Volume 295, number 1,2,3, 195-199 FEBS 10542 0 1991 Fcdcration of Europcdn Biochemical Sociclics 00145793i9l/S3.50

December 1991

Binding of inositol phosphates

to arrestin

Krzysztof Palczewski’, Alexander Pulvermiiller”, Janina Buczylko’*‘, Caroline Gutmann’ and Klaus Peter Hofmann2 ‘R. S. Dow

and

Neurologicul ‘InsWr~ fiir

Sciences Ricp!:ysik

ltls~i~ute of Good Satnariran Hospital md SMrietrbiologic der Uuiversiriil.

Received 25 October

and Medical Cenrer. Porrland, Alberrstr. 23, D-7800 Freiburg,

OR 97209. Germany

USA

1991

Arrcstin binds to phosphorylavzd rhodopsin in its light-activated form (melarhodopsin II), blocking thcrcby its interaction with the G-protein, mmsducin. In this study, WCshow thal highly phosphorylatcd forms of inositol compete against the arrcslin-rhodopsin inlcraction. Competition curves and direct binding assays with free arrcslin consislcntly yield affinities in the micromolar range; for example, inositol 1.3.4.5-telrakisphospharc (InP,) and inosilol hcxakisphosphalc (InP, bind to arrcstin with dissociation constants of 12pM and 5 PM. respectively. Only a small control amount or inosilol phosphates is bound. when arrcstin interacts wilh phosphorylatcd rhodopsin.This argues for a release of bound inositol phosphates by imernclion with rhodopsin. Transducin, rhodopsin kinasc, or cyclic GMP phosphodicsterasc arc noi affected by inositol phosphates. These observations open a new way 10 purify arrcsiin and to inhibh its imcraction with rhodopsin. Their physiological significance dcscrvcs further inscsligalion. Signal rransduclion; G-protein;

Rhodopsin; Arrcslin; lnositol phosphate

I. INTRODUCTION The rhodopsin/transducin/phosphodiesterase cascade of the rod is a weli-studied cxamp!e of a receptor/ G-protein/effecter system. Absorption of light leads to the formation of metarhodopsin II (MN) that activates transducin by catalysing GDP/GTP exchange [1,2]. Transducin activates a cyclic GMP phosphodiestcrase and lowers cytoplasmic cyclic GMP. Since cyclic GMP keeps Na+/Ca”- channels open for the influx of these ions, the channels close, the cell hyperpolarizes, and the Ca” level drops (reviewed in ref. 3). Quenching of the light signal occurs at least at three points. When metarhodopsin II is phosphorylated by a specific kinase (reviewed in ref. 4), it binds a regulatory protein, arrcstin, which blocks further activation of the * Prcxwr uddrcss: Technical University of WrochIw, Division of Biochemistry, 50-370 Wroclaw. Poland, Ahhreviofions: InP,. D-myo-inositol i .4,5-triphosphau; InP,, D-myoinoshol I .3,4,5+zlrakisphosphatc; InPs, D-myo-inoshol I .3.4.5,6-pcnlakisphosphatc; InP,, inosilol hcxakisphosphalc; SDS-PAGE, sodium dodccyl sulfate polyacrylamidc gel clcnrophorcsis. Corrcspotrdeme m/dre.m: K. Palczcwski, R.S. Dow Neurological Science lnstitulc of Good Samarilan Hosphal and Medical Center, I 120 N.W. 20th Avenue. Portland, OR 97209. Fax: (1)(503) 229-7229. and K. P. Hofmann, Inslitut filr Biophysik and S\rahlcnbiologic der Univcrsitat , AlbcrLslr. 23, D-7800 Frciburg. Germany, Fax: (49) (761) 203-2555.

G protein [5]. A protein with analogous function, ,?arrestin, was recently described for the/?-adrenergic system [6]. The G protein is de-activated by hydrolysis of bound GTP to GDP. In a third quenching pathway, the lowering of calcium stimulates guanylate cyciase via a calcium-binding prctein and restores the cyclic GMP level (reviewed in ref. [7]). Recently, we have found that heparin is a potent inhibitor of the arrestin-rhodopsin interaction and that heparin and phosphorylated rhodopsin induce the same distinctive pattern in the limited proteolysis of arrestin [S]. We tested other potential Iigands of arrestin, including sulfonated or phosphorylated analogues of carbohydrates, nucleotides, and sulfonated or phosphorylated inositol derivatives. A digestion pattern similar to L.,:-_.l L__..l”.,A ‘. ncparur’ Was oura~r~cu Oiiiji fei @~OSPIIV~J~O~~~ ail;!& gues of inositol. This finding, the role of inositol phosphates in invertebrate phototransduction [9] and evidence of the complete pathway in rods [lo] motivated the present study. WC present evidence that arrestin is a soluble receptor for inositol phosphates. Binding of the phosphates and of phosphorylated metarhodopsin II compete against one another. 2. MATERIALS 2. I. Rwgctm

AND METHODS

Inosilol phosphalcs were in D-myo conformalion. ‘H(N)]I,3.4,5-Tctrakisphosphatc (I 7 CiImmol) and

[inosilol-l[inositol-2-

195

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Volume 295, number I,?,3

December TABLE Binding

i protein

lnositol

I

of inoshol derivatives

to arrcstin

dcriva[ivc ZG

f protein + InP 6

con!rol

I. 2. 3. 4. 5. 6. 7. 8.

I99 I

InP, InD, InP,? InP, (I -3.4.5 isomer) InP, (I 2.5.6 isomer) InP, (I.4.5,6 isomer) IllP, InP,

IO2 000 3 360 I70 38 134 73 20 IO

$l, 3 400 I I20 57 I4 45 25 :

IGu were dctcrmincd employing die ‘cxlra Mela II’ asray(Fig. I; scclion 2. f& values wcrc rslimatcd assuming the following scheme or reactions: Ml -- Mll + A --- MII + InP,,

A

AlnP,,

0 I 8

, 10

1 PM

100

, 1000

inositol phosphate

FigI. Inhibhion of the intcracrion between melarhodopsin II (MII) and arrestin by InP, and other phosphorylated dcrivativcs of inosilol. A. Effcc~ of InP, on 111~ stabili!y of metarhodopsin II (MII! in complex with arrcslin or rransducin. The records arc the increase in absorption al 350 nm (A,,,;,, of MI1 pholoproduct). Undislurbcd inlcraclion of hill with arrcslin or transducin leads 10 enhanced MII formalion (extra MB). For arresba the cnhancemcnt is inhibited by IPr, (lcli, middle), and only lhe control amount of MII is formed. B. Lcvcl of extra Ml1 (Ml1 with arrcstin and inositol analogues, minus thecontrol MII) as a function of ligimd concentration. The symbols arc: inoshol I-phosphauc (InP,). closed square; inositol 1.4.bisphosphatc (InP,), open lrianglc; InP,, asterisk; InP,. open circle: I#,. closed triangle; and InP,. open square. The lines through the data arc computer fus whh hyperbolic functions (cxponcms between 0.8 and I. I ).

LH(N)]hcxakisphospha~c(12 Ci!mmol) were obtained from NEN (Due Porn). lnositol I-phospha~. inoshol I .I-bisphosphatc, InP1, InP,. inoshol 1.2,5,6-b~rakisphosphatc, inoshol 1.4.5,6-tctrukisphosphatc, and InP, were purchased from Bochringcr Mannhcim. InP,, and omcr chemicals were obtained from Sigma Chemical Cb..

Phosphorylatcd rhodopsin was prepared from urea-washed ROS as described previously [I I]. Arrestin was prepared from bovine rclinas using a modification of rhc previously described method [ 121. 40 bovine retinas were glass-glass homogenized whh ice-cold IO mXi HEPES buffer, pH 7.5. conmining I mM bcnxamidinc and IS pg/ml Icupcptin. Soluble proteins wcrc scparaicd by ccmrifuga\ion for 25 min at 43 700 x 6. The cxtmct was applied lo a I .6 x I4 cm column of DEAE-cellulose (Whatmann DE52) which had been cquilibraled whh IO mM HEPES, pH 7.5. The column wus washed whh HEPES buffer conmining 15 m>l NaCI, at 111~flow rate 30 ml/h umil absorb. NW el 280 nm dropped below 0. I and a major hemoglobin band Wils

196

where A is arrestin. MI and MII is mclarhodopsin I and II. rcspeclively. The equalion for IC,,, derived from the schcmc is ICI:, = ([K,,,l whcrc R IS rhodopsm; K,,, W,,, + Ilx[A,,,,,,,-R,,,,:,I)!K,,,1-I I XL =[Mll]~[Ml];n’,,~ =[Mll-A]@‘lll]x[A]: ~l,a=[A-lnP,,]![A]x[lnP,,],,,,:,. Abbreviations: D-myo-inoshol I-phosphate (InP,); D.niyo-inositol I .4-bisphosphalc ( InP1).

clutcd from the column. Arrcstin Was clutcd by a NaC! gradient (240 ml 10~1, from 0 to I50 :nM NaCl cornaining 0.5 mM benzamidinc). Arrestin was elutcd al aboul 75 mM NaCl as dc~crmincd by SDSPAGE. Fractions containing arrcsnn wcrc applied IO a I x 7 cm column of Hcparin-Sepharosc which had been cquilibra!cd with IO mM HEPES and IO0 mM NaCI, pH 7.5. The column was vvashcd with HEPES buffer containing I50 mM NaCI. at a flow ralc or I2 ml/h umil mc absorbance at 280 nm dropped below 0.01. Arrcstin was clumd by a InP, gradient (70 ml total from 0 10 8 mM InP,, in 111~washing buffer). Arrcstin was elmed at 2.5pM of InP,# and with a purhy higher than 90%. In order IO further eliminate conlaminating promins (less Ihan 5%) and InP,. arrcstin was briefly dialyzed agrins\ IO mM HEPES buff&-. pH 7.5, conmining I00 mM NaCI. Next. arrestin was applied IO a I x 3.5 cm column of Hcparin-Scpharosc, equilibrated with IO mM HEPES and 100 mM NaCI, pH 7.5. The column wus washed whh HEPES buffer comaining 200 mM NaCl (8 ml), at a flow ram of I2 ml/h. Arrcslin was clutcd by 400 mM NaCI in the HEPES bull&. Aboul 4 mg of homogeneous arrcstin wus obtained. All procedures wcrc pcrformcd at 5°C. Transducin was isolalcd from rod ouwc segments according LO Kiihn [l3].

Esrnr-.~I//-intcrac~ion with transducin [2] or arrcslin [X,14] slabilizcs the 380 nm pholoproduct mc\arhodopsin II (MII) aI the cxpensc ofits uuuomcric forms Ml and MIII, which absorb in 1hc460-480 nm spectral rang. The flash phololysis apparatus mcasurcs 111~ difference bctwccn the absorbance changes ai 380 nm (A,,,,, of MII) and 417 nm (isosbcsdc point of the Ml/MI1 transhion) [14]. Er/rri/ihrirrm dirr/J.si.r WIS pcrformcd using IO mM HEPES buffer, pH 7.5. containing 100 mM NaCI, essentially as described clscwhcrc [l5]. The dialysis cells conmin Spcctrapor I mcmbrancs (molecular weight cutoff IO 001)). I30 ~1 of arrcsiin solutions (0. l-l mg/ml) wcrc placed in one compartment and I30 ~1 of 2-705 p>l [inoshol-l‘H(N)]l.3,4.5-lclr;~kisphosphatc or 444 PM [inoshol-2-~H(N)]hcxa-

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Volume 295, number I.?,3

December 199:

kisphosphatc in the other. Dialysis wasconductcd for 50 hat S°C with continuous shaking. Controls showed thal the system rcachcd cquilibrium. lnositol derivative concentrations were dctcrmincd from spccilic radioactivity. Geljilrrcrriorr-Cicl filtration was carried out accordine to Ackcrs [I61 on a Supcrosc 6 column (Pharmacia) equilibrated iith IO mM HEPES, pH 7.5. 100 mhl NaCI. in the presence of 0.2pM [inositolI -‘H(N)]l,3.4.5-tctrakisphosphatc or 0.2 phi [inositol-2-‘H(N)]hcxakisphospatc. 0.24 mg of arrcstin was loaded per run. The protein profile was dctcrmincd by the absorption at 280 nm; inositol con-. ccntration was dctcrmincd by radioactive counting.

0.0

0.4

0.0 [III~

1.2

1.6

2.0

3. RESULTS

IJtArrl, clopsirl

15

20

25

FRACTION

30

35

40

NUMBER

Fig.‘. Binding of InP,, lo arrcstin. A. Scatchard plot of equilibrium dialysis [12.15].[inositol- ‘-‘H(N)]licxakispllosphatc and arrcstin wcrc placed in IWO dilfcrcnt compartments 01’ a dialysis cell and dialyscd IO equilibrium as dcscribcd in section 2. B. Gel filtration according to rcr. [16]. [inositol-2-‘H(N)]hcxakisphosphateand arrcstin wrc loaded on a Supcrosc 6 column as dcscribcd in scclion 2. Protein profile from 280 nm absorption (open circle), inositol from radioactive counting (closed circle).

hrerucriow

Fig. 1 shows original records (A) and competition curves (B) for the ‘extra MII’ assay. Under control conditions, a flash of light forms a small amount of metarhodopsin II (MI]). Additional MI1 arises from the interaction of arrestin or transducin with MII. The amount of extra MII (MI1 level with arresrin minus control) is a stoichiometric measure of the complexes formed [ 141. As Fig. 1A shows, only formation of the complex with arrestin, but not with transducin, is blocked by IP,. All inositol phosphates gradually suppress extra-MI1 with increasing concentration (Fig. IB), with a potency that increases roughly with the degree of phosphorylation. Among the three tested isomers of inositol tetrakisphosphate the isomer (1,3,4.5) is most effective (data not shown), tri-. di- and mono-phosphorylated derivatives of inositol are much less effective (Table I). The data for all inositol phosphates fit well to linear hyperbolic competition curves (lines through the data). The MI1 stabilization data are consistent with the interpretation that one molecule of inositol phosphate

0.72

$ o*54 T

3

0.36

0.54 P _P 3 0.36

0.16

Fig.3. Binding or InP, and InP, to phosphorylatcd mcmbrancs and arrcstin. The expcrimcnts analyst the tract amounts of inositol phosphates that bind IO phosphorylatcd mcmbrancs when arrcstin is bound IO cxccss phosphorylatcd photoactivatcd rhodopsin. The sample ofprc-phosphorylatcd rhudopsin. iu’rcstin and dilTcrcnt amounls or the inositol phosphalcs was illuminated (light) or incubated in the d;lrk (dark) and ccntrifugcd as dcscribcd in section 2. II is seen ltlal the illuminated sample (containing the rhodopsin arrcslin complex) dots IIOI bind more inositol than the dark control.

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interacts with arrestin and competes with phosphorylated metarhodopsin II for the same binding site. Like transducin, rhodopsin kinase is not inhibited by up to 1 mM InP, (nor by InP,, see ref. Palczewski et al. 1988b); cyclic GMP phosphodiesterase is not inhibited either by 250 PM InP, (data not shown). 3.2. Inosirol phosphates bind to free arres;itl The interaction between inositol phosphates and arrestin can be directly monitored by equilibrium dialysis and gel filtration. Fig. 2A shows a Scatchard plot of equilibrium dialysis data for IP,. One obtains a dissociation constant of Ko = 5.5 PM and a number of binding sites tt = 1.12 For IP,, the values are 12.5 PM and tt = 1.02 (dat not shown). These values fit to the Kn’s from the competition curves in Fig. 1 (see Table I). Elution profiles of gel filtration are shown in Fig. 2B. The positive and negative peaks are expected for binding of InP, and InPs to arrestin. A comparison of the column profiles with the standard proteins bovine serum albumin (67 000 Da) and ovalbumin (43 000) ensured that arrestin dit not aggregate in the presence of the inositols (data not shown). When Heparin-Sepharose is used as an affinity matrix for arrestin, arrestin is the only protein eluted with InP6 (see section 2). This argues for an at least partial overlap of the binding sites at arrestin for heparin and the inositol phosphates. 3.3. Inositol phosphates do nor bind to the rhodopsinarresrin complex

When an excess of pre-phosphorylated metarhodopsin II is formed, more than 90% of the arrestin in the sample is bound at ,uM concentration. As shown in Fig. 3, the amount of InP, and InP, recovered with the membranes is as small as in the dark control. Together with the data of the preceding section, this shows that bound inositol phosphates are released when arrestin binds to rhodopsin. On the other hand, the data show a small, light-independent capacity of disc membranes to bind inositol derivatives.

4. DISCUSSION Direct binding assays and inhibition of complex formation between arrestin and phosphorylated active metarhodopsin II (MII) have shown that arrestin binds inositol phosphates with micromolar affinity, The binding is specific, and is not even seen with rhodopsin kinase which shares with arrestin a high affinity for heparin. The binding site of inositol phcsphates appears to overlap the binding domain of phosphorylated rhodopsin. Identification of inositol phosphates as specific ligands of arrestin, which decouple it from phosphorylated rhodopsin, provides a simple tool for studying arrestin function in the rod cell and simplifies its isola198

December 1991

tion. /l-Arrestin [6] can be expected to be inhibited as well. Further conclusions will depend on determination of the amount of higher inositol phosphates in the rod cell. InP,, InPS and InP, are present in ,uM concentration in various mammalian cells, including neuronal cell lines [17,18]. A receptor for IP6 was recently identified in rat brain [19]. Not only IPs but also higher phosphates are involved in the control of calcium [20]. Arrestin is abundant (100-300 ,uM) in rods [21] and the binding of the inositol phosphates (Ko in the 10 ,uM range, Table I) is tight enough to complex the accessible cellular inositol with arrestin. Since the complex of arrestin with phosphorylated metarhodopsin II is much tighter (K,, in the 50 nM range [14]), a change in the InsP concentration should not influence the rhodopsinarrestin complex. However, bleaching of a large fraction of the rhodopsin (3 mM in the rod outer segment) could considerably affect the inositol pool. This is the condition of bleaching adaptation (see for example ref. [22]), a regulation of rod sensitivity that appears to bypass the G-protein [23,24]. Whether a regulatory pathway exits, which originates from the light-dependent release of arrestin-bound inositol phosphates, remains to be investigated. Acktro~Fledgenre,lrs: We thank Dr. Paul Hargrave for the valuable suggestions and comments made during the course of the work and Dr. Michael Kaplan for critical review of the manuscript. This research was supported by U.S.P.H.S. Grant EY 08061 and grant from the Oregon Lions Sight and Hearing Foundation (to K.P.), and by grants from the Dcutsche Forschungsgemcinschaft (to K.P.H; SFB 6L7 and SFB 325).

REFERENCES [I] Franke, R.R. Konig, B., Sakmar, T.P., Khorana, H.G. and Hofmann, K.P. (1990) Science 250, 123-125. [2] Hofmann. K.P. (1986) Photobiochem. Photobiophys. 13, 309327. [3] Chabre, M. and Deterre, P. (1989) Eur. J. Biochcm. 179,255-266. [4] Palczewski, K., and Benovic, J.L. (1991) Trends Biochem. Sci., in press. [S] Wilden, U., Hall, SW. and Kiihn, H. (1986). Proc. Natl. Acad. Sci. USA 83, ! :?4-! !X!. [6] Lohse, IvI.J.. Benovic, J.L., Codina, J., Caron, M.G. and Lefkowitz, R.J. (1990) Science 248, 1547-1550. [7] Stryer, L. (1991) J. Biol. Chcm. 266. 1071I-10714. [8] Palczewski, K., Pulvcmmtlllcr, A., Buczylko. J. and Hofmann, K.P. (1991) J. Biol. Chem. 266, 18649-18654. [9] Frank. T.M. and Fcin, A. (1991). J. Cicn. Physiol. 97, 697-723. [IO] Gchm, R.D. and McConnell, D.G. (1990) Biochemistry 29,54475452. [I I] Palczcwski, K.. Hargravc, P.A., McDowell. J.H. and lngcbritsen, T.S. (1989) Biochemistry 28, 415-419. [I21 Palczcwski, K. and Hargravc, P.A. (1991) J. Biol. Chcm. 266, 4201-4206. [I31 Killin, H. (1981) Ncurochcm. Int. I, 269-285. [I41 Schleichcr. A.. Kuhn. I-i. and Hofmann, K.P. (1989) Biochcmistry 28, 1770-1775. [15] Ileyduk, T. and Lee. J.C. (1989). Biochemistry 28, 6914-6924. [I61 Ackers, C.K. (1973) Methods Enzymol. 27, 441-455.

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[I71 Jackson, T.R., Hallam, T.J.. Dowries, C.P.. and Hanlcy, M.R. (1987) EMBO J. 6. 49-54. [IS] Szwergold, B.S., Graham, R.A. and Brown, T.R. (1987) Biothem. Biophys. Rcs. Commun. 149,874-881. [I91 Theiben, A.B.. Estevez, V.A.. Ferris, CD., Danoff, SK., Barrow, R.K., Prcstwich, G.D. and Snyder, S.H. (1991) Proc. Natl. Acad. Sci. USA 88, 3165-3169. 1201 Irvine. R.F. (1990) FEW Lctt. 263, 5-9.

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[Zl] Ktihn. 1-i. (1984) Progr. Retinal Res. 3. 1123-l 156. 1221 Ripps. H. and Peppcrberg, D.R. (1987) Neurosci. Res. 6,87_lOii. [23] Kahlcrt, M., Pepperberg, D.R. and Hofmann, K.P. Nature. 345 537-539. 1241 Hol’mann, K.P. and Kahlcrt, M. (1991) in: Signal Transduction in Photoreceptor Cells (P.A. Hargravc, K.P. Hofmann and U.B. Kaupp, cds.), Springer, Berlin, in press.

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