©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Carboxyl Terminus of the -Subunit of Rod cGMP Phosphodiesterase Contains Distinct Sites of Interaction with the Enzyme Catalytic Subunits and the -Subunit of Transducin (*)

Nikolai P. Skiba, Nikolai O. Artemyev(§), and Heidi E. Hamm (¶)

From the (1) Department of Physiology and Biophysics, University of Illinois, College of Medicine, Chicago, Illinois 60680

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The interaction between the GTP-bound form of the transducin -subunit (G) and the -subunit (P) of cGMP phosphodiesterase (PDE) is a key event in effector activation during photon signal transduction. The carboxyl-terminal half of P is involved in interaction with G as well as in inhibition of PDE activity. Here we have utilized a combination of synthetic peptide and mutagenesis approaches to localize specific regions of the carboxyl-terminal region of P interacting with G and P and have determined residues involved in inhibition of PDE activity. We found that synthetic peptide corresponding to residues 68-87 of P completely inhibit trypsin-activated PDE. The peptide P-63-87 bound to GGTPS with a K of 2.5 µM, whereas the binding of P-68-87 to GGTPS was approximately 15-fold less (K= 40 µM) suggesting that carboxyl-terminal P region 68-87 contains a site for interaction with P and also a part of the binding site. To map G and P sites more precisely within the carboxyl-terminal region, a set of carboxyl-terminal mutants was generated by site-directed mutagenesis. Deletion of residues 63-69 and 70-76 diminished the binding of mutants to while binding to carboxyl-terminally truncated mutants lacking up to 11 amino acid residues was unchanged. In contrast, carboxyl-terminal truncations of P from 1 to 11 resulted in a gradual decrease of its inhibitory activity. Thus, the extreme carboxyl-terminal hydrophobic sequence -Ile-Ile together with 9 adjacent residues provides inhibitory interaction of P with P. The carboxyl-terminal GGTPS binding site of P is different from but adjacent to its PDE inhibitory site. During the visual transduction process, GGTP likely binds to this region of P inducing a displacement of the extreme carboxyl terminus from the inhibitory site on P, leading to PDE activation.


INTRODUCTION

In the phototransduction cascade of enzymatic reactions, the -subunit of the rod outer segment cGMP phosphodiesterase (P) regulates the catalytic function of the large phosphodiesterase subunits, and (P) (Hurley and Stryer, 1982). The retinal rod PDE() is composed of two catalytic subunits, (99.2 kDa) and (98.3 kDa), and two identical inhibitory -subunits (9.7 kDa) (Baehr et al., 1979; Ovchinnikov et al., 1986, 1987; Lipkin et al., 1990b; Deterre et al., 1988; Fung et al., 1990). Activation of the effector enzyme occurs upon binding of GGTP (G*) to P complexed with P and displacement of P from its inhibitory site on the catalytic subunits (Wensel and Stryer, 1986, 1990; Deterre et al., 1986). The activated PDE rapidly hydrolyzes cytoplasmic cGMP resulting in closure of plasma membrane cationic channels and hyperpolarization of the rod cell (for review, see Liebman et al.(1987) and Stryer(1991)). In the absence of photoreceptor membranes or at a low membrane concentration, the complex of G* with P dissociates from P (Deterre et al., 1986; Yamazaki et al., 1990; Wensel and Stryer, 1990). In the presence of high ROS membrane concentrations and under physiological ionic conditions the G*PP complex remains membrane-bound (Clerc and Bennett, 1992; Catty et al., 1992).

Despite intensive investigation, the molecular mechanism of the regulation of PDE activity involving interactions between G, P, and P, is still unclear. Significant progress has recently been achieved in the understanding of P functional structure. Peptide studies have demonstrated that the central positively charged region of P located within residues 24-45 (P-24-45) interacts with both G (Lipkin et al., 1988; Morrison et al., 1989; Artemyev et al., 1992, Artemyev et al., 1993a) and P (Artemyev and Hamm, 1992). The carboxyl-terminal tryptic peptide of P corresponding to residues 46-87 (P-46-87) also interacts with G (Artemyev et al., 1992) and inhibits P (Artemyev and Hamm, 1992). The last several carboxyl-terminal residues are required for the inhibitory interaction of P with P (Lipkin et al., 1988; Brown and Stryer, 1989; Brown, 1992). Mutational analysis of P has revealed that the positively charged residues Arg, Arg, Lys, and Lys are important for binding to P (Lipkin et al., 1990a; Brown, 1992; Lipkin et al., 1993). Despite the number of P mutants with impaired PDE inhibition activity, only two mutants so far have been reported to have diminished G binding properties: the triple mutant K41Q,K44Q,K45Q (Brown, 1992) and P W70F (Otto-Bruc et al., 1993). It is likely that the length of the spacer sequence linking the central and carboxyl-terminal sites for P interaction as well as Pro are important for their optimal orientation (Brown, 1992; Lipkin et al., 1993). Much less data have been accumulated concerning functional regions of G and P that are involved in interaction with P. We have recently reported that the synthetic G peptide corresponding to residues 293-314 activates PDE (Rarick et al., 1992) through interaction with a carboxyl-terminal fragment of P, residues 46-87 (Artemyev et al., 1992). This region of GGTPS is a part of a surface that, according to its recently solved crystal structure (Noel et al., 1993), forms a contiguous patch of exposed residues. Another three adjacent regions that are localized on one face of G and change conformation upon GDP-GTP exchange, switch I (Ser-Thr), switch II (Phe-Thr), and switch III (Asp-Arg) (Lambright et al., 1994) may also be potential PDE binding sites of G. Mutational analysis of recombinant G produced in Sf9/baculovirus expression system have shown that tryptophan 207 is involved in the activation-dependent binding to P (Faurobert et al., 1993). The information regarding regions of PDE catalytic subunits interacting with P is limited to the finding that synthetic peptides, representing distinct amino-terminal sites of P and P, exhibited P binding in solid-phase radioimmunoassay (Oppert et al., 1991; Oppert and Takemoto, 1991).

In this study we have combined synthetic peptide and mutagenesis approaches to map the functional sites of P located within its carboxyl-terminal region. We have determined the ability of the peptides P-63-87 and P-68-87 as well as several carboxyl-terminal P mutants to inhibit PDE activity and bind to G. We have assigned the carboxyl-terminal hydrophobic sequence -Ile-Ile plus nine adjacent residues as a P inhibitory site and a region internal to this, residues 63-76, as a carboxyl-terminal G binding site. Binding of G* to this site relieves the inhibition normally imposed on PDE by its -subunit and leads to PDE activation.


EXPERIMENTAL PROCEDURES

Materials

GTP, GTPS, cGMP, calf intestinal alkaline phosphatase, and all restriction and DNA modification enzymes were obtained from Boehringer Mannheim. Blue-Sepharose CL-6B and Fast Flow SP-Sepharose were products of Pharmacia Biotech Inc. Trypsin and soybean trypsin inhibitor were from Worthington. [-P]ATP (600 Ci/mmol) was from ICN Inc. All other analytical grade reagents were from Sigma or Fisher.

Preparation of GGTPS, PDE, and tPDE

Bovine ROS membranes were prepared by the method of Papermaster and Dreyer(1974) with some modifications (Mazzoni et al., 1991). The GGTPS was extracted from ROS membranes with GTPS and purified using chromatography on a Blue-Sepharose CL-6B column as described by Kleuss et al. (1987). PDE was extracted from ROS membranes according to the procedure described by Baehr et al.(1979). PDE was purified and tPDE was prepared using the protocol of Artemyev and Hamm(1992). The purified proteins were kept in 40% glycerol at -20 °C for several months with no loss of functional activity.

Expression of P in E. coli

Recombinant P was expressed under the control of the T7 promoter in E. coli BL21/DE3. The P expression vector was constructed and generously provided by J. Sondek (Yale University, New Haven, CT). This vector is a derivative of pET11a, where polymerase chain reaction-amplified P gene flanked with NdeI and BamHI sites was inserted between corresponding sites of the parent plasmid. The pLcIIFXSG plasmid DNA containing the synthetic P gene (Brown and Stryer, 1989) was the PCR template. The expression product was authentic full length P at 87 amino acids.

Construction of P Mutants

The genes encoding P3, -4, -5, -8 and P1, -2, -1I86F, 1I86N were constructed by inserting degenerate phosphorylated oligonucleotides I or III, correspondingly, into the expression vector cut with SacI and BamHI. P11 and -17 were constructed by replacement of the NcoI-SacI fragment of the P gene with degenerate phosphorylated oligonucleotide II. P70-76 and P63-69 were made by insertion of the synthetic duplex I or II, respectively, into the expression vector cut with KpnI and SacI. PG85A was prepared by replacing the P gene SacI-BamHI fragment with phosphorylated oligonucleotide IV (see for a list of the oligonucleotides). All DNA manipulations were performed using standard techniques (Maniatis et al., 1989). The sequences of all mutant P genes were confirmed by dideoxy DNA sequencing (Sanger et al., 1977).

Purification of Recombinant P and Mutants

E. coli cells, after a 4-h induction with 0.5 mM isopropyl-1-thio--D-galactopyranoside, were spun down, resuspended in 1:20 of a cell culture volume of buffer containing 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 50 mM NaCl, 1 mM dithiothreitol, 10 µg/ml pepstatin A, 5 µg/ml aprotinin, and 100 µM phenylmethylsulfonyl fluoride (buffer A). The cell suspension was sonicated, and insoluble material was spun out at 100,000 g for 60 min. The supernatant was loaded onto a SP-Sepharose fast flow cation exchange column equilibrated with buffer A and eluted off the column with buffer A containing 0.5 M NaCl. Recombinant proteins were additionally purified by reversed-phase chromatography on HPLC Vydac C-4 column using a gradient concentration of acetonitrile in 0.1% trifluoroacetic acid/HO. The purified proteins were lyophilized, dissolved in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM MgCl, 1 mM dithiothreitol, aliquoted and stored at -80 °C. The final yield of the different Ps ranged from 15 to 30 mg of more than 95% pure protein/liter of culture.

Peptide Synthesis

Peptides corresponding to residues 63-87 and 68-87 of P were synthesized by the solid-phase Merrifield method on an Applied Biosystems automated peptide synthesizer. The amino and carboxyl termini of the peptides were not modified. Peptides were purified by reversed-phase chromatography on a preparative Aquapore octyl column (25 1 cm) (Applied Biosystems). The purity and chemical formula of peptides were confirmed by fast atom bombardment mass spectrometry, analytical reversed-phase HPLC, and amino acid analysis.

PDE Activity Assay

Different amounts of P peptides or mutants were added to 50 pM tPDE in 200 µl of 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM MgCl, and 1 mM dithiothreitol. The reaction was initiated by the addition of 0.5 mM cGMP. Bacterial alkaline phosphatase (1 unit) was added to the reaction mixture immediately after substrate addition, and incubation was continued at 37 °C for 10 min. The reaction was stopped by adding 800 µl of 0.012% Triton X-100. Inorganic phosphate release was determined using ammonium molybdate as described by Eibl and Lands (1969). To prevent adsorption of proteins to the walls during the enzymatic reaction, siliconized glass test tubes were used.

Fluorescent Assay of Competition between PLY and P Mutants or Peptides for Binding to GGTPS

For fluorescent measurements, P was specifically labeled at its single Cys using the sulfhydryl specific reagent lucifer yellow vinylsulfone as described by Artemyev et al.(1992). Fluorescent assays were performed on a Perkin Elmer LS5B spectrofluorimeter in 10 mM HEPES pH 8.0, 100 mM NaCl, and 1 mM MgCl (buffer B) with excitation at 430 nm and emission at 520 nm. Fluorescence of PLY (50 nM) in the presence of GGTPS (100 nM) was measured before and after additions of increasing concentrations of P mutants or synthetic peptides. K values for all P mutants and peptides were determined from their competition curves based on calculated EC values and K= 36 nM for the PLYGGTPS complex (Artemyev et al., 1992). The affinity of the unlabeled recombinant P to GGTPS was slightly higher then that for PLY (K= 14 nM, see ``Results'' for details).

Miscellaneous Methods

Protein concentration was determined by the method of Bradford(1976) using bovine serum albumin as a standard. The concentrations of P and its mutants containing both Trp and Tyr were determined spectrophotometrically using = 6,700. The extinction coefficients for Tyr lacking mutants (P4, -5, -8, -11, -17) and Trp lacking mutant P70-76 were 5,300 and 1,400, respectively. Curve-fitting of the experimental data was performed with nonlinear least squares criteria using GraphPad InPlot 4.0 software.


RESULTS

The Carboxyl-terminal PDE Inhibitory Site of P Is Located within Region 68-87-Previous experiments have shown that the carboxyl-terminal half of P corresponding to residues 46-87 possesses PDE inhibitory activity (K= 0.8 µM) (Artemyev and Hamm, 1992; Artemyev et al., 1992), and the last 5 carboxyl-terminal amino acid residues are necessary for inhibitory binding to P (Brown, 1992). To understand how PDE inhibitory function is distributed within this region of P, we studied the functional properties of the synthetic peptides P-63-87 and P-68-87. Peptide P-68-87 was capable of completely inhibiting tPDE with K 0.8 µM (Fig. 1A). The K values obtained for both P-63-87 and P-46-87 were similar to that of peptide P-68-87 (data not shown), indicating that the carboxyl-terminal PDE inhibitory region lies mainly within amino acid residues 68 and 87. The Region 63-87 of P Contains the Carboxyl-terminal GBinding Site-The binding of P-63-87 and P-68-87 to GGTPS was estimated by competition with PLY for binding to GGTPS. The synthetic peptides had no effect on the fluorescence of PLY (data not shown). However, addition of either peptide reversed the fluorescent increase of PLY upon their binding to GGTPS due to their ability to compete with PLY for binding to GGTPS (Fig. 1B). A K of 2.5 µM for the P-63-87GGTPS complex was calculated based on the K for the PLYGGTPS complex (36 nM) (Artemyev et al., 1992) and the concentration of GGTPS in the assay (50 nM). This K value was similar to that of P-46-87 (K= 1.5 µM) and a shorter carboxyl-terminal peptide P-58-87 (K= 2 µM) (Artemyev et al., 1993b) indicating that the P region 46-62 does not contribute significantly to binding to GGTPS. The peptide P-68-87 showed 15-fold weaker binding to GGTPS (K= 40 µM) compared to P-63-87. Mutational Analysis of P Functional Regions within Residues 63-87-To determine carboxyl-terminal sites of P interaction with P and G more precisely, we generated a set of mutants containing deletions and substitutions of amino acid residues at the carboxyl terminus of P (see for details). The P mutants were analyzed for their ability to inhibit tPDE. The calculated K value for recombinant P from fitting of the inhibition curve (Fig. 2A) was approximately 10 pM, similar to the K reported earlier (Wensel and Stryer, 1986, 1990). Removing even one carboxyl-terminal amino acid residue (Ile) resulted in a 7-fold decrease of P inhibition activity (K= 75 pM); however, this mutant was still able to inhibit tPDE completely. Subsequent truncations of 2 to 5 amino acids not only further reduced the ability of P mutants to inhibit tPDE (K values ranged from 220 to 250 pM), but also gradually decreased their maximal inhibitory activity from 80% (P2) to 50% (P5) (Fig. 2B). The mutants with more profound truncations, P8 and P11, showed an additional decrease in PDE inhibition activity (K of 330 pM and 650 pM and a maximal effect of 50 and 40%, respectively). The P17 had the weakest inhibitory activity, and its maximal inhibitory effect was only 20% (Fig. 2, A and B). The mutants P70-76 and P63-69, containing internal deletions of amino acid residues but an intact extreme carboxyl-terminal sequence, were more than 20-fold weaker PDE inhibitors compared to P (K values of 230 and 270 pM correspondingly), but were able to inhibit tPDE completely (I).


Figure 1: A, effect of synthetic peptide P-68-87 on tPDE activity. The activity of tPDE (50 pM) was determined in the presence of increasing concentrations of synthetic peptide. Percent of maximal tPDE activity is plotted as a function of peptide concentration. B, competition between PLY and synthetic peptides P-63-87 or P-68-87 for binding to GGTPS. The fluorescence of PLY (50 nM) in the presence of GGTPS (100 nM) was measured before and after addition of increasing concentrations of P-63-87 (triangles) or P-68-87 (circles), and the relative fluorescent change (F/F) is plotted as a function of the peptide concentration.




Figure 2: Inhibition of PDE activity by P mutants. The activity of tPDE (50 pM) in the presence of increasing concentrations of P (filled circles), P1 (filled diamonds) (A), and also P2 (open circles), P4 (filled triangles), P8 (open diamonds), and P11 (open triangles) (B) was determined. Percent of maximal tPDE activity is plotted as a function of P or P mutant concentration.



The hydrophobicity of the carboxyl-terminal Ile-Ile cluster implies a hydrophobic interaction of this region with corresponding site(s) on the catalytic subunits that may form a complementary pocket. To test this idea, we constructed a set of carboxyl-terminal mutants to vary hydrophobicity as well as the steric pattern at this inhibitory site. Replacing -Ile-Ile with Phe partially restored the inhibitory activity of P2 and made this mutant very similar to P1 (I). Substitution of the Ile-Ile with hydrophilic Asn revealed similar inhibitory properties of this mutant as P2 (I). The role of the small flexible glycine at the third position from the carboxyl terminus was examined by substitution with the less flexible alanine. This mutant showed a 7-fold decrease in the potency of PDE inhibition (I) suggesting that for maximal inhibition of PDE the Ile-Ile hydrophobic tail of P must be flexible. The Carboxyl-terminal Site of Interaction of P with G Is Located within Amino Acid Residues 63-76-To determine the ability of P mutants to bind to G, we studied their ability to compete with PLY for binding to GGTPS in a fluorescent assay. In control experiments, the calculated K for P binding to GGTPS in competition with PLY was 14 nM. The binding of carboxyl-terminally truncated P mutants 1, 2, 3, 4, 5, and 8 to GGTPS was not changed (I), indicating that the carboxyl-terminal eight amino acid residues of P are not involved in binding to GGTPS. The binding of P11 to GGTPS was only slightly decreased (K= 30 nM, Fig. 3), suggesting that residues 77-79 are also not very significant for G binding. A more significant change in affinity to GGTPS occurred when 17 carboxyl-terminal residues were deleted (P17), resulting in a 6-fold decrease in affinity to GGTPS (K= 90 nM, Fig. 3). Peptide binding studies had implicated residues 63-67 as well as adjacent residues 68-87 in binding to G (Fig. 1B). Data on binding of carboxyl-terminally truncated P mutants to GGTPS allow the G binding site within region 68-87 to be restricted to residues 68-76. To test the role of amino acids in the 63-76 region, a series of internal deletion mutants were constructed. Deletion of amino acids 63-69 caused a drastic decrease in affinity for G (K= 230 nM, Fig. 3 ), while deletion of residues 70-76 resulted in a further drop in affinity (K= 460 nM, Fig. 3). Comparison of the primary structure of P70-76 and P17 () would suggest that the only meaningful difference in their sequence, assuming that region 77-87 is not critical for transducin binding, is Trp which is the last amino acid residue in P17 and is deleted in P70-76. The 5-fold difference in the affinity for GGTPS between P70-76 and P17 would implicate Trp in binding of P to GGTPS. The decreased affinity of P17 and P63-69 to GGTPS indicates that amino acids surrounding Trp within region 63-76 compose the carboxyl-terminal G binding site of P.


Figure 3: Competition between PLY and P mutants for binding to GGTPS. The fluorescence of PLY (50 nM) in the presence of GGTPS (100 nM) was measured before and after addition of increasing concentrations of P (open squares), P11 (filled squares), P17 (open triangles), P63-69 (filled triangles), and P70-76 (diamonds). The fluorescent change (F/F) is plotted as a function of P mutant concentration. The K values for all mutants were calculated from the competition curves as described under ``Experimental Procedures.''




DISCUSSION

The -subunit of PDE is a small molecule consisting of 87 amino acid residues. Despite its small size, P possesses three essential functions that play critical roles in visual signal transduction: interaction with P and PDE inhibition; interaction with transducin and PDE activation; and, finally, in a membrane complex G*PP, stimulation of transducin GTPase activity (Arshavsky et al., 1994; Angleson and Wensel, 1994). The important functional roles of P in the regulation of the light-induced enzymatic reactions in ROS combined with the simplicity of its structure make P a very attractive object for the study of molecular mechanisms of protein-protein interaction and provides insight into the detailed mechanism of PDE activation.

Biochemical, peptide, and mutagenesis studies have revealed two functional regions of P (central, residues 24-45, and carboxyl-terminal, residues 46-87) involved in interaction with both P and G. In solution, P by itself displays both sites for interaction with G. From the peptide studies it appears that the binding energy of PG interaction in solution (K= 14 nM) is distributed approximately equally between these two sites based on K values for the central and carboxyl-terminal peptides (0.7 µM and 1.5 µM respectively, Artemyev et al. (1992)). However, in the complex with P, the affinity of P to activated G, based on its K for PDE activation by GGTPS (1 µM), is significantly lower. This can be explained by an interaction of one or both of these sites with P in the P complex, leading to inaccessibility for GGTPS interaction. Our earlier data suggest that binding of G-293-314 to the carboxyl-terminal half of P causes PDE activation (Rarick et al., 1992; Artemyev et al., 1992) while the central region binds both PDE catalytic subunits (Artemyev and Hamm, 1992) and G. This suggests that the central region may not be available for G binding in the PDE holoenzyme, contributing to the low K of PDE activation in solution.

Because the interaction of G with the carboxyl-terminal region leads to PDE activation, in this study we investigate in more detail the binding sites within this region with P and G. We utilized synthetic peptides corresponding to residues 63-87 and 68-87 of P to gradually narrow down P and G binding sites within the carboxyl-terminal region 46-87. Both peptides completely inhibited tPDE with a K similar to that of the tryptic peptide P-46-87. This observation defines the carboxyl-terminal inhibitory domain within residues 68-87 and suggests that residues 46-67 are not critical in this function. The fluorescent studies on competition between PLY and synthetic peptides for binding to GGTPS have revealed a significant difference in their affinity to GGTPS. The binding of P-63-87 was similar to that of the previously reported longer carboxyl-terminal peptides P-58-87 (Artemyev et al., 1993b) and P-46-87 (Artemyev et al., 1992) suggesting that the P region between residues 46-62 is not critical for binding to GGTPS. The peptide P-68-87 bound to GGTPS approximately 15-fold weaker in comparison with the P-63-87. Thus, we conclude that P region 63-67 participates in the interaction with GGTPS; however, part of the G binding site is located within region 68-87.

To map the carboxyl-terminal sites of P interaction with P and G more precisely, we constructed 13 mutants containing systematic deletions and substitutions of carboxyl-terminal amino acid residues. We determined their ability to inhibit tPDE and bind to GGTPS in a competition fluorescent assay. All P mutants had impaired inhibitory function. A deletion of even one amino acid residue (P1) caused a 7-fold decrease in inhibitory activity. Subsequent deeper truncations of carboxyl-terminal residues resulted in a gradual decrease in the inhibitory activity of mutants. P11 and P17 were the weakest inhibitors of tPDE. Earlier data have shown that correct spacing and orientation of the central region (residues 24-45) and carboxyl-terminal inhibitory site are required for full inhibitory activity of P. The P mutant P69L had impaired inhibitory activity, presumably because of a change in the orientation of two sites of binding to P (Lipkin et al., 1993). Pro is a part of the deleted region in mutant P63-69 and may be responsible for the decrease in its inhibitory activity. The decrease in the inhibitory activities of P63-69 and P70-76 may also be caused by shortening of the necessary spacer length similar to the very weak inhibitor P54-61 (Brown, 1992). These observations indicate that the P carboxyl-terminal site interacting with P is composed of the last 11 residues and support data obtained with synthetic peptides.

The last two isoleucine residues of P are important for its inhibitory binding to P. Their removal causes more than a 20-fold decrease in inhibitory activity. Replacing the double isoleucine for phenylalanine did not completely restore the authentic inhibition of P, indicating that there is a tight hydrophobic fit of the -Ile-Ile cluster, presumably into a hydrophobic pocket on P. Previous data on the decreased affinity of a P variant, containing six additional hydrophobic residues on the carboxyl terminus, to P (Brown, 1992) supports this idea. Decreased inhibitory activity of the G85A mutant to P may mean that Gly provides flexibility of the carboxyl-terminal hydrophobic region necessary to match its partner site. Another explanation of this effect might be that Gly is involved in -turn formation. Glycine residues are frequently found in the second and third position of -turns which are often present in regions of molecular recognition (Wilmot and Thorton, 1988, 1990). Protein termini are also often sites of recognition. For example, the carboxyl termini of G-subunits are involved in recognition of receptors (Conklin et al., 1993). A recent NMR study on the structure of the carboxyl-terminal peptide 340-350 has identified that a similar structural motif -G-L-F-COOH has a -turn critical for its binding to rhodopsin (Dratz et al., 1993).

Our data on the inhibitory binding of P mutants make it attractive to predict some of the molecular events that take place upon PDE inhibition. The carboxyl-terminal 11 amino acid residues of P function as an inhibitory domain. The region 77-87 interacts with P likely in close proximity to its catalytic site(s) and induces a conformational change that imposes an inhibitory constraint. The Ile-Ile domain anchors in a complementary hydrophobic pocket on P and locks the inhibitory conformation of the P complex. Such a model satisfactorily explains the functional properties of the truncated mutants.

Studies on competition between PLY and P mutants for binding to GGTPS revealed that the carboxyl-terminal site interacting with is located in close proximity to the P binding site, but is not identical or overlapping. While GGTPS binding of mutants with truncations up to 11 residues was practically unchanged, deeper truncation (P17) and deletion of regions 70-76 and 63-69 significantly diminished their affinity to GGTPS. The truncation of amino acid residues 70-76 can be viewed as a deletion of a carboxyl-terminal part of the G binding core, and, thus, decreased affinity of P70-76 to G reflects the removal of a part of the carboxyl-terminal G binding site. The decreased affinity of P63-69 may be partially caused by a shortening of the distance between the 70-76 and the central regions of P. We cannot distinguish whether diminished affinity of P63-69 is simply a result of the partial removal of one G binding site or a composite effect of a change in the geometry of two binding sites combined with a deletion of a part of the G binding site. These data, together with data on synthetic peptide functions, also indicate that the residues within region 63-76 form the carboxyl-terminal site of P important for interaction with G. This observation explains why peptide P-68-87 has more than a 15-fold lower affinity to GGTPS compared to peptides P-46-87 and P-63-87, because it lacks several amino acid residues from the G binding site. A diminished affinity of a PW70F mutant to GGTPS (Otto-Bruc, 1993) is in good agreement with our data.

In our earlier studies defining sites on G of interaction with PDE we determined that a synthetic peptide encompassing residues 293-314 of G can activate PDE (Rarick et al., 1992) while directly binding to the carboxyl-terminal half of P (Artemyev et al., 1992). Our current data allow us to narrow the interface of such an activating interaction between G and PDE to residues 63-76 of P. This region of P is located in close proximity to its inhibitory site but does not overlap it and thus is available for coupling with GGTPS. The primary event of PDE activation appears to include interactions between the G region 293-314 and P region 63-76. We suggest that this interaction impairs the inhibitory binding of P region 77-87 to P, resulting in the physical removal of the carboxyl terminus of P from its inhibitory site on PDE. Thus, of the two G binding sites on P, our current and previous data indicate that the carboxyl-terminal site is sufficient for activating interaction with G. Alternatively GGTPS may bind to a composite site which includes also lysine residues 41, 44, and 45 (Brown, 1992) of the central region. The close agreement between the peptide binding and activation studies and the mutant studies make it unlikely that the carboxyl-terminal region is only secondarily involved in the PDE activation process. A conformational change at carboxyl terminus may cause a more profound perturbation in P structure leading to exposure of its central region for binding to GGTPS or reorientation of the central and carboxyl-terminal sites in an optimal way for G binding. Under some conditions, GGTPS can completely displace P from its complex with P in the absence of ROS membranes (Wensel and Stryer, 1990). In the presence of ROS membranes, the residual interaction of P with P via the central region as well as the anchoring of GGTPS and P on the membrane can hold the GGTPSPP complex together (Clerc and Bennet, 1992; Catty et al., 1992).

Given this information about the molecular details of activation of cGMP phosphodiesterase, an intriguing question is whether there is a conserved molecular mechanism of effector activation by G proteins. Similarities in amino acid sequence make it likely that -subunits of all heterotrimeric G proteins share the same basic three-dimensional structure. The recent finding of overall structural similarity of G and G (Noel et al., 1993; Lambright et al., 1994; Coleman et al., 1994) support this idea. The fact that one of the G regions involved in activation of adenylyl cyclase (residues 349-356) (Berlot and Bourne, 1992) corresponds to residues 307-314 of G important in PDE activation (Rarick et al., 1992) may indicate that the cognate regions of all G-subunits form effector interacting surfaces. Available information does not indicate any primary structure similarity among known effectors; however, conservative mechanisms of G protein activation/inhibition of effector via structurally similar recognition surfaces may be considered as a working hypothesis. Thus, the molecular details of G protein-effector interaction that are best understood for the visual signal transduction system may be applied to study relevant partners in other signaling systems.

  
Table: Oligonucleotides used for construction of carboxyl-terminal P mutants

The degeneracy of nucleotides at indicated positions (letters above and below the line) was utilized for simultaneous statistical introduction of terminator codons (oligonucleotides I, II, and III) and also for statistical substitution of Ile for phenylalanine or asparagine (oligonucleotide III).


  
Table: Carboxyl-terminal amino acid sequence of P mutants


  
Table: Functional properties of P mutants



FOOTNOTES

*
This work was supported by National Institutes of Health Grant 06062. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a National Alliance for Research on Schizophrenia and Depression Young Investigator Award.

To whom correspondence and reprint requests should be addressed: Dept. of Physiology and Biophysics, University of Illinois, College of Medicine, Box 6998, Chicago, IL 60680. Tel.: 312-996-7151; Fax: 312-996-1414.

The abbreviations used are: PDE, phosphodiesterase; tPDE, trypsin-activated PDE; ROS, rod outer segment; GTPS, guanosine 5`-3-O-(thio)triphosphate; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. John Sondek for the gift of P expression vector, Dr. John Mills for valuable discussions, and Ferdinand Belga for technical assistance.


REFERENCES
  1. Angleson, J. K., and Wensel, T. G.(1994) J. Biol. Chem. 269, 16290-16296 [Abstract/Free Full Text]
  2. Arshavsky, V. Y., Dumke, C. L., Zhu, Y., Artemyev, N. O., Skiba N. P., Hamm, H. E., and Bownds, M. D.(1994) J. Biol. Chem. 269, 19882-19887 [Abstract/Free Full Text]
  3. Artemyev, N. O., and Hamm, H. E.(1992) Biochem. J. 283, 273-279 [Medline] [Order article via Infotrieve]
  4. Artemyev, N. O., Rarick, H. M., Mills, J. S., Skiba, N. P., and Hamm, H. E.(1992) J. Biol. Chem. 267, 25067-25072 [Abstract/Free Full Text]
  5. Artemyev, N. O., Mills, J. S., Thornburg, D. R., Knapp, D. R., Schey, K. L., and Hamm, H. E. (1993a) J. Biol. Chem. 268, 23611-23615 [Abstract/Free Full Text]
  6. Artemyev, N. O., Skiba, N. P., Mills, J. S., and Hamm, H. E. (1993b) Methods (Orlando) 5, 220-228 [CrossRef]
  7. Baehr, W., Delvin, M. J., and Applebury, M. L.(1979) J. Biol. Chem. 254, 11669-11677
  8. Berlot, C. H., and Bourne, H. R.(1992) Cell 68, 911-922 [Medline] [Order article via Infotrieve]
  9. Bradford, M. M.(1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  10. Brown, R. L.(1992) Biochemistry 31, 5918-5925 [Medline] [Order article via Infotrieve]
  11. Brown, R. L., and Stryer, L.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4922-4926 [Abstract]
  12. Catty, P., Pfister, C., Bruckert, F., and Deterre, P.(1992) J. Biol. Chem. 267, 19489-19493 [Abstract/Free Full Text]
  13. Clerc, A., and Bennett, N.(1992) J. Biol. Chem. 267, 6620-6627 [Abstract/Free Full Text]
  14. Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G., and Sprang, S. R.(1994) Nature 265, 1405-1412
  15. Conklin, B. R., Farfel, Z., Lustig, K. D., and Bourne, H. R.(1993) Nature 363, 274-276 [CrossRef][Medline] [Order article via Infotrieve]
  16. Deterre, P., Bigay, J., Robert, M., Pfister, C., Kuhn, H., and Chabre, M.(1986) Proteins Struct. Funct. Genet. 1, 188-193 [Medline] [Order article via Infotrieve]
  17. Deterre, P., Bigay, J., Forquet, F., Robert, M., and Chabre, M.(1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2424-2428 [Abstract]
  18. Dratz, E. A., Furstenau, J. E., Lambert, C. G., Thireault, D. L., Rarick, H., Schepers, T., Pakhlevaniants, S., and Hamm, H. E.(1993) Nature 363, 276-280 [CrossRef][Medline] [Order article via Infotrieve]
  19. Eibl, H., and Lands, W. E. M.(1969) Anal. Biochem. 30, 51-57 [Medline] [Order article via Infotrieve]
  20. Faurobert, E., Otto-Bruc, A., Chardin, P., and Chabre, M.(1993) EMBO J. 12, 4191-4198 [Abstract]
  21. Fung, B. K.-K., Young, J. H., Yamane, H. K., and Grisword-Prenner, I. (1990) Biochemistry 29, 2657-2664 [Medline] [Order article via Infotrieve]
  22. Hurley, J. B., and Stryer, L.(1982) J. Biol. Chem. 257, 11094-11099 [Abstract]
  23. Kleuss, C., Pallat, M., Brendel, S., Rosenthal, W., and Schultz, G. (1987) J. Chromatogr. 407, 281-289 [CrossRef][Medline] [Order article via Infotrieve]
  24. Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B.(1994) Nature 369, 621-628 [CrossRef][Medline] [Order article via Infotrieve]
  25. Liebman, P. A., Parker, K. R., and Dratz, E. A.(1987) Annu. Rev. Physiol. 49, 765-791 [CrossRef][Medline] [Order article via Infotrieve]
  26. Lipkin, V. M., Dumler, I. L., Muradov, K. H., Artemyev, N. O., and Etingof, R. H.(1988) FEBS Lett. 234, 287-290 [CrossRef][Medline] [Order article via Infotrieve]
  27. Lipkin, V. M., Udovichenko, I. P., Bondarenko, V. A., Yurovskaya, A. A., Telnykh, E. V., and Skiba N. P. (1990a) Biomed. Sci. (Lond.) 1, 305-308
  28. Lipkin, V. M., Khramtsov, N. V., Vasilevskaya, I. A., Atabekova, N. V., Muradov, K. G., Li, T., Johnston, J. P., Volpp, K. J., and Applebury, M. L. (1990b) J. Biol. Chem. 265, 12955-12959 [Abstract/Free Full Text]
  29. Lipkin, V. M., Bondarenko, V. A., Zagranichny, V. E., Dobrynina, L. N., Muradov, K. G., and Natochin, M. Y.,(1993) Biochim. Biophys. Acta 1176, 250-256 [Medline] [Order article via Infotrieve]
  30. Maniatis, T., Fritsch, E., and Sambrook, J.(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  31. Mazzoni, M. R., Malinski, J. A., and Hamm, H. E.(1991) J. Biol. Chem. 266, 14072-14081 [Abstract/Free Full Text]
  32. Morrison, D. F., Cunnick, J. M., Oppert, B., and Takemoto, D. J.(1989) J. Biol. Chem. 264, 11671-11681 [Abstract/Free Full Text]
  33. Noel, J. P., Hamm, H. E., and Sigler, P. B.(1993) Nature 366, 654-663 [CrossRef][Medline] [Order article via Infotrieve]
  34. Oppert, B., and Takemoto, D. J.(1991) Biochem. Biophys. Res. Comm. 178, 474-479 [Medline] [Order article via Infotrieve]
  35. Oppert, B., Cunnick, J. M., Hurt, D., and Takemoto, D. J.(1991) J. Biol. Chem. 266, 16607-16613 [Abstract/Free Full Text]
  36. Otto-Bruc, A., Antonny, B., Vuong, T. M., Chardin, P., and Chabre, M. (1993) Biochemistry 32, 8636-8645 [Medline] [Order article via Infotrieve]
  37. Ovchinnikov, Yu. A., Lipkin, V. M., Kumarev, V. P., Gubanov, V. V., Khramtsov, N. V., Akhmedov, N. B., Zagranichny, V. E., and Muradov, K. G.(1986) FEBS Lett 204, 288-292 [CrossRef][Medline] [Order article via Infotrieve]
  38. Ovchinnikov, Yu. A., Gubanov, V. V., Khramtsov, N. V., Ischenko, K. A., Zagranichny, V. E., Muradov, K. G., Shuvaeva, T. M., and Lipkin, V. M. (1987) FEBS Lett. 223, 169-173 [CrossRef][Medline] [Order article via Infotrieve]
  39. Papermaster, D. S., and Dreyer, W.(1974) Biochemistry 13, 2438-2444 [Medline] [Order article via Infotrieve]
  40. Rarick, H M., Artemyev, N. O., and Hamm, H. E.(1992) Science 256, 1031-1033 [Medline] [Order article via Infotrieve]
  41. Sanger, F., Nicklen, S., and Coulson, A. R.(1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  42. Stryer, L.(1991) J. Biol. Chem. 266, 10711-10714 [Free Full Text]
  43. Wensel, T. G., and Stryer, L.(1986) Proteins Struct. Funct. Genet. 1, 90-99 [Medline] [Order article via Infotrieve]
  44. Wensel, T. G., and Stryer, L.(1990) Biochemistry 29, 2155-2161 [Medline] [Order article via Infotrieve]
  45. Wilmot, C. M., and Thorton, J. M.(1988) J. Mol. Biol. 203, 221-232 [Medline] [Order article via Infotrieve]
  46. Wilmot, C. M., and Thorton, J. M.(1990) Protein Eng. 3, 479-493 [Abstract]
  47. Yamazaki, A., Hayashi, F., Tatsumi, M., Bitensky, M. W., and George, J. S.(1990) J. Biol. Chem. 265, 11539-11548 [Abstract/Free Full Text]

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