Proc. Natl. Acad. Sci. USAVol. 93, pp. 5407-5412, May 1996Biochemistry

Mechanism of photoreceptor cGMP phosphodiesterase inhibitionby its y-subunitsNIKOLAI 0. ARTEMYEV*tt, MICHAEL NATOCHIN*, MARK BUSMAN§, KEVIN L. SCHEY§, AND HEIDI E. HAMMt*Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, IA 52242-1109; §Department of Cell and MolecularPharmacology, Medical University of South Carolina, Charleston, SC 29425-2251; and tDepartment of Physiology and Biophysics, University of Illinois College ofMedicine, Chicago, IL 60680

Communicated by Henry R. Bourne, University of California, San Francisco, CA, January 22, 1996 (received for review November 17, 1995)

ABSTRACT cGMP phosphodiesterase (PDE) is the keyeffector enzyme of vertebrate photoreceptor cells that regu-lates the level of the second messenger, cGMP. PDE consistsof catalytic a and 13 subunits (Pa and PIS) and two inhibitory'y subunits (Py) that blockPDE activity in the dark. The majorinhibitory region has been localized to the C terminus of Py.The last C-terminal residues -IleIle form an important hy-drophobic domain critical for the inhibition of PDE activity.In this study, mutants of Py were designed for cross-linkingexperiments to identify regions on Pa and P18 subunits thatbind to the Py C terminus. In one of the mutants, the cysteineat position 68 was substituted with serine, and the last fourC-terminal residues ofPy were replaced with a single cysteine.This mutant, Py83Cys, was labeled with photoprobe 4-(N-maleimido) benzophenone (MBP) at the cysteine residue. Thelabeled Py83CysMBP mutant was a more potent inhibitor ofPDE activity than the unlabeled mutant, indicating that thehydrophobic MBP probe mimics the Py hydrophobic C ter-minus. A specific, high-yield cross-linking of up to 709% wasachieved between the Py83CysMBP and PDE catalytic sub-units. Pa and the N-terminally truncated P,B (lacking 147 aaresidues) cross-linked to Py83CysMBP with the same efficiency.Using mass spectrometric analysis of tryptic fragments fromthe cross-linked PDE, we identified the site of cross-linking toaa residues 751-763 of Pa. The corresponding region of P18,P13-749-761, also may bind to the Py C terminus. Our datasuggest that Py blocks PDE activity through the binding to thecatalytic site of PDE, near the NKXD motif, a consensussequence for interaction with the guanine ring of cGMP.

In retinal rod cells, the visual receptor rhodopsin is activatedby the absorption of a photon that leads to GTP-GDPexchange on the retinal GTP-binding protein, transducin (Gt)and to the dissociation of the a-subunit of Gt (GtaGTP) from13yt and rhodopsin. GtaGTP then activates the effector en-zyme, cGMP phosphodiesterase (PDE), by relieving the in-hibitory constraint of the PDE 'y-subunits (P-y). cGMP hydrol-ysis by active PDE results in closure of cGMP gated channelsin the plasma membrane (for reviews, see refs. 1-3).To better understand the mechanism of photoactivation of

PDE, it is critical to determine the structural details of how PTyinteracts with and inhibits the PDE catalytic subunits in thedark. The inhibitory role of Py was originally shown usingproteolysis of PDE with trypsin (4, 5). Tryptic proteolysissimultaneously destroys Py and activates PDE. The addition ofexogenous Py reinhibits the trypsin-activated PDE (5). Thestructural elements of the Pa(Pf3)-Py interaction on Py sub-unit have been extensively studied (6-9). It appears that theprimary regions of Py involved in the interaction with Pap arethe central polycationic region Py-24-45 and the C-terminalregion of Py. Both of these regions participate in the binding

of Py to Pap3, but the Py C terminus is the most important forPDE inhibition. The last two C-terminal residues (-Ile-Ile)form an important hydrophobic domain that is critical for theinhibition of PDE activity (10, 11). Much less information isavailable about the sites of Pa(P3)-Py interaction on the PDEcatalytic subunits. It was reported that the major sites of Painteraction with Py are located within the Pa N terminus(16-30 and 78-90) (12), whereas sites on P,B of interaction withPy are in other regions (91-110 and 211-230) (13). TheN-terminal regions ofPa and P,B have practically no homology,despite an overall identity of 72% (14). The observation thatdifferent sites within Pa and P,B interact with Py appearsunusual in light of the evidence that Pa and P3 interact withidentical sites on Py (7, 8). It is also inconsistent with thefindings that both cone Py and rod Py inhibit cone PDE (Kiof 200pM and 600 pM, respectively) and rod PDE (Ki of 80 pMfor either Py) (15) since the N-terminal regions of cone Pa'and Pa (or P,3) also are very different (14, 16). Therefore, webegan to further investigate the inhibitory PFy binding sites onPa and P13. Specifically, we used a photo cross-linking ap-proach combined with the analysis of the cross-linked frag-ments using mass spectrometry to identify sites on PDEcatalytic subunits that bind to the Py C-terminal region.

MATERIALS AND METHODSMaterials. All restriction and DNA modification enzymes

were obtained from Pharmacia, Boehringer Mannheim, orGIBCO/BRL. Trypsin and soybean trypsin inhibitor wereobtained from Worthington. cGMP was purchased fromBoehringer Mannheim. 4-(N-maleimido)benzophenone(MBP) was from Sigma. The matrix material, a-cyano-4-hydroxycinnamic acid, was purchased from Aldrich. All otherchemicals were from Sigma or ICN.

Preparation of PDE and Trypsin-Treated PDE (tPDE).Bovine ROS membranes were prepared by the method ofPapermaster and Dreyer (17). PDE was extracted frombleached ROS membranes as described (18). The PDE-containing extract was concentrated by ultrafiltration using aYM-30 membrane (Amicon). Proteolysis of PDE with trypsinwas carried out in 20 mM MOPS buffer (pH 7.5), containing1 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, and 25%glycerol (buffer A). Typically, concentrated PDE extract (1mg/ml) was digested with trypsin (50 Ag/ml) for 2 h at room

Abbreviations: PDE, cGMP phosphodiesterase; Pa, P,B, and Py, sub-units of PDE; tPDE, trypsin-treated PDE, consisting of 90-kDa and70-kDa polypeptides; taPDE, trypsin-activated PDE, activated withlimited trypsin digestion to remove y-subunit; MBP, 4-(N-maleimido)benzophenone; Gt,,, a subunit of the photoreceptor GTP-binding protein transducin; P'y83Cys and Py85Cys, mutants of PDEy-subunit with Cys68 replaced for Ser and C-terminal four or twoamino-acid residues replaced with Cys; MALDI, matrix-assisted laserdesorption ionization.*To whom reprint requests should be addressed at: Department ofPhysiology and Biophysics, The University of Iowa College of Med-icine, 5-660 Bowen Science Building, Iowa City, IA 52242-1109.

5407

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

5408 Biochemistry: Artemyev et al.

temperature. The reaction was stopped by the addition ofsoybean trypsin inhibitor (300 Ag/ml). This tPDE containedequimolar amounts of 88 kDa and 70 kDa polypeptides.Trypsin-activated PDE (taPDE) was obtained by digestion ofPDE under the conditions described above for 15 min. Suchdigestion fully activated PDE (by cleaving Py subunits) withoutproducing the 70-kDa band. PDE, tPDE, or taPDE waspurified by ion-exchange HPLC on a MonoQ column (Phar-macia). The column was equilibrated with buffer A (withoutglycerol). Proteins were eluted with a NaCl gradient (100-800mM). PDE preparations obtained by these procedures were>95% pure, based on densitometric scanning of Coomassieblue-stained gels.

Sequencing of the 70-kDa Band of the tPDE. The 70-kDaband of tPDE was separated using a Tris-Tricine SDS/PAGE,electroblotted onto polyvinylidene difluoride paper, and se-quenced with an Applied Biosystems model 475A gas phasesequencer with on-line phenylthiohydantoin amino acid ana-lyzer and a 900A data station in the Protein Structure Facility(The University of Iowa).

Preparation ofPy and Its Mutants. Py and Py mutants wereobtained based on the Py expression vector (11). The Pycoding sequence flanked with NdeI and BamHI sites wasPCR-amplified from the synthetic gene (19) and was insertedbetween the corresponding sites of pET-lla vector (Novagen)under the control of the T7 promoter (11). To obtain the Pygene encoding the substitution of Cys68 for Ser, the fragmentof Py gene was PCR-amplified with an upstream primercontaining the NdeI site and the downstream primer contain-ing the NcoI site and the mutation. This fragment was insertedinto the NdeI-NcoI fragment of the Py expression vector. ThePTy mutants with the C-terminal two or four residues replacedwith cysteine were constructed by PCR-amplification of thePyCys68-Ser mutant gene with an upstream primer containingthe NdeI site and downstream primers containingBamHI sitesand the mutations. These mutant genes were inserted into theNdeI-BamHI fragment of the Py expression vector. Thesequences of all mutant Py genes were confirmed by dideoxyDNA sequencing (20). All DNA manipulations were per-formed using standard techniques (21). Py and its mutantswere expressed in Escherichia coli BL21/DE3 and purified tohomogeneity as described (11).

Labeling ofPy and Its Mutants with MBP. During reversed-phase HPLC purification on a Vydac (Hesperia, CA) C-4column Py or the mutants were eluted in -45% acetonitrile/0.1% trifluoroacetic acid. Typically, the Py or the mutantconcentration in the fractions after HPLC was 200-300 AM.Hepes buffer (200 mM; pH 8.4) was added to the fractions toadjust pH to 7.3. Then MBP (10 mM) in acetonitrile was added(final concentration, 400-600 ,uM) and the reaction wasallowed to proceed for 20 min at room temperature. MBP-labeled proteins were separated from the unreacted MBPusing a G-25 desalting column (Pharmacia) equilibrated with20 mM Mops buffer (pH 7.5), containing 100 mM NaCl and1 mM dithiothreitol. All operations were carried out in dimlight. The efficiency of the MBP-labeling was usually 70-75%based on the absorption spectra of the unlabeled and labeledproteins. The concentrations of the MBP labeled proteins weredetermined using 8260 = 23,000 for MBP.

Cross-Linking of Py or Its Mutants to tPDE. tPDE wasmixed at a final concentration of 2 ,uM with one of the proteins(PyCys68MBP, Py85CysMBP, or Py83CysMBP; 10 ,uM each)in a polypropylene microcentrifuge tube and irradiated for 4min at a distance of 4 cm with a Transilluminator ultravioletlamp (Ultraviolet Products, San Gabriel, CA). After photol-ysis, the proteins were analyzed by SDS/PAGE. tPDE cross-linked with P-y83CysMBP was repurified on a MonoQ columnas described above to remove uncross-linked Py83CysMBPand to enrich the preparation with the cross-linked products.Proteins purified by HPLC were stored at - 17°C.

Proteolysis of Proteins for Mass Spectrometric Analysis.Aliquots of 10-15 ,ul (0.1-0.3 mg/ml) of tPDE (control) or thecross-linked tPDE were digested by adding trypsin (0.5 mg/ml)in 10 mM NH4HCO3 to provide a 10:1 protein/trypsin molarratio. Digestions were carried out at room temperature andaliquots were taken in 30-min intervals up to 6 hrs for analysisby matrix-assisted laser desorption ionization (MALDI) massspectrometry. For electrospray mass spectrometry, the HPLC-purified tPDE was desalted by rinsing extensively with deion-ized water over an Amicon model Microcon micrococentratortube with a 10,000 molecular weight cutoff membrane. Theprotein was then digested by adding trypsin in 10 mMNH4HCO3 as described above for 90 min, after which thesample was lyophilized.Mass Spectrometry. MALDI analysis was carried out on a

custom-built time-of-flight mass spectrometer equipped with anitrogen laser [Laser Science (Cambridge, MA) model VSL-337ND] (22, 23). Digestion aliquots (1 ,l) were prepared foranalysis by mixing with 3 ,ul of matrix solution/50mM a-cyano-4-hydroxycinnamic acid in 70% acetonitrile/water, 0.1% tri-fluoroacetic acid. Analysis of the 16-kDa product from thetPDE preparation was carried out by mixing 1 ,ul proteinsolution with 3 ,uI of 50 mM sinapinic acid in 70% formic acid.For low mass range calibration, substance P (Mr 1348.6) and

bovine ubiquitin (Mr 8565) were used, whereas hen egglysozyme (Mr 14306) and bovine 13-lactoglobulin B (Mr 18277)were used as higher molecular weight calibration standards.Low mass ions were deflected away from the ion detector bya delayed voltage pulse to enhance sensitivity in the mass rangeof interest.

Electrospray ionization tandem mass spectrometry experi-ments were carried out on a Sciex (Thornhill, ON, Canada)model API III triple quadrupole mass spectrometer. Thelyophilized digest was redissolved in methanol/water/aceticacid (47:47:6) and sprayed at a flow rate of 0.3 ml per h. Thedoubly charged precursor ion of interest (m/z 772.5) wasselected by MS-1 (the first quadrupole) and, after collisionwith a mixture of argon and nitrogen in the collision quadru-pole, product ions were mass analyzed by MS-2 (the thirdquadrupole).

Analytical Methods. The PDE activity was measured usingthe proton evolution assay (24). The pH was monitored witha pH microelectrode (Microelectrodes, Londonderry, NH). Pro-tein concentrations were determined by the Coomassie blue

AkDa

29 - i._

f:.202-3'123

BklDa90-70-

PyNI 31P-

cMBP

a

0

MBP

1. Pyl-67Cys69-87

2. Pyyl -67Ser69-85C s-Nl 3P

1 2 3 4 3. Py1-67Scr619-X3C's-N1M3

FIG. 1. (A and B) SDS polyacrylamide gels stained with Coomassieblue. (A) Proteolysis of PDE by trypsin with the formation of the=70-kDa polypeptide. Lanes: 1, PDE extract; 2, tPDE; 3, tPDEpurified on a MonoQ column. (B) Cross-linking between tPDE andP.yCys68MBP or P-y85CysMBP. tPDE was mixed at a final concentra-tion of 2 ,uM with PyCys68MBP or P-y85CysMBP (10 ,uM each) in apolypropylene microcentrifuge tube and irradiated for 4 min at adistance of 4 cm with an ultraviolet lamp. After photolysis, the proteinswere analyzed by SDS/PAGE. Lanes: 1 and 3, tPDE; 2, tPDEcross-linked with PyCys68MBP; 4, tPDE cross-linked withPy85CysMBP. (C) Chemical structure of MBP and schematic repre-sentation of the MBP-labeled proteins.

Proc. Natl. Acad. Sci. USA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 5409

kD)a

90 - _70~-0 1oi-

I 3 4 8)

FIG. 2. Cross-linking between tPDE and Py83CysMBP. tPDE wasmixed at a final concentration of 2 ,uM with P-y83CysMBP (10 ,uMeach) in a polypropylene microcentrifuge tube and irradiated for 4 minat a distance of 4 cm with an ultraviolet lamp. The PDE cross-linkedproducts were then purified on a MonoQ column and treated withtrypsin in a 20:1 protein/trypsin for 15 min. SDS/PAGE analysis of thecross-linked products. Lanes: 1, tPDE; 2, tPDE cross-linked withPy83CysMBP; 3, the MonoQ flow-through fraction; 4 and 5, fractionscontaining the cross-linked tPDE after MonoQ column; 6, controltPDE; 7 and 8, fractions of the cross-linked PDE (as in lanes 4 and 5,respectively) treated with trypsin.

binding method (25) using bovine serum albumin as a standardor using the calculated extinction coefficients at 280 nm.SDS/PAGE was performed by the method of Laemmli (26).

RESULTS AND DISCUSSIONIdentification of the PDE Tryptic Cleavage Site that Pro-

duces the 70-kDa Band. The formation of a 70-kDa polypep-tide after proteolysis ofPDE with trypsin has been reported (5,27). It was suggested that the 70-kDa peptide could be aproteolytic product of the P13 subunit because the increase inintensity of the 70-kDa band correlated with decreased inten-sity of the P13 band during the proteolysis (27). However, thecleavage site on PDE that produces this polypeptide has never

8505456

574458

750

5 650-

been identified. To identify this site, we performed thetrypsinization of PDE under the conditions described. Afterpurification over MonoQ column (Fig. 1A), this tPDE exhib-ited maximal enzymatic activity of 3500 mol cGMPs-s'-(mol ofPDE)-', identical to the activity of taPDE, which is in agree-ment with the data of Catty and Deterre (27).

Microsequencing of the 70-kDa polypeptide resulted in theN-terminal sequence MVNVQDVMECPHF, which corre-sponds to the residues 148-160 of Pp. The first sequencingcycle yielded Lys together with Met, indicating that trypsincleaves after both K146 and K147. Interestingly, the Pa subunitalso contains this pair lysines, K148 and K149, correspondingto the K146 and K147 of P3. However, trypsin does not cleavePa after these lysines, implying that the Pa and P13 subunitsmay have conformational differences near the cleavage site.tPDE and taPDE showed no difference when tested for

inhibition of their catalytic activity by P-y (K, < 100 pM). Thissuggests either that the N-terminal 146 residues of P13 are notinvolved in binding of Py, or that P1-2-146 remain bound to andcomigrate with the C-terminal 70-kDa fragment of P,8 on theMonoQ column. It appears that only a small portion of P13-2-146comigrates with the 70-kDa polypeptide. This fragment is notseen in tPDE on the Coomassie blue stained gel (Fig. lA, lane 3).However, MALDI mass spectrometric analysis of tPDE purifiedon a MonoQ column has shown the presence of a small signal atm/z 16342 ± 29, which is consistent with the predicted molecularmass 16326 Da of P,3-2-146 (N-terminal methionine clipped andthe P13 N terminus is acetylated).The identification of the tryptic site that produces the 70-kDa

fragment of PDE was very instrumental for the cross-linkingexperiments designed to identify the P'y binding site on Pa or P13.First, the use of tPDE allowed us to monitor the cross-linking ofindividual PDE catalytic subunits with Thy because Pa and the70-kDa fragment of P13 are well-separated on SDS/PAGE.Second, early on, we would be able to see whether N-terminalregion of P13 cross-links with the Py subunit or not.

Cross-Linking Approach. Chemical cross-linking is a widelyused tool in studying sites of protein-protein interactions. Theaccuracy of this method often depends on the size of thecross-linker used. To localize the major Py inhibitory site on

mlz

FIG. 3. MALDI mass spectra in the 5000-7000 Da range of tryptic digests of tPDE cross-linked with PTY83CysMBP and control tPDE. Proteinswere digested with trypsin in 10:1 molar ratio of protein/trypsin for 90 min. A 1-lI aliquot was mixed 1:3 with a-cyano-4-hydroxycinnamic acid(70% acetonitrile, 0.1% trifluoroacetic acid) and 0.5 p,l of the mixture spotted on the sample probe. Masses were calibrated using m/z 5456, 5743,and 5895 as internal standards that were calibrated with ubiquitin in a separate experiment.

Biochemistry: Artemyev et al.

5410 Biochemistry: Artemyev et al.

A 21'

a 16,

3-._

y 11,

6

30CB

2500-

4

I-

._I"

2000-

1500-

10001

10(

mlz

D0 1500 2000 2500 3000 3500

mlz

FIG. 4. MALDI mass spectra in the 1000-3500 Da range of tryptic digests of control tPDE (A) and tPDE cross-linked with Py83CysMBP (B).Digestion and MALDI preparation were carried out as described in the legend to Fig. 3. Masses were calibrated usingm/z 1779 and 1950 as internalstandards that were calibrated with substance P in a separate experiment.

Pap4, we used a photoactivatable cross-linker MBP (Fig. 1C).Our cross-linking experiments were designed to minimize theerror related to the size of the cross-linker. The design was

based on the finding that the last two residues of Py (-Ile-Ile)are likely to form an important hydrophobic domain that canfit into a binding pocket on Pan, thus inhibiting PDE activity(10, 11). The idea behind our approach is that if the photo-probe has a size similar to the hydrophobic tail of Py, it maymimic this tail and bind in the same pocket on Paf3. C-

terminally truncated PTy mutants with Cys at the C terminus(Cys68 replaced with Ser) have been made and labeled withMBP at the Cys residue (Fig. 1C). Taking into account the sizeof the photoprobe plus an extra Cys (-12 A), two or fourC-terminal amino-acid residues of Py were truncated. If theidea is correct, then the labeling with MBP would restore theability of the truncated mutants to effectively inhibit tPDE.

Indeed, labeled mutants P'y83CysMBP and P'y85CysMBP weremore potent inhibitors ofPDE activity than unlabeled mutants(not shown). Py83Cys and Py83CysMBP maximally inhibited50% (Kj -1.5 nM) and 90% (Ki -0.4 nM) of tPDE activity,while Py85Cys and Py85CysMBP maximally inhibited 75% (Kj-0.4 nM) and 90% (Ki -0.25 nM) of tPDE activity, respec-tively. In control experiments, PTy and PyCys68MBP bothcompletely inhibited tPDE with Ki < 0.1 nM.

Cross-Linking of MBP-Labeled Py and Its Mutants to Paand Pp3. tPDE was mixed with PyCys68 MBP, Py85CysMBP, orP-y83CysMBP and, after photolysis, proteins were analyzed bySDS/PAGE. Fig. 1B demonstrates the cross-linking betweentPDE and PyCys68MBP (lane 2) or Py85CysMBP (lane 4).P,yCys68MBP and Py85CysMBP cross-linked to both Pa andthe 70-kDa fragment of P,3 with a similar efficiency (yield15-20%), resulting in formation of the cross-linked products

2278

1212 1436

19491779 2192

15462304

/ ~~~~~2557 3165

Proc. Natl. Acad. Sci. USA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 5411

with molecular masses of -100 kDa and -80 kDa, respec-tively. Use of the Py83CysMBP mutant dramatically improvedthe efficiency of cross-linking (Fig. 2). Py83CysMBP wascross-linked to Pa and the 70-kDa fragment of Po with thesame yield, typically 60-70%. In control experiments,P-y83CysMBP showed no cross-linking to purified holoPDE,which contains intrinsic Py subunits (not shown). These resultsindicate that the N-terminal region of P,B does not containbinding site for the Pry C terminus.

Localization of the Py Inhibitory Domain on PDE CatalyticSubunits. tPDE cross-linked to P-y83CysMBP was additionallypurified on a MonoQ column. This purification step removeduncross-linked Py83CysMBP, and the cross-linked and uncross-linked catalytic subunits were partially separated, allowing us toobtain a fraction highly enriched in the cross-linked products witha small amount of Pa (Fig. 2, lane 4). Trypsin was selected toproteolyze the cross-linked tPDE-P'y because full digestion withtrypsin would leave a large Py-tag attached to the tryptic frag-ments of PDE catalytic subunits. As we have shown (7), fulldigestion with trypsin generates two C-terminal fragments of Py,Py45-87 and Py46-87. MALDI mass spectra of the Py83CysMBPtryptic digest (data not shown) indicate two signals at m/z 4461and m/z 4584 corresponding to the predicted tryptic peptidesPy46-83CysMBP (Mr 4461) and P-y45-83CysMBP (Mr 4590).Having a large tag with a known molecular mass significantlysimplifies identification of the cross-linking site using mass spec-trometric analysis. Limited treatment of the aliquots from thecross-linked PDE fractions with trypsin visualized the attachmentof the P-y fragments (Py46-83CysMBP and/or Py45-83CysMBP)to Pa and the 70-kDa PB on the SDS/PAGE. The proteolysis ledto the formation of -95-kDa and -75-kDa bands, which reflectsthe tag attachment (Fig. 2, lanes 7 and 8).

Next, the cross-linked tPDE and control tPDE were extensivelydigested with trypsin and the fragments were analyzed withMALDI mass spectrometry. Fig. 3 shows the MALDI massspectra in the molecular mass range of 5000-7000 Da of bothcontrol tPDE and cross-linked tPDE samples after tryptic diges-tion. A new peak (m/z 6136 ± 26) is observed in the mass

spectrum of the cross-linked protein, which differs in mass fromPy45-83CysMBP by 1547 Da. Fig. 4 shows the MALDI massspectra in the molecular mass range of 1000-3500 Da for bothcontrol tPDE and cross-linked tPDE samples after tryptic diges-tion. The abundant signal at m/z 1546.5 in the control PDEspectrum clearly decreases in intensity after cross-linking. Thereare five possible tryptic peptides from Pa, P,B, and P-y that havemasses within the experimental error (Pa-262-274, Mr 1542.8;Pa-318-330, Mr 1543.81; Pa-751-763, Mr 1543.86; P,B-316-328, Mr1543.81, and Pf3-749-761, Mr 1544.84). Therefore, tandem massspectrometry was pursued to sequence this peptide. No othersignificant changes in signal intensities were observed in the lowor high mass range on cross-linking.The electrospray mass spectrum of the control tPDE tryptic

digest indicated that the most abundant ion in the 1546 massrange appeared as the doubly charged ion (m/z 772.5), whichis typical of small tryptic peptides. The tandem mass spectrumof this ion is shown in Fig. 5. The dissociation pattern producedon collisional activation directly identifies Pa-751-763 as theabundant peptide in this mass range, the sequence of which isshown in Fig. 5. This sequence is in the catalytic domain of Pa.The tandem mass spectrum indicates a single set of signalscorresponding to fragment ions from a single peptide. (Thesmall signal at m/z 988 could be indicative of the presence ofP,B-749-761; however, the low intensity suggests that very littleof this peptide is present in the digest.) We were unable toconclusively identify the peptide fragment from the P,B subunitthat cross-link with Py. Perhaps the P,B polypeptide is not cleavedby trypsin as efficiently as Pa and the cross-linked fragment is toolarge in mass to be seen well by mass-spectrometry.Mechanism of Inhibition of Photoreceptor PDE Activity by

the Inhibitory PTy Subunits. Because the rod PDE has beenshown to have two identical inhibitory P-y subunits, the ques-tion of whether the two molecules of Py have different affinitiesfor each catalytic PDE subunit has been intensively studied. Thework has yielded essentially opposite conclusions. One study hasconcluded that the affinities of P-y for the two binding sites aresimilar (-10 pM) and binding of each Py subunit inhibits about

PDEa 751-763 TVLQQNPIPMMDR7

649.0

TV LIl

201.5314.5

... -1 II I 1 1- I

200

536.0478.5j

111

400

IP

683.5

600 800

m/z

859.5

- N Q I LI

973.5 1101.5 1228.0

l | | ~~1342.0

|.t -I-I'.I,IIJkl,, 1 ,A1,, 1.

1000 1200 1400

FIG. 5. Tandem mass spectrum of the PDE peptide at m/z 772.5. Desalted tPDE was digested for 90 min as described in the legend to Fig.3 and lyophilized. The dried sample was resoubilized in 47:47:6 methanol/water/acetic acid (100 ,l). The ion at m/z 772.5 was mass selected byMS-1 and collided with an argon/nitrogen mixture. Product ion signals were mass analyzed by MS-2. The sequence of the peptide identified is shownabove the product ion signals.

12.5

9.4

6.2

3.1

0.0

Biochemistry: Artemyev et al.

'722.5

5412 Biochemistry: Artemyev et al.

half of PDE activity (28). In contrast, it has been suggested thatthere are two classes of Py binding sites on Pap3 (29).The two putative classes of Py binding sites may have

different origins including: (i) cooperative binding of Pysubunits or (ii) structural differences in the regions of Pa andP,B that interact with Py. Studies of the sites on Pa and P,B ofinteraction with Py, performed using synthetic peptides asprobes, seem to provide a structural basis for the two Pybinding sites. Different N-terminal sites with highest dissimi-larity between Pa and Po3 were implicated as major regions ofinteraction with Py (12, 13). However, the lack of any signif-icant homology within these regions on the PDE catalyticsubunits fails to explain the tight binding of Py even to a loweraffinity site on PaI3. This also appears to contradict the datathat the two inhibitory subunits most likely play equivalentroles in PDE inhibition (7, 28, 30, 31).Using the photo cross-linking approach and analysis of the

cross-linked products with mass spectrometry, we identifiedthe site on the Pa catalytic subunit that interacts with theinhibitory C-terminal domain of Py. This site corresponds tothe residues 751-763 of Pa. Although our data do notprovide conclusive evidence for the site on P13, the corre-sponding domain P,B-749-761 is highly homologous (Fig. 6),and we speculate that it binds the Pry C terminus as well.Moreover, both rod and cone photoreceptor PDEs havealmost identical sequences in this region, consistent with theevidence that the photoreceptor PDEs have similar mecha-nisms of inhibition by the y-subunits (Fig. 6). The Pa-751-763 (or 749-761 for P13 and Pa') region has a sequence-PIPM- that might form a hydrophobic pocket for bindingthe C-terminal residues of Py.Our findings indicate that the affinities of the Py C terminus

for Pa or P1 are probably similar; however, they do not ruleout the model of two classes of Py binding sites on Pap,because there is another region on Py, Py-24-45, that binds toPap3 and its partner regions on Pa and P13 are as yet unknown.Another interesting implication comes from the alignment

of Pa-751-763 sequence with the corresponding sequences ofother cGMP binding PDEs: cGMP-specific PDE from bovinelung (32) and cGMP stimulated PDE from bovine heart (33)(Fig. 6). These PDEs are significantly dissimilar to photore-ceptor PDEs in this site. Furthermore, a BLAST search at theNational Center for Biotechnology Information (Bethesda) tocompare the Pa-751-763 sequence against protein sequencedatabases did not reveal any homologous sequences other thanthose of photoreceptor PDEs from various species. Based onthis analysis, one would predict that inhibition by Py is a uniquefeature of photoreceptor PDE and that other PDEs may notbe effectively inhibited by this protein.The most important result of this study is the finding that Py

inhibits PDE activity by interacting with the catalytic domainsof Pa and P13 near the NKXD motif, which is thought to specifybinding of the cGMP guanine ring by analogy with the NKXDguanine ring binding of GTP to G-proteins (16, 34, 35). Thisinteraction provides a simple explanation for the effectiveinhibition of PDE activity. The Py subunit might eitherphysically block binding ofcGMP to the catalytic site or induce

rod PDEarod PDEPcone PDEa'cGS-PDEcGB-PDE

+ NKXD-R-TVLQQNP I PMMDR-NKAD--R-TVLDQQP I PMMDR-NKAA--R-TVLQQQP I PMMDR-NKKD--A. MGNRPMEMMDR--KELNI E PADL MNR-

FIG. 6. Comparison of the Pa-751-763 region with the correspond-ing regions of P13, cone Pa', cGB-PDE, and cGS-PDE (from themultiple sequence alignment in ref. 32).

local conformational changes of the catalytic site such thatalthough cGMP still can bind, its hydrolysis is disabled. It hasbeen shown that the addition of Py to taPDE causes very littlechange in the apparent Km value (5), indicating that Py may bea noncompetitive inhibitor of PDE. Py binds to Pa13 verytightly (Kd 10 pM) compared with cGMP binding (Km forcGMP is in the range of 20-80 ,uM), and off-rates of Py fromPa13 are very slow (>10 min) (5, 8). Such differences in affinitymay not allow cGMP to compete effectively with Py bound toPa13. Therefore, the question of whether Py blocks cGMPhydrolysis competitively or allosterically needs further inves-tigation. Our results make the first alternative an attractivepossibility. The structural information about the Py-Pa13 in-teraction obtained in this study is an important step forunderstanding the mechanisms of regulation of rod PDEactivity on transduction of the light signal, as well as under-standing the general regulation of catalysis by cyclic nucleotidephosphodiesterases.

We thank Dr. Alan Bergold and the Protein Structure Facility at TheUniversity of Iowa for the protein sequencing and Dr. Peter Moellerof the National Marine Fisheries Laboratory (Charleston, SC) for useof the Sciex API III instrument. This work was supported by theNational Institutes of Health Research Grant EY-10843 to N.O.A.N.O.A. is a recipient of a National Alliance for Research on Schizo-phrenia and Depression Young Investigator Award.

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