data suggest that while the N-terminal region has some direct participation in rhodopsin binding, the middle portion of the molecule determines the overall binding characteristics of the chimera.

We also constructed a series of 6 chimeras containing two central binding domains of different origin. The first pair of these (BBAA and AABB) demonstrates virtually no preference between Rh and Rh and have P-Rh/P-Rh binding ratios of 4.4 and 4.9, respectively (i.e. intermediate between betaarr and arr). While the binding of AABB to P-Rh is 70% that of arr, BBAA binding to P-Rh is only 25% that of arr and is even lower than betaarr binding. The chimeras ABAA and BABB demonstrate P-Rh/P-Rh binding ratios of 5.6 and 5.5; however, their binding to P-Rh was reduced compared to the binding of BBAA and AABB. Chimera AABA has a P-Rh/P-Rh binding ratio of 3.7 while the symmetric chimera BBAB binds very poorly to P-Rh and demonstrates no preference for P-Rh over P-Rh. Thus, most of the chimeras that contain one binding domain from betaarr and one from arr demonstrate a binding selectivity between that of betaarr (2-fold) and arr (14-fold). The three chimeras with the structure XBAX (where X can be either A or B) demonstrate an unexpectedly low binding to P-Rh (i.e. even lower than the corresponding arrestins with the structure XBBX) (compare BBAA with BBBA). In contrast, in all other chimeras a change from XXBX to XXAX enhances binding to P-Rh as might be expected (compare AABB with AAAB).

When chimera binding to the m2 mAChR is compared, the most noticeable difference between betaarr and arr is the extremely low level of arr binding to P-mAChR (Fig. 5B). This is reflected in both a higher affinity of betaarr for P-mAChR (K(d) = 0.48 nM) compared to arr (K(d) = 7.2 nM) and a higher B(max) for betaarr (16.3 fmol) compared to arr (7.0 fmol) (Fig. 4). However, while the levels of betaarr and arr binding to the m2 mAChR are very different, the selectivity of the binding (P-mAChR/P-mAChR ratio) is similar. Substitution of the arr C terminus into betaarr (BBBA) increases binding to all forms of the m2 mAChR without significantly affecting the affinity, while substitution of the N terminus (ABBB) has minimal effect on the binding but decreases the affinity 2.4-fold ( Fig. 4and Fig. 5B). When both the N- and C-terminal regulatory domains are changed (ABBA) the binding is decreased 2-fold without a change in affinity. The symmetric series of substitutions in arr (AAAB, BAAA, and BAAB) do not significantly improve arr binding to the m2 mAChR and AAAB actually binds with an affinity 1.7-fold lower than that of arr. Overall, substitutions of either the C- or N-terminal regulatory regions lead to modest changes (leq2-fold) in affinities and B(max) for binding to the P-mAChR.

Chimeras BBAA and AABB demonstrate comparable binding to P-mAChR (between that of betaarr and arr), with BBAA being more selective than AABB (Fig. 5B). BBAA also has a modestly higher affinity for the P-mAChR compared to AABB (K(d) = 1.38 versus 2.11 nM, respectively) (Fig. 4). Chimera BABB is similar to BBAA in terms of selectivity profile and affinity (K(d) = 1.25 nM) even though these proteins have the opposite binding domain combinations ( Fig. 4and Fig. 5B). In contrast, chimera ABAA, which has the same combination of binding domains as BBAA, demonstrates very poor selectivity, a low affinity (K(d) = 3.1 nM) and a low B(max) (2.3 fmol). Thus, the structure XBAX is as unfavorable for binding to the m2 mAChR as it is for binding to rhodopsin. The binding of chimera BBAB to the P-mAChR most resembles the binding profile of BBAA although the affinity of BBAB is 2-fold lower. While the chimera AABA most resembles AABB structurally, the B(max) of AABA is significantly increased over that of AABB although there is a modest reduction in affinity (Fig. 4). Interestingly, AABA demonstrates almost as poor a discrimination between P-mAChR and P-mAChR as did the symmetric chimera BBAB between P-Rh and P-Rh (Fig. 5, A and B). Thus, both central binding domains play an important role in arrestin interaction with the m2 mAChR, in agreement with the rhodopsin binding studies.

In general, the binding of betaarr and arr to the P-beta(2)AR most resembles the P-mAChR binding profiles (Fig. 5). This is seen in both the selectivity profile (P-beta(2)AR/P-beta(2)AR binding ratio of 2) and the 15-fold higher affinity of betaarr (K(d) = 0.14 nM) compared to arr (K(d) = 2.1 nM) (Fig. 4). However, in contrast to the m2 AChR binding studies, the B(max) for arr and betaarr binding to the P-beta(2)AR are similar (Fig. 4). The chimeric arrestin studies demonstrate that substitution of the C terminus of betaarr (BBBA) again modestly increases the B(max) without changing the affinity (K(d) = 0.12 nM) ( Fig. 4and Fig. 5C). Substitution of the arr C terminus (AAAB), N terminus (BAAA), or both (BAAB) again does not improve the binding to the beta(2)AR (Fig. 5C). However, substitution of the arr N terminus into betaarr (ABBB) dramatically reduces the affinity (11-fold) and increases the B(max) (8-fold) compared to betaarr (Fig. 4). Substitution of both the N and C terminus of arr into betaarr (ABBA) has a less dramatic reduction in affinity (3-fold) and increase in B(max) (3-fold) (Fig. 4). These results suggest that the N terminus plays a direct role in betaarr interaction with the beta(2)AR. An examination of the central binding domains reveals that, as expected, chimeras containing both betaarr-derived binding domains bind better to the P-beta(2)AR as compared to chimeras containing a combination of betaarr- and arr-derived binding domains (Fig. 5C). The structural pattern XBAX appears to be even less favorable for binding to the beta(2)AR than it was for rhodopsin and the m2 mAChR, since BBAA, ABAA, and BBAB all demonstrate lower binding to the beta(2)AR than the corresponding symmetric chimeras AABB, BABB, and AABA.

In an attempt to more directly assess the role of the N-terminal half of arr and betaarr in receptor interaction, we compared the binding of truncated arr(1-191), betaarr(1-217), ABBB(1-221), and BAAA(1-187) (Fig. 6). In contrast to the corresponding full-length proteins (Fig. 5A), there is no dramatic difference between the ability of arr(1-191), betaarr(1-217) and the truncated chimeras to bind to rhodopsin, although the chimeras appear to have a reduced preference for the phosphorylation state of the receptor (Fig. 6A). However, the binding levels of all truncated arrestins are lower than that of the corresponding full-length proteins (compare Fig. 5A and Fig. 6A), underscoring the important role of the C-terminal binding domain. Binding of the truncated arrestins to the m2 mAChR reveals that only betaarr(1-217) demonstrates high binding and selectivity, while ABBB(1-221) and BAAA(1-187) do not significantly differ from arr(1-191). These data suggest that both the N terminus and N-terminal binding domain are required for high affinity interaction with the P-mAChR. ABBB binding to the P-mAChR is not significantly different from betaarr (Fig. 5B), again underscoring the important role of the C-terminal binding domain. The binding of all four truncated proteins to the beta(2)AR is also surprisingly similar (Fig. 6C), unlike the binding of the full-length proteins (Fig. 5C). The selectivity profiles of both truncated chimeras resemble that of betaarr(1-217), while arr(1-191) is less selective. Interestingly, while the binding of betaarr(1-217) and ABBB(1-221) to the P-beta(2)AR is lower than that of the full-length proteins, the binding of arr(1-191) and BAAA(1-187) is comparable to the full-length proteins (compare Fig. 5C and 6C). This suggests that the C-terminal binding domains of arrestin and BAAA do not significantly interact with the beta(2)AR. In general, both truncated chimeras and arr(1-191) demonstrate a greater specificity for rhodopsin and the beta(2)AR compared to the m2 mAChR. This may correlate with the localization of the relevant phosphorylation sites in these receptors and suggests that the specificity of wild type and chimeric arrestins is determined to a large extent by the C-terminal binding domain.


Figure 6: Truncated arr, betaarr, and chimeric arrestin interaction with receptors. 50 fmol of the indicated truncated arrestins were incubated with the indicated functional form of rhodopsin (A), human m2 mAChR (B), or human beta(2)AR (C), as described under ``Experimental Procedures.'' The specific binding (mean ± S.D. from two to three independent experiments performed in duplicate) is shown.




DISCUSSION

Rhodopsin, beta(2)AR, and m2 mAChR represent three distinct subfamilies of G protein-coupled receptors. Rhodopsin via its specific light-promoted interaction with the G protein transducin activates cGMP phosphodiesterase, the beta(2)AR via interaction with G(s) activates adenylyl cyclase, and the m2 mAChR via its association with G(i) inhibits adenylyl cyclase and activates K channels. There are also distinct structural differences among these receptors (reviewed in (31) ) that likely play a role in arrestin binding. One distinction is the localization of relevant phosphorylation sites on the receptors. Another significant difference is the size and amino acid sequence of the third cytoplasmic loop and C-terminal domains. These domains have previously been implicated in receptor interaction with G proteins (32, 33, 34, 35, 36, 37, 38) , G protein-coupled receptor kinases(39, 40, 41, 42, 43, 44) , and more recently in visual arrestin interaction with rhodopsin(30) . Rhodopsin has a relatively short third cytoplasmic loop (29 residues) and a C terminus (39 residues) that contains all of the rhodopsin kinase phosphorylation sites. The third cytoplasmic loop of the beta(2)AR is intermediate in size (54 residues) and most (if not all) of the betaARK phosphorylation sites are localized in the C terminus (84 residues). In contrast, the third cytoplasmic loop of the m2 mAChR is much longer (181 residues) and it is this loop, rather than the short C terminus (23 residues), that appears to contain all of the betaARK phosphorylation sites(45) .

Receptor Specificity of Arrestins

Arrestin was found to bind very specifically to phosphorylated/light-activated rhodopsin, with only weak binding to P-mAChR and P-beta(2)AR (Fig. 1). Interestingly, the affinity of arrestin for P-beta(2)AR (K(d) = 2.1 nM) is about 3.5-fold higher than its affinity for P-mAChR (K(d) = 7.2 nM), which correlates with the structural similarities between the beta(2)AR and rhodopsin. Both beta-arrestin and arrestin 3 demonstrate very high affinities for the beta(2)AR and m2 mAChR (0.14-0.51 nM). However, betaarrestin has a 3-4-fold higher affinity for the P-beta(2)ARversus the P-mAChR suggesting that beta-arrestin may be more specific for the beta(2)AR and other structurally related receptors. Arrestin 3 has a similar affinity for P-mAChR and P-beta(2)AR (0.33-0.38 nM) suggesting that arrestin 3 may be less specialized than arrestin and beta-arrestin, perhaps serving as a generic arrestin in some non-visual cells. The most striking specificity observed is that of arr toward P-Rh. This suggests that there are certain features shared by the beta(2)AR and m2 mAChR that preclude high affinity binding of visual arrestin. Since the cytoplasmic domains of both the beta(2)AR and m2 mAChR are substantially bulkier than the corresponding domains of rhodopsin, perhaps the receptor-binding pocket of visual arrestin is too small for the beta(2)AR and m2 mAChR. This hypothesis is further corroborated by the characteristics of arr/betaarr chimeras ( Fig. 4and 5). Both the size of crucial arrestin binding cytoplasmic domains and the localization of relevant phosphorylation sites may also determine the relative specificity of beta-arrestin toward the beta(2)AR, while arrestin 3 appears to be less sensitive to structural differences between receptors. In these studies we did not detect any striking functional differences between the variant forms of beta-arrestin or arrestin 3, although both betaarrS and arr3S appear to bind to a higher percentage of the P-beta(2)AR compared to the longer polypeptide forms (Fig. 1, Table 1).

Receptor Phosphorylation Levels Required for High-affinity Arrestin Binding

Receptor phosphorylation appears to be a prerequisite for arrestin interaction. Previous studies have demonstrated that only 2 mol of phosphate/mol of receptor are critical for high affinity arrestin binding to rhodopsin(26) , the m2 mAChR(24) , and the beta(2)AR (this study). Interestingly, 2 residues (serines 338 and 343) were recently identified as the major rhodopsin kinase phosphorylation sites in the rhodopsin C terminus(46) . These data suggest that there may well be only a few basic residues within the phosphorylation-recognition domain of the arrestins that are involved in phosphoamino acid interaction. The extensive additional receptor phosphorylation demonstrated in vitro (each of these receptors can be phosphorylated by their respective receptor kinases to stoichiometries of 8-12 mol/mol) might conceivably serve as a backup mechanism in vivo to directly impair receptor/G protein coupling.

The Role of Arrestin Functional Regions in Receptor Interaction

The general molecular architecture of betaarr and arr3 appears to be similar to that of arr ( Fig. 2and Fig. 3, Table 1). Thus, the construction and characterization of chimeric arrestins served as a feasible approach to further elucidating the regions involved in determining receptor specificity. Moreover, the large number of chimeric arrestins studied enabled an assessment of the role of each individual region by comparison of the binding of several pairs of chimeras that differed only in a particular region.

Regulatory C-terminal Region

There are 5 pairs of chimeras that result from a substitution of this region of betaarr into arr (arr to AAAB, BBAA to BBAB, BAAA to BAAB, ABBA to ABBB, and BBBA to betaarr). The effects of this substitution do not appear to follow any particular pattern since this change can lead to a decrease (BBAA/BBAB and BBBA/betaarr), an increase (ABBA/ABBB), or no change (arr/AAAB and BAAA/BAAB) in binding to P-Rh ( Fig. 4and Fig. 5). The P-Rh/P-Rh binding ratio also either does not change (arr/AAAB, BBBA/betaarr, and BAAA/BAAB) or decreases (ABBA/ABBB and BBAA/BBAB). Similar non-discriminate differences in the binding patterns, affinities, and B(max) of these chimeras for the P-mAChR and P-beta(2)AR are also observed. Thus, the effect of substitution of this region appears to be determined by the combination of the other functional domains, suggesting that the C-terminal regulatory region does not directly participate in receptor interaction. However, the C-terminal region clearly plays an important role in arrestin binding since partial deletion of this region dramatically changes the functional characteristics of both arr (25, 26) and betaarr (Fig. 3, Table 2). Moreover, the similar binding of betaarr and BBBA on the one hand, and arr and AAAB on the other, suggests that the C terminus of betaarr is capable of fulfilling the functions of its arr counterpart and vice versa. While the C terminus is the most divergent region in the arrestins(5, 7, 8, 9, 10, 11, 12) , these results suggest that the majority of the functionally important residues in this region are conserved between arr and betaarr.

Regulatory N-terminal Region

There are also 5 pairs of chimeras that differ only in the origin of this region (arr to BAAA, AABB to BABB, ABAA to BBAA, ABBA to BBBA, and ABBB to betaarr). Comparison of these chimeras reveals a more uniform pattern of changes. While binding to P-Rh either decreases (arr/BAAA, AABB/BABB, and ABBB/betaarr) or slightly increases (ABBA/BBBA and ABAA/BBAA), no changes in the P-Rh/P-Rh and Rh/Rh binding ratios are observed (Fig. 5A). Binding to the P-mAChR either does not change (AABB/BABB and ABBB/betaarr) or increases (arr/BAAA, ABAA/BBAA, and ABBA/BBBA) due to an increased affinity and/or B(max) ( Fig. 4and Fig. 5B). While the affinity for P-beta(2)AR increases for ABBA/BBBA and ABBB/betaarr, the B(max) decreases such that the binding at a fixed concentration (1 nM) also decreases or remains the same (ABAA/BBAA) ( Fig. 4and Fig. 5C). Although this pattern suggests that the N terminus is not a major direct participant in receptor binding, it appears to play some role in arrestin-receptor interaction.

N-terminal Binding Domain

Five pairs of chimeras differ only in the origin of this region (arr to ABAA, BAAA to BBAA, AABA to ABBA, BAAB to BBAB, and BABB to betaarr). The functional consequences of this substitution are remarkably similar in all cases with decreased binding to P-Rh, decreased P-Rh/P-Rh and Rh/Rh binding ratios, and increased binding to both P-mAChR and P-beta(2)AR due to an increased affinity ( Fig. 4and Fig. 5). However, no consistent effect of the origin of this region on the B(max) was observed. Nevertheless, these results are consistent with this region being one of the most important domains of both arr and betaarr involved in receptor interaction.

C-terminal Binding Domain

The effects of the substitution of this domain can also be analyzed using 5 pairs of chimeric arrestins (arr to AABA, BBAA to BBBA, ABAA to ABBA, BAAB to BABB, and BBAB to betaarr). This change results in either no change in the binding (BAAB/BABB), decreased binding (arr/AABA), or increased binding (BBAA/BBBA, ABAA/ABBA, and BBAB/betaarr) to P-Rh. It should be noted that the increased binding occurs only when the first chimera has an apparently unfavorable XBAX structure. In contrast, other functional consequences are exceptionally uniform.

Intramolecular Interactions in Arrestins

The proposed multistep mechanism of arr interaction with rhodopsin(26, 27) , which involves the mobilization of an additional binding site in response to the simultaneous occupancy of the phosphorylation- and activation-recognition sites, suggests the participation of intramolecular interactions in arrestin. Based on comparisons of the effects of C- and N-terminal deletions in arr (26) and the analysis of polyanion inhibition of arrestin-rhodopsin interaction(27) , we previously proposed that the regulatory C terminus interacts with the basic N-terminal region of arr (residues 1-29). Thus, one might expect that the functional effect of the substitution of either of these regions would depend on whether a ``matching'' (i.e. from the same arrestin) pair of these regions is created or destroyed. When chimeras with N- and C-terminal substitutions are analyzed from this perspective, in all but one case where the substitution destroys the match the binding to P-Rh increases, while substitutions creating the match decrease binding. Similarly, matched N and C termini decrease the B(max) while mismatched termini increase the B(max) for binding to the P-mAChR and P-beta(2)AR. These results are consistent with an interaction between the N- and C termini controlling the chimeras transition from a ``low'' to a ``high'' affinity binding conformation, as previously proposed for visual arrestin(26, 27) .

The low level of binding of all XBAX chimeras suggests that it is this particular structure per se, rather than the origin of the binding domains, that makes these arrestins bind poorly. In fact, all XBAX chimeras have even lower binding than betaarr(1-217), which lacks the C-terminal half of the molecule (Fig. 3Fig. 4Fig. 5, Table 2). This demonstrates that the C-terminal binding domain in XBAX not only does not contribute to the binding, but actually seems to inhibit receptor interaction. These results suggest that there may be an obligatory interaction between the N- and C-terminal binding domains in arrestin, which is dramatically impaired in chimeras with the XBAX structure but not impaired in XABX chimeras. The reason for this apparent lack of symmetry may be that a portion of one binding domain (the key) must fit into a pocket in the other binding domain (the keyhole). In this scenario a ``small key/big keyhole'' mismatch is less detrimental for interaction than a ``big key/small keyhole'' mismatch.

Molecular Architecture of Arrestins

The results of previous structure-function studies of arr (24, 25, 26, 27, 30) and betaarr(24, 27) , the data on truncated and chimeric arrestins presented in this study (Fig. 3Fig. 4Fig. 5Fig. 6, Table 2), and the comparison of the primary structures of the arrestins enables identification of the major functional elements within the arrestin molecule (Fig. 7). The very N-terminal region of 25-29 amino acids (R1) of all arrestins is highly positively charged (+8 in arr, +6 in betaarr and arr3), and is most likely involved in interaction with the highly negatively charged C-terminal domain (R2). The next region of 130 residues contains a domain involved in recognition of the activation state of the receptor (activation-recognition) (A). This appears to involve a multisite interaction between the arrestin and receptor that at least in the case of arr binding to P-Rh and Rh involves the first and third cytoplasmic loops of the receptor(30) . The next segment of all arrestins contains a positively charged stretch of 20 residues (net charge of +8 in arr and +7 in betaarr and arr3) that appears to serve as a major phosphorylation-recognition site (P). The next 10-30-residue segment in visual arrestin may also participate in activation-recognition (A) (26) . The next 120 to 150 residue stretch on the C-terminal half of the molecule contains secondary binding sites (S) that are involved in receptor interaction in response to the occupancy of the primary phosphorylation- and activation-recognition binding sites. The C-terminal 60-85 residues serve as a regulatory region (R2) which appears to take part in intramolecular interaction with the N terminus (R1). This interaction serves to maintain the overall conformation of the arrestin molecule and controls its transition into the high-affinity binding state. Interestingly, the polypeptide variants that have been identified for arr, betaarr, and arr3 are within the C-terminal regulatory region. These variants might be expected to differ primarily in their sensitivity to regulatory effects (such as potential interaction with non-receptor proteins, phosphorylation, or other post-translational modifications).


Figure 7: Molecular architecture of arrestins. The putative major functional regions in the arrestins are shown with different fill patterns and are designated as follows: R1, N-terminal regulatory region (25-29 residues); R2, C-terminal regulatory region (60-80 residues); A, activation-recognition region; P, phosphorylation-recognition region (residues 163-182 in arr, residues 157-177 in betaarr and arr3); S, region containing secondary binding site(s) (equivalent to the hydrophobic booster site in visual arrestin(26) ). Arrowheadsabove show the borders between regions used in the chimera constructions (residue numbers are indicated in the legend to Fig. 4). Arrowheadsbelow show the position where differences between polypeptide forms of arr (I and II), betaarr (I), and arr3 (III) were found. See text for details.



The Mechanism of Arrestin-Receptor Interaction

Our proposed kinetic model of non-visual arrestin interaction with receptors is based on several experimental findings (Fig. 8). These include the finding that: 1) wild type non-visual arrestins demonstrate very low binding to unphosphorylated receptors (Fig. 1); 2) the binding of arrestins to phosphorylated receptors is activation-dependent (Fig. 1, Fig. 3, Fig. 5, and Fig. 6); 3) the interactions involved in arrestin binding to phosphorylated and phosphorylated-activated receptors are the same (no change in affinity or salt sensitivity) ( Fig. 2and Table 1); 4) the B(max) for betaarr and arr3 binding to a given receptor is different and is always lower than the number of receptors available (Table 1, Fig. 4, (24) ); 5) abolition of the strict control of arrestin binding by mutagenesis increases the B(max) (Table 2, Fig. 3and Fig. 4); 6) receptor transition into an active state involves its conformational rearrangement(48, 49, 50) ; and 7) high-affinity arrestin binding to phosphorylated-activated receptors requires a conformational transition in the arrestin molecule(26, 47) .


Figure 8: a, general model of arrestin-phosphoreceptor interaction in the presence of agonist (L). Wild type non-visual arrestin (A) binds to receptor-agonist complex (RL) in which phosphoreceptor (R) is in the basal (RL) or activated (ℝL) conformation, to form ARL or AℝL. Arrestin can then undergo a transition into its high-affinity binding conformation (𝔸), giving rise to 𝔸RL and 𝔸L complexes. While all four arrestin-phosphoreceptor-agonist complexes are at equilibrium, the 𝔸L complex has considerably higher affinity for arrestin and therefore represents the experimentally measured B(max). b, C-terminal truncated arrestin binding to receptor. C-terminally truncated arrestins such as betaarr(1-367) and arr3(1-375) more readily assume the high-affinity binding conformation (𝔸) effectively eliminating one of the limiting steps leading to 𝔸L complex formation. c, binding of arrestins lacking their secondary binding sites. Arrestins lacking the C-terminal half of the molecule such as betaarr(1-217), betaarr(1-206), and arr3(1-183) also lack the secondary binding sites mobilized during the A 𝔸 transition. d, arrestin binding to receptor in the presence of antagonist(N). The same model for full-length arrestin binding in the presence of an antagonist(N), which does not induce receptor activation. In this case the 𝔸N complex can be formed only via 𝔸RN due to the ability of non-visual arrestins to assume a high-affinity binding conformation (𝔸) while interacting with non-activated receptors (R). The resulting percentage of receptors in an 𝔸N complex (B(max)) would be lower than an 𝔸L complex. See text for details.



In the presence of agonist there is an equilibrium between free receptor (R) (here and below the receptor is presumed to be phosphorylated since this is a prerequisite for high affinity arrestin binding), free agonist (L), receptor-agonist complex (RL), and active receptor-agonist complex (ℝL). While a high concentration of agonist converts virtually all receptors into RL, the number of ℝL complexes depends on the equilibrium between RL and ℝL. Conceivably, arrestin (A) can bind to both forms of the receptor forming either an ARL or AℝL complex. Bound arrestin then undergoes a conformational transition from a low to a high affinity state (A 𝔸) giving rise to 𝔸RL and 𝔸L complexes, which are again at equilibrium. The linear Scatchard plots obtained for the binding of all arrestins to the beta(2)AR and m2 mAChR indicate that one of these 4 arrestin-receptor-agonist complexes has a significantly higher affinity than the others. Therefore it is the formation of this particular complex, which is most likely 𝔸L, that we are measuring. By increasing the arrestin concentration we can drive all of the receptor-agonist complexes to bind arrestin, however, the actual number of 𝔸L complexes (the experimental B(max)) still depends on the equilibrium between ARL, AℝL, 𝔸RL, and 𝔸L (Fig. 8). The RL to ℝL equilibrium depends on the intrinsic characteristics of agonist binding to a particular receptor, while the ARL to AℝL and 𝔸RL to 𝔸L equilibrium may be shifted compared to RL to ℝL. The magnitude of this shift is arrestin-dependent and gives rise to the observed differences in B(max) for different wild type and mutant arrestins.

The deletion of the C terminus of arrestin promotes its transition into the high-affinity binding conformation (𝔸)(25, 26) , thus eliminating one of the limiting steps in the sequence of events leading to 𝔸L and increasing the observed B(max) (Table 1). While deletion of the entire C-terminal half of the arrestin molecule produces an arrestin that cannot form a high affinity binding conformational state(27) , it still appears capable of forming an AℝL complex that is stable enough for detection (25-27, Fig. 3and Fig. 6). This truncation also shortens the chain of events leading to the high-affinity complex and therefore also increases the B(max) (Table 1). While antagonists(N) do not induce receptor activation, arrestins are still capable of assuming a high-affinity binding conformation (𝔸RN), albeit with a lower probability, and consequently induce a conformational change in the receptor to form 𝔸N. Thus, while a lower percentage of the receptors will be present in the 𝔸N complex at equilibrium (compared to 𝔸L), the complex itself is essentially the same as in the presence of agonist (at least as far as arrestin-receptor interaction is concerned). This would translate into no change in the K(d) but a lower B(max) as was experimentally observed (Table 1). Truncated arrestins would be expected to shift the equilibrium toward 𝔸N in the presence of an antagonist more effectively than the full-length arrestins, again in agreement with the experimental data (Fig. 3).

The kinetic model of arrestin-receptor interaction suggests that analogous to visual arrestin, non-visual arrestins are selective toward the activated/phosphorylated form of receptors. The major difference between the visual and non-visual arrestins appears to be in their ability to induce an activation-like conformational change in the phosphorylated inactive receptor. Thus, betaarr and arr3 appear to be able to promote formation of an 𝔸 (L) complex even in the absence of ligand. The data with truncated arrestins ( Fig. 3and Fig. 6) suggest that both rhodopsin and the beta(2)AR are rather rigid while the m2 mAChR appears to be more flexible, since even the shortest arrestin species are able to induce an active conformation in a large proportion of the P-mAChR (Table 1). Arr3S appears to possess the best ability to shift the equilibrium toward a high-affinity complex since it has the highest B(max) with both P-beta(2)AR and P-mAChR, and is second only to visual arrestin in binding to P-Rh (Fig. 1, Table 1).

The model of arrestin-receptor interaction is analogous to the current model of G protein-receptor interaction in several important aspects (48, 49, 50) . Both arrestins and G proteins preferentially bind to agonist-activated receptors, although both are capable of interacting with the receptor in its basal state(49, 50) . In the presence of a saturating concentration of agonist, only a certain percentage of the receptors are able to form a high-affinity complex with either an arrestin (Table 1) or G protein(48, 49) . Since the same functional state of the receptor is capable of interacting with both G protein and arrestin, this suggests that direct competition between the two is the most plausible mechanism of arrestin action. Since receptor associated with G protein has a higher affinity for agonists than the receptor alone(48, 49) , the 𝔸L complex might also be expected to have a higher affinity for agonists. In the visual system, where all-trans-retinal bound to metarhodopsin II is analogous to the agonist-occupied activated form of the receptor, transducin interaction stabilizes the metarhodopsin II state. This is fully analogous to the stabilization of the receptor-agonist complex by other G proteins. Interestingly, visual arrestin was also shown to stabilize the metarhodopsin II state of phosphorylated rhodopsin(47) . The affinity of P-beta(2)AR and P-mAChR for agonists in the presence and absence of non-visual arrestins remains to be determined.

The other implication of these data and the model of arrestin-phosphoreceptor interaction is a very slow dissociation of arrestin. While dissociation of visual arrestin from P-Rh is facilitated by the decay of phosphometarhodopsin II to phosphoopsin (26) , dissociation of the agonist from 𝔸L only modestly facilitates arrestin dissociation (Table 1). Thus, either non-visual arrestin dissociation is indeed a relatively slow process in the cell or it might be facilitated by either receptor sequestration or modification (e.g. phosphorylation) of the bound arrestin. In any event the arrestins are likely to stay bound to the phosphoreceptor at least until agonist dissociation, thus ensuring high fidelity of the signal termination.

Conclusions

This series of studies utilizing various truncated and chimeric arrestins demonstrates that the relatively high degree of amino acid homology among the arrestins translates into a similar localization of their functional domains. The mechanisms ensuring the selectivity of visual arrestin (26, 27) appear to be operational in the case of non-visual arrestins as well. Two centrally localized domains are primarily involved in receptor interaction and therefore are major determinants of arrestin specificity. Both domains seem to play comparable roles in arrestin interaction with receptors. These data corroborate the hypothesis (26, 27) that there is an interaction between N- and C-terminal regulatory regions in arrestins. The significant interchangeability of the arr and betaarr regulatory regions suggests that future mutagenesis studies should target conserved residues within these domains.


FOOTNOTES

*
This research was supported in part by Grants GM44944 (to J. L. B.), GM47417 (to J. L. B.), GM47120 (to J. J. O.), and HL31601 (to M. M. H.) from the National Institutes of Health, a grant-in-aid from the American Heart Association (to M. M. H.), and a grant from the National Kidney Foundation of Wisconsin (to J. J. O.). 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.

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

(^1)
The abbreviations used are: beta(2)AR, beta(2)-adrenergic receptor; arrestin, a generic term that includes all arrestins; arr, visual arrestin; betaarr, beta-arrestin; arr3, arrestin 3; betaARK, beta-adrenergic receptor kinase; G protein, guanine nucleotide binding protein; m2 mAChR, m2 muscarinic cholinergic receptor; mAChR, carbachol-activated mAChR; P-mAChR, phosphorylated mAChR; P-mAChR, phosphorylated carbachol-activated mAChR; beta(2)AR, isoproterenol-activated beta(2)AR; P-beta(2)AR, phosphorylated beta(2)AR; P-beta(2)AR, isoproterenol-activated phosphorylated beta(2)AR; Rh, dark rhodopsin; Rh, light-activated rhodopsin; P-Rh, phosphorylated rhodopsin; P-Rh, phosphorylated light-activated rhodopsin; S, short; L, long.

(^2)
We use the term activated to refer to the agonist-occupied receptor while non-activated refers to the antagonist-occupied receptor. However, this does not necessarily imply that all of the receptors are in a completely activated or non-activated conformation. We use the term specificity to refer to the ability of a given arrestin to bind to different receptors, while the term selectivity refers to the ability of an arrestin to discriminate among the different functional forms of a given receptor.


ACKNOWLEDGEMENTS

We are grateful to Dr. T. Shinohara for providing the visual arrestin cDNA and R. Bodine and X. Cheng for technical assistance.


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