Identification of a binding site on retinal transducin a for thephosphodiesterase inhibitory y subunitJess CUNNICK, Christian TWAMLEY, Igor UDOVICHENKO, Karen GONZALEZ and Dolores J. TAKEMOTO*Department of Biochemistry, Kansas State University, Manhattan, KS 66506, U.S.A.

Transducin alpha (Ta) activates retinal rod cyclic GMP phospho- We have identified a region, residues 250-275 of Ta, which showsdiesterase (PDE) by interacting with and removing the inhibitory a high affinity for PDEy and for the PDEy (24-45) bindingPDEy subunit. A Ta-PDEy complex can be isolated in vitro, and peptide. The peptide did not bind to the C-terminal residuesour previous work [Morrison, Rider and Takemoto (1987) FEBS 50-87 of PDEy. Likewise, a region of Ta, 1-25 did not exhibitLett. 222, 266-270; Morrison, Cunnick, Oppert and Takemoto high-affinity binding to PDEy or to the 24-45 PDEy peptide.(1989) J. Biol. Chem. 264, 11671-11681] has identified a region Specific binding of the 250-275 peptide to PDEy was confirmedof PDEy, residues 24-45, that binds to Ta. The C-terminal by its ability to compete with Ta binding to PDEy, although aregion of PDEy is the site that interacts with PDEa//B and higher concentration was required (10 x). The Ta-(250-275)inhibits catalytic function. The site on Ta that binds to the PDEy peptide activated a fully inhibited PDE a/3y2 complex in a dose-24-45 region has not been identified. Synthetic peptides (15- dependent manner. These results suggest that a region on Ta thatmers) which span the bovine Ta sequence were tested for binding recognizes the PDEy-binding site is found within residuesto purified recombinant PDEy using a solid-phase assay. The 250-275 of Ta.peptides were also tested for ability to activate a PDE complex.

INTRODUCTION [23]. The phosphorylated PDEy has increased ability to inhibitthe PDE activity.

In photoreceptor cells, visual transduction is realized through the It has been postulated that, like the PDE catalytic subunitsphotoexcitation of rhodopsin and the resultant exchange of [24,25], the Ta subunits may have two sites for interaction withGDP for GTP on the retinal G-protein, transducin (T) [1-7]. The PDEy [26]. One region which interacts with the C-terminus ofTa-GTP then binds to the inhibitory subunit of the phospho- the PDEy is included in residues 293-314 of Ta [26]. The regiondiesterase (PDE), forming a soluble Ta-PDEy complex [8-101 or of interaction of Ta that recognizes the basic PDEy region ofa Ta-PDEOcqy2 complex [11,12]. The nature of this complex residues 24-45 has not been identified. By use of syntheticdepends on the occupancy of the non-catalytic cyclic GMP peptides and a direct binding assay, this region has been identified(cGMP)-binding sites found on the PDE a and , catalytic as including residues 250-275 of Ta.subunits [13]. If these sites are occupied, then the Ta forms acomplex with the PDEa/1y2; if the sites are empty, the Ta EXPERIMENTALremoves the PDEy from the PDEa/x [13]. On activation of thePDE, the resulting lowering ofcGMP levels results in changes in Materialsthe cGMP-gated channel protein. This results in membrane Bovine eyes were obtained immediately after slaughter fromhyperpolarization [14,15]. Iowa Beef Packers (Emporia, KS, U.S.A.). [8-3H]cGMP

Physiologically, this means that at high cGMP levels, in the (15 Ci/mmol) was obtained from ICN Radiochemicals anddark, the non-catalytic sites would be occupied and PDEy would purified further by anion-exchange chromatography. Carrier-be bound tighter to the PDEa,/, resulting in low basal activity, free 1251 was purchased from Amersham International. AminoWhen cGMP is low, in the light, the PDEy would bind less acids (t-butoxycarbonyl and resin forms) were obtained fromtightly. The GTPase activity that terminates the PDE activity is Sigma. Aquacide III was purchased from Calbiochem, h.p.l.c.slower when these sites are occupied and the Ta is in a complex columns from P. J. Cobert (St. Louis, MO, U.S.A.), nitrocel-with the PDEy [16,17]. lulose from Schleicher and Schuell, X-ray film from DuPont, andThe PDEy subunit plays a pivotal role in the visual trans- developing solutions from Kodak. All other buffers and reagents

duction cascade as it must have binding sites for both PDE a and were from Sigma. Reagents used for peptide synthesis were,/ and for the Ta. The sites have been identified, and mutants h.p.l.c. grade from Fisher or Sigma, or Septuenal grade fromhave been tested to verify these functional residues [18-22]. The Pierce.basic region of PDEy encompassing residues 24-45 contains abinding site for Ta and for the PDEa/3, whereas the inhibitorysite and a lower-affinity site for Ta can be found within the C- PDE purficationterminus. Recently, a site for phosphorylation by rod outer ROS were prepared by the method of Papermaster and Dreyersegment (ROS) protein kinase C has been identified on Thr-35 [27]. Purified ROS discs were washed with buffer containing

Abbreviations used: ROS, rod outer segments; PDE, cyclic GMP phosphodiesterase from rod outer segments; Tax,y, subunits of the rod outersegment guanine nucleotide-binding protein, transducin; PDEa/3y, subunits of the phosphodiesterase; GTP[S], guanosine 5'-[y-thio]triphosphate;cGMP, cyclic GMP; PMSF, phenylmethanesulphonyl fluoride; DTT, dithiothreitol.

* To whom correspondence should, be addressed.

Biochem. J. (1 994) 297, 87-91 (Printed in Great Brftain) 87

88 J. Cunnick and others

10 mM Tris/HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1.0 mMdithioerythritol and 0.2 mM phenylmethanesulphonyl fluoride(PMSF). Soluble PDE (non-activated) was eluted from thewashed membranes with 10 mM Tris/HCl, pH 7.4, containing1.0 mM dithiothreitol (DTT), 0.2 mM PMSF and 2,uM leu-peptin. Soluble PDE was dialysed against h.p.l.c. buffer (20 mMsodium phosphate, pH 6.8, 100 mM Na2SO4, 1 mM DTT) and

concentrated against Aquacide III or by Amicon ultrafiltration.PDE was further purified by h.p.l.c. on a TSK G3000SW columnusing the same h.p.l.c. buffer. Fractions were monitored byabsorbance at 280 nm and PDE was identified by SDS/PAGE[28] and Western blotting [10] using peptide antisera to bothcatalytic and inhibitory subunits of ROS PDE.

PDEy purificationPDEy was expressed in Escherichia coli and purified as described[20]. The PDEy was utilized as a fusion protein with 38 residuesof AcIl and the clotting-proteinase (factor Xa) cleavage site andthe PDEy sequence (87 residues) [20]. PDEy was purified byreversed-phase chromatography on a h.p.l.c. Vydak C-4 columnusing a 10-900% acetonitrile gradient in 0.1% trifluoroaceticacid.

Ta-GTP[S] purfflcationPDE-depleted ROS membranes were then used as a source of Taby elution of these proteins with 10 mM Tris/HCl, pH 7.4, buffercontaining 1 mM DTT, 100 ,tM GTP[S], 0.2 mM PMSF and2 ,uM leupeptin. The crude Ta-GTP[S] was concentrated againstAquacid III or by Amicon ultrafiltration and further purified byh.p.l.c. on a TSK G2000 SW column using a running buffercontaining 20 mM sodium phosphate (pH 6.8), 50 mM Na2SO4,10 mM MgSO4 and 1 mM dithioerythritol. T,/y was purifiedunder the same conditions. Purity of all proteins was assessed onCoomassie-Blue-stained SDS/polyacrylamide gels and on

Western blots.

Isolation of the Tx 33 kDa fragmentPurified Ta-GTP[S] was incubated for 45 min on ice with 16 ,ug

of Ta/unit of insoluble trypsin (Tos-Phe-CH2Cl 'TPCK '-treatedtrypsin/acrylamide; Sigma) in 50 mM Tris/HCl, pH 7.2, 5 mMMgCl2. Insoluble Tos-Phe-CH2CI-treated trypsin was removedby centrifugation. After proteolysis, the amount of 33 kDacompared with whole Ta was assessed by gel scanning andscanning of autoradiograms using antiserum to the 15 C-terminalresidues of Ta (aTcC), and to residues no. 52-66 of Ta [aTa-(52-66)]. The preparation was usually less than 50% Ta. Toremove Ta, the 33 kDa piece was separated by h.p.l.c. gelfiltration, resulting in pure 33 kDa (no detectable Ta). The33 kDa piece still contained GTP[S] as assessed by the use of[35S]GTP[S]. The fragment still activates PDE [29], but does notbind to rhodopsin or to Tfly [30-33].

POE activity assay

PDE activity was determined using the assay of Thompson andAppleman [34]. The final reaction mixture was 50 mM Tris/HCI, pH 7.4, 5 mM MgCl2, 40,uM cGMP and [3H]cGMP(40000 c.p.m./tube, specific radioactivity 15 Ci/mmol) in a finalvolume of 400 ,ul. Reaction mixtures were incubated at 30 0C.Purified PDEy and/or peptides were added as indicated.

In some cases, PDE was first activated with trypsin for 1 minon ice using 20 ,ug of purified PDE and 40 ,tg of trypsin in 400 ,ul

final volume in buffer containing 50 mM Tris/HCl (pH 7.4) and20 mM MgCl2. The reaction was stopped by the addition of2 x excess trypsin inhibitor. The trypsin-treated PDE was thenrepurified on an h.p.l.c. TSK G3000 SW column as describedabove to remove PDEy fragments. After h.p.l.c. the PDE wasassayed by radioimmunoassay to determine if all PDEy had beenremoved using antisera to residues 1-49 [aPy-(1-49)], 50-62[aPy-(50-62)] and 63-87 (aPyC) of bovine PDEy [35]. PDEwhich had all regions of PDEy removed was used in bindingassays.

Peptide synthesis and antiserum productionPeptides corresponding to indicated bovine amino acid sequenceswere synthesized by the method of Merrifield [36] as modified byGorman [37]. Cleavage of the peptide from the resin andprotecting groups was accomplished with anhydrous HF [38].Peptides were purified and quantified using reversed-phase h.p.l.c.[10]. Sequences were verified using amino acid analyses [39] andmass spectrometry.

Peptide antisera were produced by cross-linking peptides tokeyhole limpet haemocyanin by the procedure ofTakemoto et al.[40].

Protein determinationProtein concentrations were determined by the method ofBradford [41] or by scanning gel densitometry of CoomassieBlue-stained SDS/polyacrylamide gels [28] using BSA standards.Gels were scanned on a Gilford multimedia densitometer usinga Shimadzu integrator.

Radiolmmunoassay/binding assaySolid-phase radioimmunoassay was a modification of the methodof Suter [42]. Polystyrene tubes (12 mm x 75 mm) were incubatedwith 0.20% glutaraldehyde in 0.5 ml of buffer A (0.1 M sodiumphosphate, pH 5.0) and agitated at room temperature for 3 h.After the tubes had been washed three times with buffer A, initialpeptides or proteins were added as described in the text in avolume to 200 ,ul of buffer B (0.1 M sodium phosphate, pH 8.0).Tubes were incubated at 37 °C for 1 h, followed by two washeswith buffer C (0.15 M NaCl, 0.50% Tween 20) and one withbuffer B. Purified proteins or peptides were then added to a finalvolume of 300 ,ul in buffer B and incubated while being agitatedat room temperature. Tubes were then rinsed twice with buffer Band incubated with 0.2% glutaraldehyde in buffer A for 15 minwith agitation at room temperature. This linked the protein orpeptide covalently to the initial binding protein. After twowashes with buffer C and one with buffer D (0.1 M sodiumphosphate, pH 7.4, 0.15 M NaCl, 0.05 % Tween 20), antisera asindicated were added at 1:100 in buffer D and tubes wereincubated with agitation at room temperature overnight. Tubeswere then washed three times with buffer E (10 mM Tris/HCl,pH 8.0, 0.05% Tween 20), and twice with water, and then 0.5 mlof buffer D containing 1251-labelled protein A (106 c.p.m./tube)was added to each tube and the tubes were agitated for 1 h atroom temperature. After two washes with buffer E and two withwater, tubes were counted in a Beckman y counter. The signifi-cance of binding of a secondary polypeptide to a tube-boundpolypeptide was determined by comparison with the binding ofthat secondary polypeptide to a blocked blank tube (P = 0.05).

Western blotProteins were separated by SDS/PAGE [28] and transferred tonitrocellulose using a Genie transfer apparatus (Idea Science,

Binding site on transducin a for phosphodiesterase y 89

Corvallis, OR, U.S.A.). After being blocked in 2% BSA, blotswere incubated with appropriate antisera, followed by washingand reaction with 1251-Protein A.

RESULTSWe have previously reported that the three exons of PDEy thatencode residues 1-49, 50-62 and 63-87 [43] represent distinctfunctional regions of the protein [10]. The region with the most

Table 1 Binding of retinal visual transduction proteins to PDEyBinding assays were performed as described in the text using 2 pM/assay tube of Ta-GTP[S],Ta 33 kDa or PDEy. Antisera were used at 1:100 dilution and were quantified using 1251_Protein A at 106 c.p.m./tube. Polystyrene tubes were saturated at above 1 ,ug/tube. Background(302 + 21 c.p.m.) is without protein or peptides and is subtracted from the samples. Values aremeans+ S.E.M. of three samples. Antiserum aPy(1-49) is used. Reaction of antiserum with2 pM of PDEy, directly bound to the tube, was 19160+ 950 c.p.m. The reaction of aPy(1-49)to PDEy added to a Tween-20-blocked tube without bound protein was 840 + 156 c.p.m. PDEywas added to bound protein.

Radioactivity boundSample (c.p.m.)

Ta-GTP[S] aloneTa-GTP[S] + PDEyTa 33 kDa aloneTa 33 kDa+ PDEy

681 +17210907 + 782

329 + 425235 + 804

Table 2 Binding of PDEy to Ta peptidesBinding assays were as described in Table 1 using Ta-GTP[S] at 2 pM/tube and peptides at30 pM/tube. PDEy was added as the second (binding) protein at 0.22 pM/tube. Antiserum[aPy-(1-49)] was used at 1:100 dilution with 1251-Protein A at 106 c.p.m./tube. PDEy beforeblocking = 4500+190 c.p.m.; PDEy after blocking = 430 +23 c.p.m. Numbers refer toamino acids residues of the bovine Ta sequence [44]. Background (92 + 30 c.p.m.) is withoutprotein or peptide and is subtracted from the samples. Values are means+ S.E.M. of threesamples.

Radioactivity boundPeptide (c.p.m.)

Ta-(1-1 5)Ta-(1 6-30)Ta-(31-45)Ta-(46-60)Ta-(61-75)Ta-(76-90)Ta-(91-105)Ta-(96-1 10)Ta-(1 06-120)Ta-(1 21-1 35)Ta-(136-1 50)Ta-(1 51-165)Ta-(1 66-180)Ta-(1 81-195)Ta-(196-21 0)Ta-(211-225)Ta-(226-240)Ta-(241-255)Ta-(256-270)Ta-(271-285)Ta-(286-300)Ta-(301-31 5)Ta-(316-330)Ta-(331-345)Ta-(336-350)

405 +19231 + 78118 +9131 +22612 +186730 + 8614 +158896 + 186123 +19718 + 30315 + 32348 +13754 + 3778 + 201102 + 24774 + 268610+ 62

1046 + 3172625 + 659767 +16608 + 208715 +146838 + 33654 +184731 + 51

Table 3 Binding of PDEy to To peptidesBinding assays were as described in Table 1. Ta-GTP[S], PDEy and peptides were added asindicated in parentheses. Order of samples indicates order of addition to tube. Antiserum [ccPy-(1-49)] was used at 1 :100 dilution with 1251-Protein A at 106 c.p.m./tube. 'Before' indicatessample added before blocking, i.e. bound to the tube. 'After' indicates sample added afterblocking with Tween 20. Values are means+ S.E.M. of three samples.

Radioactivity boundSample (c.p.m.)

PDEy - before (0.02 nM)PDEy - after (0.02 nM)Ta-GTP[S] alone - before (5.4 nM)Ta-GTP[S] + PDEy (as above)Ta-(1-45) alone - before (5.4 nM)Ta-(1-45) + PDEyTa-(293-314) alone - before (9.6 nM)Ta-(293-314) + PDEyTa-(250-275) alone - before (1.2 nM)Ta-(250-275) + PDEy

30674 + 30261665 + 685778 +146

25305 +117263 + 35

1896 + 847263 + 35

4195 + 3192886 + 276

13 447 + 332

energy of binding for both Ta and PDEa, is found within a basicregion, residues 24-45, and the inhibitory region is within residues80-87 [10]. It appears that two sites are also utilized by the a andf, subunits of the PDE for interaction with PDEy [24,25]. It wasof interest to see if Ta, which also interacts directly with PDEy,has two sites of interaction. One site has been identified withinresidues 293-314 of Ta [26], but this region interacts with the C-terminus of PDEy. In order to identify the region of Ta thatinteracts with the basic region of PDEy (residues 24-45), wholeTa-GTP[S] was tested in a direct binding assay as well as the33 kDa fragment of Ta. This Ta fragment has been shown toactivate PDE but does not bind to rhodopsin or to T/Jy [29-33].Binding to PDEy was observed using this direct binding assaywith both Ta and the 33 kDa fragment (Table 1). However,binding was reduced for 33 kDa Ta indicating that either cleavagedisrupts the native structure or that the N- and C-termini whichare not included within the 33 kDa piece are also required foroptimum binding of PDEy.We have previously used a rapid screening technique to identify

potential regions of proteins that bind to PDEy [24]. This isfeasible for PDEy because the binding site within the basicregion is linear and has little secondary structure (J. Cunnick,C. Twamley, I. Udovichenko, K. Gonzalez and D. J. Takemoto,unpublished work). This approach was also used to tentativelyidentify regions of Ta that bind to whole PDEy (Table 2).As the initial screen used peptides that were 15 amino acids in

length, larger peptides were used to rescreen the regions that werethought to contain a binding site for PDEy. These included Ta-(1-45), Ta-(293-314) and a larger region of a tentative bindingsite, Ta-(250-275). Very high binding was observed for the Ta-(250-275) peptide whereas lower binding was observed for thepreviously identified region (Table 3). In contrast, the N-terminalpeptide Ta-(1-45) did not exhibit binding to PDEy (comparewith 'PDEy -after', i.e. PDEy added after the tube with nobound protein or peptide was blocked).A major binding region of PDEy is within residues 24-45 of

PDEy [10] and we have used the peptide that corresponds toresidues 1-49 of PDEy to determine if this is the region that isrecognized by Ta-(250-275). This peptide, PDEy-(1-49), wasfound to bind to whole Ta-GTP[S] as well as whole PDEybound to Ta-GTP[S] (Table 4). PDEy-(1-49) bound both to Ta-(250-275) and to whole Ta (Table 5). In contrast, PDEy-(1-49)did not bind to Ta-(293-314) or Ta-(1-45) peptides [compare

90 J. Cunnick and others

Table 4 Binding of Toe to PDEy or PDEy-(1-49)Binding assays were described in Table 1. Ta-GTP[S] was added first at 25 nM/tube followedby addition of PDEy or PDEy-(1 -49) as indicated. Antiserum [aPy-(l -49)] was used at 1 :100dilution with 1251-Protein-A at 106 c.p.m./tube. PDEy (114 nM/tube) before block =27246± 285 c.p.m.; PDEy (114 nM/tube) after block = 1675 ±357 c.p.m.; PDEy-(1-49)(204 nM/tube) before block = 43021 ±1019 c.p.m.; PDEy-(1-49) (204 nM/tube) afterblock = 1407 + 185 c.p.m. Background (333 + 14 c.p.m.) is without protein or peptide and issubtracted from the samples. Values are means+ S.E.M. of three samples.

Radioactivity boundSample (c.p.m.)

Ta-GTP[S] aloneTa-GTP[S] + PDEy

Tax-GTP[S] + PDEy-(1-49)

(11 nM)(23 nM)(90.0 nM)(10 nM)(20 nM)(102 nM)

908 +1557821 +115

10976 + 21115448 + 8507491 + 197

11931 +10419319 + 383

Table 5 Binding of To peptides to PDEy peptidesBinding assays were as described in Table 1. Tax-GTP[S] was added at 0.02 nM/tube, Ta-(1-45) at 5.4 nM, Tac-(293-314) at 9.6 nM, Ta-(250-275) at 5.9 nM, PDEy-(1-49) at5.9 nM. Antiserum [aPy-(1-49)] was added at 1:100 dilution with 1251-Protein-A at106 c.p.m./tube. The backgrounds for each antiserum in tubes without protein or peptide havebeen subtracted from the samples. Background for aPy-(1-49) was 291 ±96 c.p.m. andbackground for oxPyC was 410+158 c.p.m. Values are means+ S.E.M. of three samples.

Radioactivity boundAntiserum Sample (c.p.m.)

Table 6 Competition by Ta peptides for binding of PDEy by Ta-GTP[SJBinding assays were as described in Table 1 using aPDEy-(1-49) at 1:100 dilution and106 c.p.m./tube for 1251-Protein A. 'Before' and 'After' refer to additions before or after blockingwith Tween 20. PDEy and Ta-GTP[S] were used at 0.02 nM/tube. Peptides were added atthe same time as the PDEy to compete, concentrations are indicated in parentheses.Background (302± 21 c.p.m.) is without protein or peptide and is subtracted from the samples.Values are means+ S.E.M. of three samples.

Radioactivity boundSample (c.p.m.)

PDEy alone - beforePDEy alone - afterTa4-GTP[S] alone - beforeTa4-GTP[S] + PDEyTa-(1-45) - beforeTa-(293-314) - beforeTax-(250-275) - beforeTa-GTP[S] + PDEy + Ta-(1-45) (1 nM)

(2 nM)(3.2 nM)(4.3 nM)(5.4 nM)(27.0 nM)

Ta-GTP[S] + PDEy + Ta-(293-314) (0.96 nM)(2.8 nM)(5.7 nM)(9.6 nM)(19.2 nM)(28.8 nM)

Ta4-GTP[S] + PDEy + Tx-(250-275) (0.12 nM)(0.24 nM)(0.60 nM)

19160+ 949842 +156681 +172

10907+782255 +112246 + 53

1470 + 6189270 + 16129669

1094590869176

11 87410818 +140211 773 + 238496 + 5267896 + 1097843 + 6889281 + 789421 +160552+104

1005 + 242

aPy-(1-49) PDEy-(1-49) - beforePDEy-(1-49) - afterPDEy-GTP[S] alone - beforeTa4-GTP[S] + PDEy-(1-49)Ta-(1-45) alone - beforeTa-(1-45) + PDEy-(1 -49)Ta-(293-314) alone - beforeTx-(293-314) + PDEy-(1-49)Ta-(250-275) alone - beforeTax-(250-275) + PDEy-(1-49)

aPyC PDEy-(50-87) alone - beforePDEy-(50-87) alone - afterTa-(250-275) alone - beforeTa-(250-275) + PDEy-(50-87)

35253 + 7281745 + 39897 + 54

13195 + 2453430 + 228

1492 + 123261 + 27

1505 + 2391817 + 2618282 + 18012096 + 47565 + 131830 + 59680 + 53

with 'PDEy-(1-49) -after' in Table 5, i.e. binding of PDEy-(1-49) to a blocked tube without bound protein or peptide]. Ta-(250-275) did not bind to the C-terminal peptide, PDEy-(50-87).This indicates that the Ta-(250-275) binds only to the bindingregion [PDEy-(1-49)] of PDEy.

If Ta-(250-275) binds specifically to PDEy, then it shouldprevent or decrease the binding of Ta-GTP[S] to PDEy. Com-petition binding assays were conducted using the ability of theadded peptide to prevent the binding ofPDEy to the Ta-GTP[S]which was bound to the assay tube (Table 6). No competitionwas observed using the peptides Ta-(1-45) or Ta-(293-314).However, Ta-(250-275) completely prevented the binding ofTa-GTP[S] to PDEy at 0.12 nm. This was about 10-fold excess

of peptide over Ta and PDEy.If the Ta-(250-275) peptide competes for Ta-GTP[S] binding

to PDEy, then it may activate the inhibited PDE throughinteraction with the inhibitory PDEy subunits. In order to test

this, a PDE activity assay was carried out using PDE that wasfully activated, then re-inhibited by the addition of excess PDEy.PDE inhibited by adding PDEy could be activated by theaddition of Ta-GTP[S] (Table 7). The addition of an equalconcentration of Tfy or an excess of Ta-(1-45) (27000 pM) didnot activate the PDE. However, the peptide Ta-(250-275)activated the PDE at 1200 pM. This was about 50 times as muchas was required for activation by Ta-GTP[S] (27 pM).

DISCUSSIONThe structure of transducin, the GTP-binding protein of theretinal ROS, has not been determined in detail. It consists of x,, and y subunits of molecular mass 39 kDa, 37 kDa and 6 kDarespectively in a Ta,xy complex [1-7].During visual transduction, the photoexited rhodopsin under-

goes a conformational change which results in exchange ofGDPfor GTP on the Ta subunit. Tac is then released from T/Jy whichremains membrane associated. The Ta subunit activatesPDEafly2 by directly interacting with and removing the con-straints of the inhibitory PDEy subunits [8-12].A structural model has been proposed for Ta which consists of

domains for interactions with Mg2"-GDP and -GTP, T,/y,rhodopsin and PDE [45]. This model was developed usingpreviously known sequence data and information obtained frompartial proteolysis of Ta [30-32]. Ta can be functionally dividedinto a 1-2 kDa region on the N-terminus, which is needed forbinding to T/?y [30-33], a 21 kDa region (approximately residues19-204), which interacts with the PDE [33], a 12 kDa region,which binds guanine nucleotides, and a C-terminus, which bindsto rhodopsin [32,46]. This model has, thus far, not been confirmedby tertiary structural analyses.

Binding site on transducin a for phosphodiesterase y 91

Table 7 tifect of Ta peptides on phosphodiesterase activityPDE was measured as described in the Experimental section using 0.78 pM pure PDE and22 pM PDEy. PDE was either not activated (control) or was trypsin activated to remove PDEy,reconstituted with PDEy (22 pM), and repurified on h.p.l.c. Trypsin activation was as describedfor i min on ice. Assay was for 10 min at 30 OC with 20 ,uM cGMP. Total protein concentrationis in parentheses. Results are means+S.E.M. of three samples. The blank (i.e. background;4358 + 640 c.p.m.) without protein or peptide was subtracted from the samples. The maximumradioactivity possible was 18550 + 588 c.p.m.

PDE activitySample (c.p.m.)

Control PDETrypsin-activated PDETrypsin-activated PDE+ PDEy+ Ta-GTP[S] (27 pM)+ Tfly (20 pM)+ Ta-(1-45) (542 pM)

(27000 pM)+1%x-(250-275) (60 pM)

(120 pM)(1200 pM)

9304 + 92613 224 + 5798905 + 571

11 437 + 2628253 + 8218626 + 11038315+ 6988778 + 9699580 + 552

14 772 +1045

The identity of the site for interaction of Ta with PDEy is ofinterest because this interaction constitutes the initial activationof PDE by Ta-GTP. We have previously functionally mappedthe PDEy-inhibitory protein using partial proteolysis, mutantsand synthetic peptides [10,18-21]. The 87-amino-acid-lengthprotein has a high-affinity binding region in residues 24-45 andan inhibitory region at the C-terminus. This protein interactswith the PDEa/, at both sites [10]. Likewise, both PDEa and-fl have two interaction domains for PDEy [24,25].The location of the Ta-binding sites in the 21 kDa piece is an

approximation. In fact, recent structural studies have identifiedone site of Ta interaction with the C-terminus of PDEy on Taresidues 293-314 [26]. We now report that another site is foundwithin Ta residues 250-275. This region binds to PDEy at the'basic region' binding site, but not at the C-terminus. Thepeptide, Ta-(250-275), also competes for Ta-GTP[S] binding toPDEy and activates PDE. These results imply that, like PDEa//.,Ta also has two interaction domains for PDEy. The bindingmechanism is not known, but the major energy of interaction ofPDEy with Ta is within the 24-45 region of PDEy [22]. Aspreviously suggested [26], perhaps the initial interaction of Ta-(293-314) at the C-terminus of PDEy lowers the binding affinityat the second site [Ta-(250-275) for PDEy-(24-45)]. There arealso modifications on both PDEa/,# [13] and PDEy [23] whichcontrol the binding affinity of Ta for PDEy. The occupancy ofthe non-catalytic cGMP-binding sites on PDEa/,8 determineswhether Ta removes PDEy or remains in a Ta-PDEa/fly2complex [13]. In addition, we have recently reported [23] thatPDEy is phosphorylated at Thr-35 by retinal protein kinase C.The phosphorylated PDEy has increased ability to interact withand inhibit PDEa/fl [23]. When phosphorylated, the PDEy alsoshows decreased binding to Ta-(250-275) (J. Cunnick,C. Twamley, I. Udovichenko, K. Gonzalez and D. J. Takemoto,unpublished work).Thus Ta appears to have two sites for interaction with PDEy,

and the interactions of these domains are under multiple controlmechanisms.

This project is supported in part by a grant from the American Heart Association,National (to D. J. T) and by the Great Plains Diabetes Research, Inc., Wichita, KS, U.S.AK. G. is a predoctoral fellow supported by a training grant from NIH, CA-0941 8. I. U.is a Scholar of the Kansas Health Foundation, Wichita, KS, U.S.A.

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Received 4 May 1993/28 July 1993; accepted 3 August 1993