Identification of the retinal cyclic GMP phosphodiesterase inhibitory

THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1991 by The American Society for Biochemistry and Molecular Biology, Inc

VOl. 266, No , 25, Issue of September 5 , pp. 16607-16613,1991 Printed in U.S.A.

Identification of the Retinal Cyclic GMP Phosphodiesterase Inhibitory 7-Subunit Interaction Sites on the Catalytic a-Subunit*

(Received for publication, January 22, 1991)

Brenda Oppert, Jess M. Cunnick, Daniel Hurt, and Dolores J. Takemoto From the Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506

Retinal rod outer segment phosphodiesterase (PDE) consists of two similar catalytic subunits (a and 8) and two identical inhibitory subunits (yz). A trypsin-activated soluble PDE exhibiting the ability to be reinhibited by PDEr was shown by peptide antisera to retain both N and C termini. Synthetic peptides corresponding to residues 16-30, 78-90, 389-403, and 535-563 of PDEa used in a PDE activity assay with trypsin-activated PDE partially prevented inhibition by exogenous PDEr; however, only competitions by peptides 16-30 and 78-90 (corresponding to PDEa 16-30 and 78-90) were concentration-dependent below 100 nmol of peptide. Binding studies using radioimmunoassays and PDEa peptides confirmed that peptides 16-30 and 78-90 (corresponding to PDEa 16- 30 and 78-90, respectively) were able to bind PDEr. Additionally, peptides corresponding to the PDEa region 453-534 bound PDEr in the binding assay. This suggests that several regions on PDEa interact with the PDEr inhibitor. While some regions may be involved in binding to PDEr, other sites may be involved in PDEr inhibition of catalytic activity. Our results suggest that the major regions of PDEa that interact with PDEr reside within the N terminus (16-30 and 78-90), with weaker interaction regions within or near the hypothesized catalytic domain (453-563). Se- quence analysis of three retinal phosphodiesterases (rod outer segment a, B, and cone outer segment a’) revealed the highest region of dissimilarity in the N and C termini.

Retinal rod outer segment phosphodiesterase (PDE)’ (3’,5’- cyclic-nucleotide phosphodiesterase, EC 3.1.4.17), activated by light through a signal transmitted from rhodopsin to transducin, functions by controlling ion channels in the rod outer segment plasma membrane (3). These ion channels are closed upon hydrolysis of cyclic GMP by active PDE (4), whereas the process can be reversed when cyclic GMP is regenerated by guanylate cyclase (5). The catalytic subunits of bovine retinal PDE consist of an a- (88-kDa) and p- (84- kDa) subunit (6), and recently published sequences of a (7)

* This project supported in part by National Institutes of Health/ National Educational Institute Grant EY06490 and by Grant G28 from the American Heart Association, Kansas Affiliate (to D. J. T.). This is Contribution 91-34-5 from the Kansas Agriculture Experi- ment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ’ The abbreviations used are: PDE, phosphodiesterase; KLH, keyhole limpet hemocyanin; PMSF, phenylmethylsulfonyl fluoride; ROS, rod outer segment; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

and B (8) reveal strong homology between the two (9). When bound to two inhibitory subunits (PDEy2, 11 kDa each), the entire PDE complex is inactivated (10, 11).

Retinal PDEs have homology with a family of PDEs in a proposed catalytic region (30). Recently, a cGMP-stimulated PDE, isolated from a variety of mammalian tissues, was shown by photoaffinity labeling to contain a cGMP binding region in the proposed catalytic region (33).

The inhibitory constraints on PDE appear to be relieved in uitro (and possibly in uiuo) by the interaction of transducin a-GTP with PDEy (1, 12-14). PDE can be activated in uitro by a limited trypsin digestion that presumably destroys the inhibitory PDEyz subunits (10). This activated PDE can be reinhibited by the addition of purified PDEy, resulting in a loss of 97% of catalytic activity (10).

The two PDEys exhibit different binding affinities for PDEaP (15). In bovine retina, only a partial activation (5- 17%) occurs with the loss of a low affinity PDEy. The PDEy complexes with transducin a-GTP, resulting in a soluble GTP-transducin-a-PDEy complex (15). Full activation is achieved at high concentrations of transducin a-GTP, but this activation does not result in removal of the high affinity PDEr from the membrane (15). The site on PDEr that interacts with PDEaB is located within residues 24-45 and at the C terminus (residues 54-87) of PDEy. This was identified using synthetic peptides as probes of function (1, 2), proteo- lytic digestion to remove the C terminus (2, 16), and deletion mutants lacking the C terminus (17, 18).

In this report we present evidence for the location of the binding regions on PDEa for PDEy. These regions are located within PDEa residues 16-30 and 78-90, with other possible interaction sites within residues 453-563. Sequence comparisons of rod outer segment PDEa (7) and f i (8), together with cone outer segment a’ (19), indicate that the regions of greatest dissimilarity are at the N and C termini.

EXPERIMENTAL PROCEDURES

Materials-Bovine eyes were obtained immediately following slaughter from Iowa Beef Packers (Emporia, KS). [8-3H]cGMP (15 Ci/mmol) was obtained from ICN Radiochemicals and purified further by anion exchange chromatography. Carrier-free lZ5I was purchased from Amersham International. Amino acids (t-butoxycarbonyl and resin forms) were obtained from Vega Biochemicals, United States Biochemical Corp., or Sigma. Aquacide I11 was purchased from Behring Diagnostics, HPLC columns from Phenomenex, nitrocellulose from Schleicher and Schuell, x-ray film from Du Pont-New England Nuclear, and developing solutions from Kodak. All other buffers and reagents were from Sigma. Reagents used for peptide synthesis were HPLC grade from Fisher or Sigma, or Sequenal grade from Pierce.

Rod Outer Segment Purification-Rod outer segments were pre- pared by the method of Papermaster and Dreyer (20). Bovine eyes were kept in the dark on ice during transport (about 3 h), and retinas were removed under dim red light and stored at -70 “C in the dark. All procedures were performed under dim red light unless noted

16607

16608 Identification of PDEr Binding Sites on PDEa otherwise. Approximately 50 thawed retinas were suspended in 50 ml of ROS 1 (5 mM Tris, pH 7.4, 65 mM NaCl, 2 mM MgCl,, 1 mM dithioerythritol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 30% sucrose (w/v)) and shaken vigorously for 1 min (resulting in rod outer segments being sheared from the inner segments). After centrifugation at 4,000 rpm for 5 min (Sorvall SS-34 rotor), the pellet containing ROS was resuspended in 15 ml of ROS 1 and recentrifuged as above. Pooled supernatants were diluted 1:2 in ROS 2 (10 mM Tris, pH 7.4, 1 mM MgCI,, 1 mM dithioerythritol, 0.5 mM PMSF) and centrifuged at 7,000 rpm for 10 min (Sorvall SS-34 rotor). Pelleted ROS were resuspended in 12 ml of ROS 3 (10 mM Tris, pH 7.4,l mM MgC12, 1 mM dithioerythritol, 0.5 mM PMSF, and 26.3% sucrose (w/ v)), homogenized with a 26-gauge needle, and layered (2 ml) onto a discontinuous sucrose density gradient of 1 ml each of 1.11,1.13, and 1.15 g/ml sucrose in ROS 2. After centrifugation at 24,000 rpm (55,000 X g) for 45 min (Beckman SW50.1 rotor), ROS discs were collected at the 1.11-1.13 interface. Purified ROS discs were then washed with a 3-fold volume of ROS buffer 7 (10 mM Tris, pH 7.4, 100 mM NaCl, 5 mM MgCI,, 0.1 mM dithioerythritol, 1 mM PMSF) and centrifuged at 15,000 rpm for 15 min (Sorvall SS-34 rotor). After washing two more times as above, the undepleted ROS membranes were resuspended in ROS 7 and stored at -70 "C.

PDE Purification-Undepleted ROS membranes (see above) were washed five times in ROS 7 under dim red light and pelleted each time at 15,000 rpm for 15 min (Sorvall SS-34 rotor). Soluble PDE (nonactivated) was eluted from the washed membranes by resuspend- ing the pellet in 5 ml of ROS 8 (10 mM Tris, pH 7.4, 0.1 mM dithioerythritol, 0.2 mM €"SF, 10 M leupeptin, 1 M pepstatin), homogenizing with a 26-gauge needle, and incubating under bright light for 30 min on ice. Anti-proteases were omitted in preparations to be subsequently treated with proteases. This procedure was repeated three times, and the pooled supernatants containing soluble PDE were dialyzed against HPLC buffer (20 mM sodium phosphate, pH 6.8, 50 mM sodium sulfate, 1 mM dithioerythritol) and concen- trated against Aquacide 111. PDE was further purified by HPLC on a TSK G3000SW column (7.5 X 75 mm) using the same HPLC buffer. Fractions were monitored by absorbance at 280 nm, and PDE was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (21) and Western blotting (using peptide antisera to both catalytic and inhibitory subunits of ROS PDE).

PDEy Purification-Native PDEy was purified from purified PDE (see above) by the method of Hurley and Stryer (10). The pH of the PDE-containing supernatant was lowered by the addition of formic acid to a final concentration of 0.1 M. After heating at 70 "C for 5 min, the pH was readjusted to 6.5 (or until precipitate formed) with dropwise addition of 4 N NaOH. After spinning at 14,500 rpm for 15 min (Sorvall SS-34), the supernatant containing PDEy was removed, the pellet was resuspended in PDE buffer (50 mM Tris, pH 7.2, 20 mM MgCI,), and the above procedure was repeated on the pellet. Pooled supernatants were then aliquoted and stored at -70 "C until utilized as the PDEy source.

Recombinant PDE-y was kindly provided by R. Lane Brown and Lubert Stryer. A fusion protein containing PDE was expressed, purified, and cleaved as previously described (17).

EDTA-depleted Membranes-After depletion of PDE from purified ROS membranes (see above, PDE Purification), further depletion was achieved by washing ROS membranes four times in depletion buffer (10 mM Tris, pH 7.4, 0.1 mM dithioerythritol, and 1 mM EDTA). Following a final wash omitting the EDTA, membranes were suspended in depletion buffer without EDTA. These-EDTA depleted membranes were used as a source of membrane-bound PDE.

PDE Actiuity Assay-PDE activity was determined using the assay of Thompson and Appleman (22). The final reaction was 50 mM Tris, pH 7.4, 5 mM MgCl,, 40 p~ cGMP, [3H]cGMP (40,000 cpm/tube, specific activity 15 Ci/mmol) in a final volume of 400 pl. Reactions were incubated at 30 "C. Purified PDEy and/or peptides were added as indicated.

Peptide Synthesis and Antisera Production-Peptides corresponding to indicated PDEa sequences were synthesized manually by the method of Merrifield (23) as modified by Gorman (24). Cleavage of the peptide from the resin and protecting groups was accomplished with anhydrous HF (25).

Peptides were quantitated using reverse-phase HPLC with a Vydak C-18 column and a 10-50% gradient of acetonitrile in 0.01 M sodium phosphate, pH 7.0, and using o-phthalaldehyde as a detecting reagent (26). Peptides (5 pl) were hydrolyzed for 12 h under vacuum using 6 N HCI at 110 "C, followed by lyophilization to remove the HCl. Free amino acids were dissolved in 100 pl of water, and 10-p1 aliquots were

injected and quantitated by comparison to known amino acid standards using a Shimadzu peak integrator.

Peptide antisera were produced by cross-linking to keyhole limpet hemocyanin (KLH) by the procedure of Takemoto et al. (27). Briefly, 0.2 ml of KLH (15 mg/ml) was added to 0.2 ml of peptide (5 mg/ml), followed by the addition of 0.2 ml of 15 mM glutaraldehyde for cross- linking. The mixture was incubated with agitation at room temperature for 12 h. The reaction was quenched by the addition of 1 ml of sodium borohydride, then dialyzed against 1 mM ammonium bicar- bonate, pH 7.0. Rabbits were injected initially with 200 p1 of peptide- KLH and 200 pl of Freund's complete adjuvant, followed by biweekly injection of 200 pl of peptide-KLH and 200 pl of Freund's incomplete adjuvant. Sera were tested after 4 weeks by a ROS protein Western blot and radioimmunoassays using the peptide the antisera was directed against.

Protein Determination-Protein concentrations were determined by the method of Bradford (28) or by scanning gel densitometry of Coomassie Blue- stained SDS-PAGE gels (21) using bovine serum albumin standards. Gels were scanned on a Gilford multimedia den- sitometer using a Shimadzu integrator.

Binding Assay-The solid-phase radioimmunoassay (29) was adapted for a binding assay by the following procedure. Polystyrene tubes (12 X 75 mm) were incubated with 0.2% glutaraldehyde in 0.5 ml of buffer A (0.1 M sodium phosphate, pH 5.0) and agitated at room temperature for 3 h. After washing three times with buffer A, peptides were added as described in the figure legends at 10 pgltube in a volume of 200 pl of buffer B (0.1 M sodium phosphate, pH 8.0). Tubes were incubated at 37 "C for 1 h, followed by washing two times with buffer C (0.15 M NaC1,0.5% Tween 20), and one time with buffer B. Purified native PDE-y (0.1 pg) was added to a final volume of 300 pl in buffer B and agitated at room temperature for 1 h. Tubes were then rinsed twice with buffer B and incubated with 0.2% glutaraldehyde in buffer B for 15 min under agitation at room temperature. After washing two times with buffer C and one time with buffer D (0.1 M sodium phosphate, pH 7.4, 0.15 M NaCl, 0.05% Tween 201, antisera as indicated were added at 1: lOO in buffer D, and tubes were agitated at room temperature overnight. Following washes three times with buffer E (10 mM Tris, pH 8.0, 0.05% Tween 20) and two times with water, 0.5 ml of buffer E containing 1251-labeled protein A (1 X lo6 cpm/tube) was added to each tube, and tubes were agitated for 1 h at room temperature. After washing two times with buffer E and two times with water, tubes were counted in a Beckman y-counter. The results of concentration curves indicate that each tube can bind up to 1 pg of peptide before saturation occurs; therefore, the peptide was saturating. PDEy was added to give maximum binding (0.1 pg/ tube). Additional PDE-y did not increase counts/min bound regardless of peptide used. All binding curves were, therefore, at saturating concentrations of peptide and PDEy.

Western Blot-Proteins were separated by SDS-PAGE (21) and transferred to nitrocellulose using a Genie transfer apparatus (Idea Scientific, Corvallis, OR). After blocking in 2% bovine serum albumin, blots were incubated with appropriate antisera, followed by washing and reaction with lWI-protein A. Exposure of the radioactive blots (usually overnight) to Kodak XAR x-ray film and subsequent development allowed for visualization of proteins.

RESULTS

Antisera were made against specific PDEa peptide sequences. A directory of antisera used in this study is given in Table I. Nitrocellulose strips containing separated PDEa and PDEp were reacted against the various antisera (Fig. 1). Antisera a-NT and a-840 react specifically with the lower band (PDEa), while a-740 and a-810 react with both bands (PDEa and PDEP). Note that, although the amino acid com- position of PDEa predicts a molecular mass of 88 kDa (7) and that of PDEp predicts a molecular mass of 84 kDa (8), migration on the gel is reversed in this system.

A urea-depleted rod outer segment membrane preparation was digested for 30 min at 37 "C with trypsin (1 mg/ml). After SDS-PAGE of the digest, the gel was transferred to nitrocellulose and probed with anti-peptide antisera (Fig. 2). Trypsin digestion resulted in the appearance of a stable 34-kDa membrane-bound fragment of PDE. The fragment was no longer reactive to antisera a-NT, but still reacted with a-740, indi-

Identification of PDE? Binding Sites on PDEa 16609

TABLE I Directory of peptide antisera

Peptide antisera were produced by injecting rabbits subcutaneously with synthetic peptides linked to KLH (see “Experimental Proce- dures”). Bovine PDEa sequences were obtained from Ovchinnikov et al. (7); PDEy sequence was from Ovchinnikov et al. (32).

Amino acid residues Sequence Antisera

0-NT PDEa 1-15 GEVTAEEVEKFLDSN a-740 PDEa 740-754 EWEQGDLERTVLQQ a-810 PDEa 810-824 ALADEYETKMKGLEE a-840 PDEa 840-854 QHGGKQPGGGRASKS 7-1-49 PDEy 1-49 MNLEPPKAEIRSATR

VMGGPVTPRKGPPKF KQRQTRQFKSKPPKK GVQG

B - a- d

FIG. 1. Peptide antisera reactions with purified PDEcuS. HPLC-purified PDE (1 pg) was electrophoresed on a 7.5% polyacrylamide gel. The dye front was allowed to exit, and electrophoresis continued until separation of PDEa and PDEp was achieved. PDEa and PDEp are labeled accordingly. Antisera reactions are: lane a, a- NT; lane b, a-740; lane c, a-810; and lane d , a-840. Due to the high reactivity of a-NT, autoradiography of lane a was for 12 h; all other lanes were for 24 h.

a ” b C d

180- 116- a4 - 5a -

48.5 9

36.5 -

cating a partial destruction of the N terminus of PDEa in the fragment. By using peptide antisera, the 34-kDa fragment was estimated to span residues approximately 450-825 of PDEaP (data not shown). Since a-740 reacts with both PDEa and PDEP, it is probable that the 34-kDa fragment contains fragments of both subunits. Although catalysis was reduced, the incubation mixture containing the trypsin-generated fragment still exhibited activity. However, this activity was no longer responsive to (inhibited by) the addition of purified native PDEy (Table 11), indicating the loss of a PDEy- responsive region during proteolysis.

In order to identify the effect of trypsin activation on the structure of PDEaP, a trypsin-activated PDE (short term digest, 1 min) was electrophoresed and examined by Western blot. The blot was probed with antisera to both N- and C- terminal peptides of PDEa, some which react with PDEp as well (Fig. 3). Antisera to a-NT reacted with the trypsin- activated PDE, as did antisera to C-terminal peptides (a-810 and a-840) and to the proposed catalytic region (a-740). This implies that after short trypsin digestion, the PDEap catalytic subunits are relatively intact. Reaction with peptide antisera to the C terminus of PDEy indicated a loss of PDEy after short term trypsin digestion (data not shown). The activity of the trypsin-activated PDE could still be inhibited by PDEy (Table 111).

TABLE I1 PDE activity of control and trypsin-digested membrane PDE

Membrane PDE (1 pg) was digested with 0.1 pg of trypsin for 1 min on ice (control) or for 15 min at 30 “C (trypsin-digested membrane PDE) and assayed for PDE activity (see “Experimental Pro- cedures”) with increasing amounts of PDEr. Background counts/min have been subtracted from control and trypsin-digested membrane PDE samples (maximum counts/min = 25,000; background is approximately 10% of maximum counts/min).

Sample PDEr added Counts/min” % Inhibition*

Control 0 0.05 0.10 0.50 1.00

membrane PDE 0.05 0.10 0.50 1.00

Trypsin-digested 0

7,300 6,100 4,500 2,100

0 3,400 3,300 3,400 3,200 3,300

0 17 38 72

100 0 0 0 4 1

Values are the means of duplicates. ’ % Inhibition = counts/min of a trypsin-activated PDE minus counts/min of sample divided by counts/min of a trypsin-activated PDE minus counts/min of a PDEy-inhibited PDE.

a b c d e f g h

180 - 116- 84- 0 -

4a.5- 36.5-

FIG. 2. Trypsin digest of membrane-bound PDE. Membrane PDE (1 pg) was undigested (lanes a and c) or digested with 0.1 pg of soluble trypsin for 15 min, 30 “C (lanes b and d ) and separated by SDS-PAGE. After Western blotting, samples were reacted with a- NT (lanes a and b ) or a-740 (lanes c and d ) .

FIG. 3. Trypsin digest of soluble PDE. Soluble PDE (1.0 pg) was undigested (lanes a, c, e, and g) or digested with 0.3 unit of insoluble trypsin for 2 min on ice (lanes b, d, f, and h). After separation on SDS-PAGE, Western blots were reacted with a-NT (lanes a and b ) , a-740 (lanes c and d ) , a-810 (lanes e and f ) , and a-840 (lanes g and h).

16610 Identification of PDEy Binding Sites on PDEa TABLE I11

Activity of trypsin-activated PDE Soluble PDE (0.01 pg, 1.4 X 10”’ p M ) was digested with insoluble

trypsin (0.1 unit) for 2 min on ice and assayed for PDE activity (see “Experimental Procedures”) f 0.2 pg (5.5 X p ~ ) of PDEy. Background counts/min have been subtracted from all samples (maximum counts/min = 21,700, background counts/min = 1800).

Sample Counts/min“ % of maximum

Undigested PDE 5,100 (f154) 24 Undigested PDE + PDEy 5,200 (f497) 24 Trypsin-digested PDE 11,800 (f142) 54 Trypsin-digested PDE + PDEy 5,900 (f579) 27

Values are means of triplicates.

TABLE IV PDEa peptide competition

PDEa peptides (100 pg, 1.25 X p ~ ) were incubated at room temperature for 10 min with recombinant PDEy (0.04 pg, 1.1 X lo-@ pM). Soluble PDE (0.01 pg, 1.4 X 10”’ pM) was digested with 1 unit of insoluble trypsin (2 min on ice), then added to the PDEy-peptide incubation mixture, and assayed for PDE activity (see “Experimental Procedures”).

a-Peptide % Inhibited” a-Peptide % Inhibited“

1-15 97 403-416 100 16-30 53 437-449 90 30-44 95 453-467 83 46-60 100 471-485 100 63-77 100 493-507 93 78-90 31 520-534 87 91-105 93 535-549 80

112-126 97 549-563 71 134-148 92 578-592 91 148-162 89 601-615 95 164-178 91 646-660 97 180-194 90 676-689 94 195-209 99 685-699 100 223-235 99 700-714 100 229-243 98 715-729 100 246-260 100 730-744 100 260-274 100 740-755 100 273-287 100 776-780 100 287-301 100 781-795 100 301-315 100 796-810 100 339-353 100 811-825 100 351-365 100 823-837 97 366-380 100 840-854 87 389-403 75 844-858 100

a % Inhibited = counts/min of a trypsin-activated PDE minus the counts/min when peptide is included in the incubation, all divided by the counts/min of a trypsin-activated PDE minus the counts/min of a PDEy-inhibited PDE.

PDEa peptides of approximately 15 amino acids were synthesized for use in peptide competition studies. These experiments were designed to identify the regions on PDEa which interact with PDEy. Peptides which interfere with the binding of PDEy to PDEa (ie. peptides which bind to the region on PDEy that interacts with PDEa and mimic the PDEy binding site on PDEa) may prevent the inhibition of an activated PDE in a PDE activity assay. Recombinant PDEy was incubated separately with each peptide, and, after adding purified trypsin-activated PDE to the incubation, a PDE assay was performed (Table IV). A large molar excess of PDEy was used in comparison to PDEaP (approximately 80-fold) in order to achieve maximum inhibition. Peptides near the N-terminal end of PDEa, specifically peptides 16-30 and 78-90, prevented the reinhibition by PDEy of trypsin-activated PDE. Peptide 78-90 was the most effective. Peptides 389-403,535- 549, and 549-563 showed a weak competiton (Table IV) which was not concentration-dependent (Fig. 4). Prevention of PDEy inhibition of PDEa by the N-terminal PDE peptides

04 ’. ..

0.0 50 0 100.0 150.0 200.0 250.0

PDEa peptlde (pM)

FIG. 4. Competition of PDEr inhibition by increasing amounts of peptide. PDEa peptides were incubated as indicated with 0.04 pg of recombinant PDEy for 10 min, followed by addition of trypsin-activated PDE (0.01 pg) and subsequent PDE assay (see “Experimental Procedures”). PDEa peptides used were: 16-30

(0- . X I ) , and 549-563 (C - 4). (0--O), 78-90 (0---0), 389-403 (A. . . . .A), 535-549

100 Tp.,

0

0 0 0 25 0 50 0

PDEa peptide (pM)

FIG. 5. Competition of PDEr inhibition by various concentrations and combinations of PDEa peptides. Peptides were incubated as indicated (concentrations given are for each individual peptide) with 0.04 pg of recombinant PDEr for 10 min, followed by introduction of trypsin-activated PDE (0.01 pg) and subsequent PDE assay. Combinations of PDEa peptides were: 16-30 + 78-90 (0-”o), 535-549 + 549-563 (0- - -0), and 16-30 + 78-90 + 389- 403 + 535-549 + 549-563 (A . . . . .A).

was concentration-dependent for peptides 16-30 and 78-90 (Fig. 4). Peptide 78-90 is approximately 2 to 3 times more effective in preventing PDEy inhibition than peptide 16-30.

Combinations of PDEa peptides were incubated with recombinant PDEy and then added to a trypsin-activated PDE for assay of PDE activity (Fig. 5). Peptides in the N-terminal region of PDEa (peptides 16-30 and 78-90) were more effective in preventing recombinant PDEy inhibition of trypsin- activated PDE than were those near the center of PDEa (peptides 535-549 and 549-563).

As an alternate method of identifying interaction sites, binding radioimmunoassays were designed to investigate direct native PDEy binding to specific PDEa peptides. The results of these binding assays are presented in Table V. In agreement with the peptide competition experiments that measured the prevention of inhibition of PDE activity, direct PDEy binding in the N terminus was found to be within residues 16-30 and 78-90. Other peptides (approximately 471- 534) were also able to bind PDE7; however, only peptide 78- 90 completely prevented PDEy inhibition (Fig. 4) and exhib-

Identification of PDEr Binding Sites on PDEa TABLE V

Binding of PDEy to PDEa peptides Radioimmunoassays (see "Experimental Procedures") were performed with 10 pg of each peptide fixed to

polystyrene tubes and incubated with (+) or without (-) 0.1 pg of PDEr. After glutaraldehyde fixing any bound PDEr, antisera y-1-49 (1:lOO) was reacted with bound PDEy. PDEy only counts/min = 4094 f 618. PDEy background (PDEr added after first blocking with Tween 20) counts/min = 87 f 11. Background counts/min = 107 f 9.

16611

Peptide Counts/rnin (-PDEr) Counts/rnin (+PDEr) Peptide Countdrnin f-PDEr) Countdmin f+PDEr)

1-15 16-30 31-45 46-60 63-77 78-90 91-105

112-126 134-148 148-162 164-178 186-200 195-209 212-226 223-235 229-243 246-260 260-274 273-287 287-301 301-315 339-353 351-365 366-380 378-392

135 (f41) 166 (rt30) 145 (f37) 125 (f27) 84 (22)

334 (k78) 157 (f54) 233 (f18) 677 (f167) 258 (+85) 170 (rt78) 302 (+39) 302 (f45) 255 (+21) 322 (f81) 303 (f35) 209 (33) 156 (f13) 190 (+74) 116 (f36) 149 (f43) 149 (f43) 311 (f78) 363 (f167) 725 (f59)

163 (f78) 586 (f155) 120 (f36) 88 (f16)

158 ( f 5 ) 2656 (f805) 234 (f6) 199 (f75) 619 ( 5 7 ) 220 (f50) 276 (f25) 282 (f19) 368 (f83) 297 (f16) 392 (f78) 281 (f37) 148 (f13) 219 (f137) 190 (f48) 138 (f19) 288 (f62) 189 (f14) 287 (f10) 334 (f40) 755 (f53)

389-403 403-416 437-449 453-467 471-485 493-501 520-534 535-549 549-563 578-592 601-615 646-660 676-689 685-699 700-714 715-729 730-744 740-755 766-780 776-780 781-795 796-810 811-825 823-837 840-854

445 (f210) 1101 (f430) 293 (f42) 395 (f75) 301 (f37) 283 (f56) 294 (f46) 477 (f114) 880 (f36) 177 (f92) 156 (f45) 400 (f101) 338 (f124) 101 (f11) 129 (f14) 112 (f16) 164 (f10) 175 ( 5 9 ) 254 (f105) 115 (f15) 173 (f29) 173 (f48) 192 (f32) 218 (f55) 200 (f23)

463 (f42) 712 (f385) 332 (f54)

1403 (f224) 676 (f76)

799 (f46) 495 (f51) 973 (f72) 199 (f41) 234 (f18) 523 (f16) 201 (f37) 131 (f15) 188 (f79) 147 (5~4) 175 (f69) 229 ( f 7 ) 181 (f40) 163 (+lo) 232 (f52) 203 (f38) 203 (f37) 230 (f55) 374 (f35)

537 (f201)

1 10 20 30 40 50 60 70 80 90 100 1: - G E V T A E E V E K F L D S N V S F A Q Y Y N L R Y R A K V I S D L L G P R 2: - M S P S E C Q M m F L D Q N P C F ~ Y F G R K L S P E D V A N A C E D G ~ E - G C T S F R E L C Q Y E E S A A L F E L V Q D M Q E ~ R ~ K I L R R L C S I L ~ P C S H ~ 3: M C E I S Q E ~ K Y L E A N P Q F A Y F N R K L P V E V P S - - - - C G A Q N A P A S A S F P G R T L A E ~ ~ Y L E L ~ E ~ L E ~ G S V ~ ~ Q ~ Q L ~ Q ~ R C S ~ L ~ * * * * * .*

101 110 120 130 140 150 160 170 180 190 200 1: ARHCIAELATRLFNYMCDA~EECLVNAPDSEIYFPLDMGWG~~SKKIVNYPNTEEDEHFCDFMTLTEY~T~ILASPI~CKDWAIIHAVHKWC 2: Q R N G V A E U T R L F S V Q P D S ~ E D C L Y P P D S E I Y F P L D I G W G ~ A Q ~ Q D ~ C F H F S S F A D E L T D ~ R N I L A I P I ~ C ~ W A V I ~ ~ ~ 3: A R N G T P E V A S K L L D ~ P T S K F E D N L Y Y P D R W V F P L D V G I V G ~ ~ T K K T F ~ D ~ S H F S D ~ K Q T G ~ ~ L U I P I ~ ~ ~ ~ A *** * * * * "* ** * ***** * ** ** ** ** "* ** *t ** . ***** *

201 210 220 230 240 250 260 270 280 290 300 1: F H F T E N D E E I L L K Y L N F A N L I H K V F H L S Y L H N C E T R R G q I 2: FCFTVNDEDYFLKYLNFGTLNLKIYHLSYLHNCETRRGQnLWSAYFEELTDIERQFHKAFYTVRAYLNCDRYSVGLLDM-FFD~P~EAQ 3: S E F S K Q ~ ~ E v F S ~ ~ t ; S f V S I I L ~ H ~ T N ~ 4 Y f l I ~ S ~ S ? I ~ ~ ~ ~ ~ ~ ~ t ; ~ ~ ~ ~ ? ~ ~ ~ ~ T Y t ; ~ ~ E ~ ~ ~ I ~ ~ ~ ~ ~ ~ Y ~ E ~ ~ ~ ~ ~

301 310 1: PYAGPRTPM;REINFYKVIDYILHGKEDIKVIPNPPPDHWALVSGLPTWAQNGLICNILHNNAPSEDFFAFQKEPLDESG~I~S~I~EIVCV

320 330 340 350 360 370 380 390 400

2: AYSCPRTPDGREILFYKVIDYILHC1(EDIKVIPSPP~HUALASCLPTWAESGFICNIMI~MEHFNFQECPLDDSCWI~S~I~SI~V I ** ****** *** ********* ***** ** *** * ******** * *** *** * ** " " ** *.*** *.****. ****

generated using SEQALIGN2 propam. 1: A T F Y ~ G ~ F D E ~ E T L ~ S ~ Q F L G W S ~ N P D T Y E L ~ E ~ I F Q D ~ Y ~ C D N E E I Q T I L K T ~ ~ G ~ P - U E C E E E E L A E I L ~ E L P D A D 401 410 420 430 440 450 460 470 480 690 500

Exact matches are denoted by 2: A T F Y N R K D C K P F D E Q D E ~ M E S L T Q F L G W S ~ N T D T Y D ~ L E ~ K D I A Q D ~ Y ~ C D R E E l Q L I L P T R E ~ G K E P - A D C E E D E L C K I L ~ - ~ P G P A

FIG. 6. Sequence comparison be- 3 : PYKCPKTPDGREVIFYKIIDYILHCKEEIKVIPTPPNDHWTLISCLPTWAENGFICNHLNAPMEYFTFQKCPVDETG~I~SLPIVHKK~V

tween three ROS PDEs. Comparison

3 : ATFYHRKDGKPFDEYDEHIAETLTQFLGUSLLNTDTYE~N~ENRKDIAQE~LHNHTKAIPDEIKSILWKEI(LNIDVIBDCEEKQLVTILKEDLPDPR below the seauences. 1. PDEa: 2. PDEB: *******""***** ** * ****** ** *** ********** * ** "* " *.* " ** t.

3, PDEa'. , ,

501 510 520 530 540 550 560 570 580 590 600 1: K Y E I N W H F S D L P L T E L E L V K C G I Q ~ Y E L K ~ ~ H I F Q E A L ~ S L S K G ~ I T Y H N ~ G F N V G Q T W S L L ~ G ~ ~ ~ ~ D L ~ ~ C E 2: W D I Y E F H F S D L E C T E L E L V K C C I Q ~ Y E L G V V R K F Q I F Q E ~ ~ L F S V S K C ~ I T Y H ~ G F N V A Q T W T L ~ G ~ K S Y Y I D L ~ ~ ~ L C H 3 : I A D L Y E ~ P I T E H E L I K C G L ~ F F ~ I ~ K F K ~ ~ ~ T R ~ ~ G ~ V T Y H N ~ C F ~ T ~ T L ~ G ~ ~ ~ I D L ~ ~ C E

*t ** **" * * * * e * * * **** ***e*****"* **** ** ** ** *e*.. ** . ". 601 610 620 630 640 650 660 670 680 690 700

1: D I D ~ T N N L Y Q ~ S Q N P W H G S S I L E R H H L E F G K T L L R D E S L N I F Q N L ~ Q H E ~ I ~ I A I I A T D W Y C K K R ? H F Q K I V S K T Y E I ~ E ~ Q 2: DIDM(GTNNLYQ~SQNPLHGSSILERHHLEFGKFLLSEETLNIYQNL~Q~~IHLWIAIIATDLALYFKKR?HFQKIMESINYEDRKSYn 3: DIDARGTNNLYQ~STSPLARLHCSSILERHHLEYSKTLLQDESLNIFQNL~QYENIHLFEVAIIATDWYFKKRTWQKIVDACEIEEEAIK ***" .I********* *** ************* * ** * *** **** ** * ** *e******** .******e*** "

701 710 1: Y H H L D 9 T R K E I ~ T A C D L S A I T K P U E V Q S K V ~ L V ~ F W E ~ D L E R ~ ~ N P I P ~ ~ E L P ~ Q V G F I D F V C I F ~ F S ~ H E E I T P ~

720 730 740 750 760 770 780 790 BOO

2: Y L S L E T T R K E I ~ T A C D L S A I T K P U E V Q S K V A L L V ~ F U E Q G D L E R ~ ~ P I P ~ ~ L P K L Q V G F I D F V C I ~ F S ~ H E E I L P ~ 3: ~ I D P T K K E I I ~ T A C D L S A I T K P U E V Q S Q V ~ L V A N E F U E ~ D L E R ~ ~ P I F ~ ~ E L P ~ Q V G F I D F V C T ~ F S ~ ~ I I P ~ * * *** *"***********"******* ****** ************* I ********* . . . . . . . . . . . . . . . . . . . . . . . . . *t .*

801 810 820 830 840 850 860 870 880 890 900 1: D C I T N H R K E Y K A L M E Y E T K G L E E E K Q K ~ ~ Q ~ G S Q H ~ K Q P G G G ~ A S K S C C V Q > 2: DRLQNHRKEUKALADEYEAKVKALEEDQ~TT~VGTEICNGGFNAPRSSTCRIL----> 3: N G L Q N N R V E U K S L A D E Y D E K V I E E M U ( q E E C N T T E K A V - - > *** *** **.** * * f * **

ited high PDEy binding (Table V). was observed in a region which has been proposed as the By using the computer program SEQALIGN,' comparisons catalytic domain (30). Regions of greatest divergency were

were made of retinal phosphodiesterases, ROS a, p, and cone observed in the N and C termini. This includes regions we outer segment a' (Fig. 6) . The greatest degree of similarity have identified as involved in PDEy binding.

SEQALIGN is a program authored by K. L. Clark, D. C. Teller, DISCUSSION

and G. R. Reeck (manuscript in preparation), copyright 1989, Kansas The tightly membrane-bound form of PDE that was re- State University Research Foundation. peatedly observed after urea and/or EDTA treatment was

16612 Identification of PDEy Binding Sites on PDEa MODEL

PDEo DOMAINS

555-790 848-858

16-30 7 8 - 9 0

1"I

PDEY

interaction region

cGMP binding cGMP binding

region region

catalyllc regton membrane

bmding

reglon

FIG. 7. Model of PDEa domains. Line model of Droposed functional regions of the PDEa subunit. (See text for references.)

digested by trypsin to a stable 34-kDa fragment of PDE. We have utilized antisera that are specific for PDEa (a-NT) or that react with both PDEa and PDEp (a-740 and a-810) to detect the PDEa and PDEp subunits present after long term trypsin digestion. A fragment in the trypsin-digested PDE was no longer reactive to PDEa peptide antisera, a-NT, indicating a loss of the N terminus of PDEa. Reactivity with a-740 and a-810, together with its size estimation from its migration on SDS-PAGE, indicates the approximate location of the 34-kDa fragment is within PDEaP residues 450-825. Although the activity is reduced in the trypsin-digested PDE, the incubation mixture containing the 34-kDa fragment still exhibited a preference for cGMP (data not shown). The reduction in activity may be attributed to the loss of allosteric cGMP binding regions in the N terminus or may indicate a loss of comformation in the active site due to proteolysis. Since the sequence of PDEp is similar within this region, and since no other bands reactive to a-740 antisera were observed, it is probable that the 34-kDa fragment represents both PDEa and -p catalytic regions.

Activity assays show that purified native PDEy added exogenously can no longer inhibit the trypsin-digested membrane PDE produced by trypsin hydrolysis. Since the N terminus of PDEa is no longer present in the 34-kDa fragment (Fig. 2, lanes a and b) and PDEy can no longer inhibit the trypsin-digested PDE containing this fragment, it appears that a PDEy binding region is destroyed by trypsin hydrolysis of the membrane PDE, and the binding region may lie in the lost N-terminal portion of the protein. Attempts to separate the intact fragment from the digestion mixture were not successful.

Short term trypsin hydrolysis of a soluble form of PDE, which has been used as a PDE activation procedure (lo), appears to destroy the PDEy, subunits, leaving the PDEa and -p catalytic subunits relatively intact. This activated PDE can be reinhibited by reintroducing PDEy into the assay mixture. When probed with antisera to the N-terminal region of PDEa (a-NT), the antisera reactivity of the N terminus is not lost during this short term trypsin proteolysis, in contrast to the long term hydrolysis of the 34-kDa fragment of the membrane PDE. This may be an indication that an intact N terminus on PDEa is necessary for binding to PDEy (and subsequent inhibition of catalytic activity).

The ability of N-terminal PDEa peptides to prevent the inhibition of a trypsin-activated PDE indicates that a PDEy binding region may be located within an N-terminal segment of approximately residues 16-30 and 78-90 on PDEa. The linearity of the peptide concentration curves up to 100 nM using PDEa peptides 16-30 and 78-90 in peptide competition assays indicates this is the region of highest interaction between PDEy and PDEa. Radioimmunoassays indicate that

peptides 16-30 and 78-90 directly bind PDEy. Two binding sites have been identified on PDEy (1, 17).

These include an inhibitory site that includes residues 63-87 and a binding site that includes residues 24-45 of PDEy. Since two interaction sites have been identified on PDEy, two corresponding sites may be present on PDEa. From these results, it appears that a strong candidate lies within residues 78-90 of PDEa, while a weaker site may be present on residues

A model for the domains of PDEa can now be constructed using information obtained from sequence analyses and biochemical studies (Fig. 7). Two regions (16-30, 78-90) are proposed as possible PDEy interaction sites, based on the information contained in this paper. The cGMP allosteric binding regions (89-251 and 295-464) are indicated based on the work of Charbonneau et al. (31), Li et al. (19), and Stroop et al. (33). The catalytic region identified by sequence analyses (30), identification of a cGMP binding region (33), and biochemical studies revealed in this paper extend from residues approximately 555-790. The membrane binding region has been identified by sequence information and is proposed to be within the last 10 residues of the C terminus (9, 19).

16-30.

Acknowledgments-We wish to thank Kirk Clark for his assistance in sequence comparisons and R. Lane Brown and Lubert Stryer for providing the expression vector containing PDEr.

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Documents

Identification of the retinal cyclic GMP phosphodiesterase inhibitory