# The cGMP phosphodiesterase-transducin complex of retinal rods. Membrane binding and subunits interactions.

Patrice Catty, C Pfister, F Bruckert, P Deterre

► To cite this version:

Patrice Catty, C Pfister, F Bruckert, P Deterre. The cGMP phosphodiesterase-transducin complex of retinal rods. Membrane binding and subunits interactions. Journal of Biological Chemistry, 1992, 267 (27), pp.19489-19493. 10.1016/S0021-9258(18)41802-9. cea-05109356

HAL Id: cea-05109356

https://cea.hal.science/cea-05109356v1

Submitted on 12 Jun 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# The cGMP Phosphodiesterase-Transducin Complex of Retinal Rods

## MEMBRANE BINDING AND SUBUNITS INTERACTIONS*

(Received for publication, May 1, 1992)

Patrice Catty, Claude Pfister, Franz Bruckert, and Philippe Deterre‡

From the Laboratoire de Biophysique Moléculaire et Cellulaire, Unité Associée 520 du Centre National de la Recherche Scientifique, Centre d'Etudes Nucléaires, BP 85X 38041 Grenoble, France

cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is composed of two catalytic subunits (PDEα and PDEβ) and two identical inhibitory subunits (PDEγ). Native PDEαβγ₂ is peripherally bound to the membranes of ROS discs. We studied quantitatively its partition between soluble and membrane-bound fractions in ROS homogenates. In the presence of its activator, the α-subunit of transducin loaded with a triphosphate guanine nucleotide (Tα*), PDE displayed a greatly enhanced membrane binding. Neither the purified PDEγ·Tα* complex, nor the PDEαβ and PDEαβγ forms of active PDE, showed a membrane binding comparable to that of PDEαβγ₂ in the presence of Tα*. The Tα*-activated PDE is therefore an undissociated complex tightly bound to the ROS membranes. Using limited proteolysis, we showed that the membrane anchoring of the whole complex implies not only PDE (mainly by the C terminus of PDEβ) but also both termini of Tα*. The membrane binding of the purified PDEαβ species was also enhanced in the presence of Tα*; a direct link would therefore exist between the activator and the catalytic subunits. From this work emerges a plausible structural model of the Tα*-activated PDE, with its internal interactions and its sites of anchoring into the ROS membrane.

In retinal rod outer segments (ROS),<fnref n="1" /> the light signal is converted into an electrical signal through a cascade of molecular events, involving in series rhodopsin (the photon receptor), transducin (the retinal guanine nucleotide-binding protein), and a specific phosphodiesterase (PDE) hydrolyzing the cytosolic cGMP. This results in the rapid closure of cation channels in the plasma membrane, which were kept open in the dark by direct binding of cGMP molecules (Fesenko et al., 1985; for reviews see Chabre and Deterre, 1989; Stryer, 1991). The pivotal PDE enzyme is composed of two large subunits forming the catalytic core (PDEα 100 kDa, PDEβ 98 kDa) (Baehr et al., 1979; Ovchinnikov et al., 1987; Lipkin et al., 1990) and two inhibitory little PDEγ subunits (10 kDa) (Ovchinnikov et al., 1986; Deterre et al., 1988).

It is well documented that the PDE activator is the α-subunit of transducin loaded with a GTP (hereafter called Tα*), which acts by direct binding to the PDEγ subunits (Deterre et al., 1986; Fung and Griswold-Prenner, 1989; Whalen and Bitensky, 1989). However, the events following this initial interaction remain obscure, in particular, whether there is dissociation of the PDEγ·Tα* complex from the catalytic core. In bovine ROS, a PDEγ·Tα* complex can be isolated either by chromatography (Deterre et al., 1986) or by immunoprecipitation (Fung and Griswold-Prenner, 1989), suggesting that the dissociation occurs during physiological activation. Kinetics studies of the incorporation of a fluorescent-labeled PDEγ to the inactive or Tα*-activated PDE also fit with this hypothesis (Wensel and Stryer, 1990). In frog ROS, Yamazaki et al. (1983, 1990) reported the release of a soluble PDEγ·Tα* complex, while the work of Gray-Keller et al. (1990) suggested that the transducin fraction activating the PDE remained membrane-bound.

Most of the studies pointing out the dissociation model have been carried out in reconstituted systems in the presence of low concentrations of membranes or even in their absence. However, PDE is a membrane-associated protein and its enzymatic activity is enhanced in the presence of membranes (Fung and Nash, 1983; Tyminski and O'Brien, 1984; Phillips et al., 1989; Bennett and Clerc, 1989). Hence, the association to the lipid bilayer is obviously crucial for the PDE activation process. We show here that the Tα*-activated PDE is far more tightly bound to the membranes than the native PDE. This allows us to gain numerous indications about the structure of the Tα*-activated PDE in situ.

## EXPERIMENTAL PROCEDURES

*Samples Preparations*—ROS membranes were prepared from fresh bovine retina. Two flotations were carried out in 45 and 37% sucrose in a buffer containing 20 mM Hepes, 5 mM MgCl₂, 5 mM 2-mercaptoethanol, and 0.1 mM phenylmethylsulfonyl fluoride (4 retina per ml). The final pellets are stored at -80 °C under argon. The preparations were carried out either from light-adapted retina ("light" preparations) or from dark-adapted retina and under dim red light ("dark" preparations). The Rhodopsin concentration was determined, for dark preparations, by the difference at 500 nm between the absorbance spectra of the unilluminated and illuminated sample (ε = 40,000 M⁻<fnref n="1" /> cm⁻<fnref n="1" />) and, in the light preparations, by the absorbance at 365 nm of the sample in the presence of hydroxylamine (ε = 36,000 M⁻<fnref n="1" /> cm⁻<fnref n="1" />). During this preparation procedure, some soluble proteins are lost, including a subpool of PDE (Gillespie et al., 1989), which was therefore not under consideration in the present study.

After thawing, the pellets were homogenized and washed twice in buffer A (20 mM Tris, pH 7.5, 120 mM KCl, 1 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride). At this step, the soluble proteins (rhodopsin-kinase, arrestin, etc.) were removed, and transducin and PDE were sedimented with the membranes. Crude PDE preparations consisted of the supernatant of ROS membranes sedimented in buffer B (5 mM Tris, pH 7.5, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride) containing 2 mg/ml rhodopsin. A mixture of activated transducin and of transducin-activated

<footnote>* 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.</footnote>

<footnote>‡ To whom correspondence should be addressed. Present address: Laboratoire d'Immunologie Cellulaire et Tissulaire, Groupe Hospitalier Pitié-Salpétrière, Bât. CERVI, 75651 Paris, Cedex 13, France.</footnote>

<footnote n="1">¹ The abbreviations used are: ROS, retinal rod outer segment; T, transducin; Tα*, the α-subunit of transducin loaded with a GTP or a hydrolysis-resistant analog; PDE, retinal cGMP-specific phosphodiesterase; Tα*.PDE*, Tα*-activated PDE complex; PDEα, PDEβ, and PDEγ, the α-, β-, and γ-subunits of PDE; GTPγS, guanosine 5'-O-(thio)triphosphate; Rh, rhodopsin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.</footnote>
PDE was obtained by the same extraction in buffer B supplemented with 100 µM GTPγS and 100 µM MgCl₂. This extract was purified by anion-exchange chromatography (Polyanion SI, Pharmacia LKB Biotechnology Inc.), as described in Deterre *et al.* (1986, 1988), separating the three PDE species (PDEαβγ₂, PDEαβγ, PDEαβ), activated Tα (Tα*), and the PDEγ·Tα* complex. "Stripped membranes" were obtained after extraction of transducin and PDE, by subsequent washing of the ROS pellet once in buffer B containing 100 µM of GTPγS and MgCl₂, four times in buffer B alone, and twice in buffer A.

**Assay for Protein Binding to ROS Membranes**—Stripped membranes were suspended in buffer A and supplemented with PDE and the various reactants. The samples were incubated 15 min at room temperature and centrifuged 6 min at 500,000 × g (Beckman centrifuge TL100). Supernatants were removed, and each pellet (which contains membrane-bound proteins) was resuspended in 100 µl of buffer B. Samples were then incubated 15 min at room temperature and centrifuged 10 min at 500,000 × g. The Tα, PDEα, PDEβ, and PDEγ contents of the supernatants were analyzed by gel electrophoresis in the presence of SDS (SDS-PAGE). The amount of membrane-bound PDE in each condition was expressed as a percentage of the amount of membrane-bound PDE in the presence of 2 mg of Rh/ml of membranes. The amount of PDE bound to membranes at 2 mg of Rh/ml corresponds nearly to 95% of the total PDE.

**PDE Activity Assay**—Samples containing PDE (25 nM) and the various reactants were preincubated for 2 min at 25 °C, then supplemented with 2 mM cGMP (final volume, 100 µl), and the reaction was stopped after 30 s by addition of 1 ml of 10% cold perchloric acid. Samples were centrifuged (5 min, 10000× g) in the presence of 500 µg/ml bovine serum albumin to sediment proteins. The supernatants were neutralized with a solution containing 50 mM Tris, 2.5M KOH and then centrifuged (5 min, 10000× g). The GMP and cGMP content of the final supernatants were analyzed on PL-SAX column (Polymer Laboratories) equilibrated with a buffer containing 20 mM Tris-HCl, pH 8, and 1 mM MgCl₂. cGMP and GMP eluted at 2 and 3 min, respectively, with a linear gradient of NaCl (0–500 mM, 1 ml/min).

SDS-PAGE was performed on Laemmli-type slab gels using a standard method. Gels were stained by Coomassie Blue or by silver staining (Blum *et al.*, 1987) and densitometered using the CS9000 Shimadzu scanner. Protein concentrations were determined by a colorimetric Coomassie Blue assay (Bradford, 1976) using bovine serum albumin as standard.

## RESULTS

**Association of Native PDE to the ROS Membranes**—PDE is a peripherally bound protein. In isotonic buffer, it can be detached from ROS membranes by dilution of the ROS suspension just as observed for transducin (Liebman and Sitaramayya, 1984; Bruckert *et al.*, 1988): we found that half of the PDE content of a ROS suspension is in the soluble fraction at 0.1 mg of rhodopsin (Rh) per ml (Fig. 1, *open circles*). The experiment was performed either with native ROS or with reconstituted suspensions (mixture of purified PDE and stripped membranes with a stoichiometric ratio of about 100 Rh to 1 PDE, as in native conditions (Kühn, 1981)). This partition reflects the existence of an equilibrium between soluble and membrane-bound PDE, which depends upon the membrane concentration. This equilibrium was not influenced either by bleaching of the ROS membranes or by pretreating the membranes with urea (4 M, 1 h, 37 °C) or with trypsin (10 µg/ml, 1 h, 37 °C). Chelating calcium and magnesium by EGTA (10 mM) didn't have any effect on the attachment of PDE to the membranes. This is consistent with a nonspecific attachment of PDE to the lipids (Tyminski and O'Brien, 1984).

PDEαβγ and PDEαβ species (obtained from Tα*-activated PDE, see "Experimental Procedures") behaved like PDEαβγ₂ (Fig. 1, compare *open circles* with *squares* and *triangles*). This confirms previous studies (Wensel and Stryer, 1986; Catty and Deterre, 1991) showing that PDEγ has no role in the anchoring of PDE to the disc membranes.

**Formation of a Tightly Membrane-bound Complex between Tα* and Native PDEαβγ₂ (Tα*.PDE*)**—The binding curve

<figure><img src="image_2.png" /></figure>

**FIG. 1.** Binding of PDEαβγ₂, PDEαβγ, and PDEαβ to the ROS membranes. Effect of the presence of Tα*. A suspension of stripped ROS membranes at 2 mg of Rh/ml in buffer A was supplemented with 500 nM of PDEαβγ₂ (*open circles*), PDEαβγ (*squares*), or PDEαβ (*triangles*) and incubated 15 min at room temperature. Aliquots of 50 µl were then taken and diluted with buffer A to the given membrane concentration. In the case of PDEαβγ₂, this dilution was also carried out in the presence of 3 µM of purified Tα* (*closed circles* and *asterisks*). After 15 min at room temperature, the percentage of membrane-bound PDEαβ (*circles*, *squares*, and *triangles*) and PDEγ (*asterisks*) was determined (see "Experimental Procedures"). *Solid line on open symbols*, hand fitting of experimental data. These data could also be fitted by a binding curve depending of two parameters: number of PDE sites on the membranes and binding affinity. Provided the number of PDE sites was more than three times the native PDE concentration in ROS, good fitting was obtained (data not shown).

of PDE to the membranes was profoundly altered in the presence of an excess of Tα*, i.e. here Tα loaded with GTPγS (Fig. 1, compare *closed and open circles*). At all the membrane concentrations tested (from 2 mg of Rh/ml to 0.01 mg of Rh/ml) PDE in the presence of Tα* was totally recovered in the membrane fraction. We checked that this membrane-bound PDE contained not only the PDEαβ core (Fig. 1, *closed circles*) but also the two PDEγ subunits (Fig. 1, *asterisks*).<fnref n="2" /> This enhancement of PDE membrane binding was specific of Tα*: neither purified Tβγ, nor Tα loaded with a GDP, had any influence on the PDE association to ROS membranes (data not shown). Moreover, Tα* had its maximal effect at 2 µM (Fig. 4), and this dose-response curve was not modified in the presence of Tβγ (data not shown).

If the interaction of Tα* with PDE had proceeded *via* the removal by Tα* of PDEγ from PDEαβ, then necessarily (i) the PDEγ·Tα* complex formed would have had to be membrane-bound and (ii) the remaining PDE subunits (PDEαβ and/or PDEαβγ) would have had to be more tightly bound to membranes than PDEαβγ₂. Both propositions are wrong. On the one hand, Fig. 2 demonstrates that isolated PDEγ·Tα*, which remains associated during a gel filtration experiment (Fig. 2A), is soluble even in the presence of 2 mgRh/ml of ROS membranes, just as Tα* alone (Fig. 2B, *left and center*). PDEγ·Tα* can become reattached to the membranes only in the presence of PDEαβ (Fig. 2B, *right*). On the other hand, the purified PDEαβγ and PDEαβ species display strictly the same binding characteristics to the membranes as PDEαβγ₂ (Fig. 1, *open symbols*). These results provide clear evidence that the Tα*-activated PDE is an undissociated and tightly membrane-bound complex (called hereafter Tα*.·PDE*).

**Membrane-anchoring Sites of the Tα*.·PDE* Complex**—
The localization of the membrane-anchoring sites of Tα*.

<footnote n="2">² Tα* sedimentation with membranes comprises not only the PDE-bound fraction but also a fraction nonspecifically bound to the membranes. Under the present conditions of excess Tα*, this second fraction completely masks the specific one, which was therefore impossible to determine.</footnote>
<figure><img src="image_3.png" /></figure>

FIG. 2. Binding of PDEγ·Tα* to ROS membranes. A, after its purification by anion-exchange chromatography (see “Experimental Procedures”), the PDEγ·Tα* complex was dialyzed in buffer A and concentrated by Centricon 10 (Amicon). 200 µl of the purified complex at the concentration of 3.5 µM were loaded on a gel filtration column (Superose 12, fast-protein liquid chromatography system, Pharmacia) previously equilibrated in buffer A. The elution profile is displayed, with the SDS-PAGE analysis of the fractions. B, binding experiments were carried out at a membrane concentration of 2 mg of Rh/ml on samples containing either Tα* (1.6 µM) or PDEγ·Tα* (1.6 µM) or a mixture containing PDEαβ (500 nM) and PDEγ·Tα* (1.6 µM). The amount of PDEγ and Tα* in the membrane-bound fraction is expressed as a percentage of the total amount of each species.

PDE\* was examined by limited proteolysis as recently used for the native PDE (Catty and Deterre, 1991). Membrane binding and PDE activation assays were carried out on samples containing either proteolyzed PDEαβγ₂ and native Tα\* or native PDEαβγ₂ and proteolyzed Tα\* (Fig. 3). A trypsinized PDE, which had lost all of its membrane attachment without becoming activated (Catty and Deterre, 1991) (Fig. 3, A and B, compare columns 1 and 3) was tested in the presence of Tα\*. This modified PDE was still able to be slightly activated in the absence of membranes just as native PDE (Fig. 3B, columns 2 and 4). Yet, this modified PDE displayed no enhanced membrane binding in the presence of Tα\* (Fig. 3A, columns 3 and 4) as it was the case for native PDE (Fig. 3A, columns 1 and 2). Moreover, the presence of membranes produced no enhanced activity of this modified PDE (Fig. 3B, column 4), contrarily to the case of native PDE (Fig. 3B, column 2). The anchorage motifs of native PDE therefore directly participate in the membrane anchoring of Tα\*·PDE\*.

Trypsin is also known to cleave the Tα polypeptide at both of its termini (Fung and Nash, 1983). This does not affect the ability of Tα\* to activate PDE in the absence of membranes (Fung and Nash, 1983) (Fig. 3B, column 2 and 5). Yet, in the presence of membranes, this modified Tα\* induced neither an enhanced binding of PDE to membranes (Fig. 3A, column 5) nor an enhanced PDE activity (Fig. 3B, column 5). The same observation could be made after cleaving the N terminus of Tα\* by endoproteinase Glu-C (Navon and Fung, 1987) (Fig. 3, column 6) or by cleaving its C terminus by endoproteinase Arg-C (Hingorani and Ho, 1990; Mazzoni et al., 1991) (Fig. 3, column 7, in which the proteolysis was not complete after 2-h treatment). Both Tα\* termini are therefore involved in the functional binding of the Tα\*·PDE\* complex to the membranes.

We also modified the C terminus of Tα by ADP-ribosylation of its Cys-347 by the *Bordetella pertussis* toxin (Van Dop *et al.*, 1984; West *et al.*, 1985). This treatment was such that all transducins were modified. We checked that all became unable to interact with photoexcited rhodopsin and migrated on SDS-PAGE as an ADP-ribosylated species (data not shown). These modified Tα-GDP can however be artificially activated in the presence of alumino-fluoride<fnref n="3" /> (Ramdas *et al.*, 1991). These modified and AlF-activated Tα induced the PDE activation

<figure><img src="image_4.png" /></figure>

FIG. 3. Effects of proteolysis on the membrane binding of PDEαβγ₂ and on its activation by Tα\*. Solutions containing 500 nM of native or slightly trypsinized PDEαβγ₂ (1 µg/ml of L-(tosylamido-2-phenyl)ethylchloromethyl ketone-treated trypsin purchased from Worthington, 15 min, 0 °C; reaction stopped by addition of 2 mg/ml of lima bean trypsin inhibitor from Worthington, see Catty and Deterre, 1991) were prepared in the presence or not of 1 mg/ml of stripped membranes and incubated for 15 min at room temperature. Aliquots of 50 µl of either preparation were diluted 10-fold in the buffer A in the presence of native or proteolyzed Tα\* (see below) and incubated 15 min at room temperature. After homogenization, aliquots of 50 µl of these samples were taken for enzymatic PDE assay (final concentration, 25 nM PDE, 2.5 µM Tα\*, 2 mM cGMP, 0.05 mg of Rh/ml). The remaining volume was diluted twice in buffer A (final concentrations 25 nM PDE, 2.5 µM Tα\*, and 0.05 mg of Rh/ml) and was used for membrane binding assay (see “Experimental Procedures”). The purified Tα\* solution (1 mg/ml) was proteolyzed either with trypsin (40 µg/ml of L-(tosylamido-2-phenyl)ethylchloromethyl ketone-treated trypsin, 5 min, 20 °C, reaction stopped by addition of 2 mg/ml of lima bean trypsin inhibitor) or with endoproteinase Glu-C from *Staphylococcus aureus* (Boehringer Mannheim) (10 µg/ml, 1 min, 20 °C) or with endoproteinase Arg-C from mouse submaxillary gland (Boehringer Mannheim; 100 µg/ml, 240 min, 20 °C). In these two later cases the reaction was stopped by addition of 15 mM of diisopropylfluorophosphate. A, membrane-bound PDE; B, PDE enzymatic activity. In both cases, 100% refers to the experiment in the presence of native Tα\* and membranes.

and enhanced its membrane binding, just as the non-modified AlF activated transducin (data not shown), therefore Cys-347 seems not to be directly involved in the membrane attachment of Tα\*·PDE\*.

*Interactions within the Tα\*·PDE\* Complex*—The interaction of Tα\* with PDEγ is the best documented event leading to the activation of PDE. As PDEγ·Tα\* complexes can be isolated (Deterre *et al.*, 1986) (Fig. 2A), it seems likely that this PDEγ·Tα\* interaction exists in Tα\*·PDE\*. One might expect that Tα\* would also induce an enhanced membrane binding of PDEαβγ; we observed that this is the case (Fig. 4, squares). If the PDEγ·Tα\* link alone would account for the formation of Tα\*·PDE\*, one would further expect no interaction between Tα\* and PDEαβ. Surprisingly, Tα\* was able to induce the membrane binding of PDEαβ (~50% of 50 nM PDEαβ in the presence of 2 µM Tα\* (Fig. 4, triangles)). Since contamination by 25 nM of PDEγ could explain this result, we carefully checked the purity of the different reactants: (i) SDS-PAGE analysis showed no visible contamination of Tα\* by PDEγ (<1% w/w, i.e. <5 nM PDEγ) or of PDEαβ by PDEαβγ (<10% w/w, i.e. <5 nM PDEγ); (ii) washed ROS membranes could still contain some PDEαβγ₂, which would react with added PDEαβ to form PDEαβγ (Deterre *et al.*, 1988); we measured no more than 1 nM of activatable PDEαβγ₂ contaminating the washed membranes used in this

<footnote n="3">³ M. Franco and M. Chabre, personal communication.</footnote>
<figure><img src="image_5.png" /></figure>

FIG. 4. Effects of Tα* in the binding of PDEαβγ₂, PDEαβγ, or PDEαβ to ROS membranes. A suspension of stripped membranes (1 mg of Rh/ml) in buffer A was supplemented with 500 nM of PDEαβγ₂ (circles), PDEαβγ (squares), or PDEαβ (triangles) and incubated 15 min at room temperature. Aliquots of 50 µl were diluted 10-fold in buffer A in the presence of purified Tα* at the indicated concentration. The membrane-bound PDE was determined as described under "Experimental Procedures."

test. These data therefore strongly argue to the existence of a
functional interaction between Tα* and PDEαβ.

DISCUSSION

Tα\*-activated PDE Is a Membrane-bound (Tα\*)₂·PDEαβγ₂
Complex—The present study provides experimental evidences
that Tα\*-activated PDE (Tα\*-·PDE\*) in the presence of ROS
membranes is a macromolecular undissociated complex be-
tween PDEαβγ₂ and Tα\* (Fig. 1).<fnref n="4" /> Previous studies have
shown that one molecule of Tα\* is able to bind one molecule
of PDEγ (Deterre et al., 1986; Fung and Griswold-Prenner,
1989). Therefore, two Tα\* are expected to bind to one
PDEαβγ₂ molecule. Phillips et al. (1989) indeed showed that
maximal activation of one PDE is achieved when a bivalent
antibody presented to it two Tα\*. Clerc and Bennett (1992)
directly determined that two Tα\* become associated to the
membrane when activating one PDE. Our data directly prove
that PDEγ, which is not dissociated from the disc membranes
during PDE activation (Deterre et al., 1986; Wensel and
Stryer, 1986; Clerc and Bennett, 1992) remains physically
associated to the Tα\*-·PDE\* complex. These data taken to-
gether prove that this Tα\*-·PDE\* complex can be written as
(Tα\*)₂·PDEαβγ₂.

Our results demonstrate that Tα\*-PDE\* is associated with
the ROS membranes through an anchoring mode that is far
more resistant to dilution than native PDEαβγ₂ (Fig. 1) as
already observed qualitatively by Caretta and Stein (1986).
We have shown here (Fig. 3) that the anchoring of Tα\*-PDE\*
involves at least three sites: one on the PDE molecule and
one on each of the two Tα\* units involved in the complex.
The anchoring site related to PDE is degraded by slight
trypsinization, before any functional degradation of the PDEγ
subunits, and therefore is localized on PDEαβ, like in native
PDEαβγ₂ (Catty and Deterre, 1991). It therefore primarily
implicates the C terminus of PDEβ and probably involves the
cysteine of its terminal C-X-X-X motif (Catty and Deterre,
1991), which has been indeed demonstrated to be posttrans-
lationally modified by a geranylgeranyl moiety (Anant et al.,
1992) and methylated (Catty and Deterre, 1991; Anant et al.,
1992). The anchoring site of Tα\*-PDE\* related to Tα\* in-

volves both of its termini (Fig. 3) probably by peptides Met-1
to Lys-19 and Arg-310 to Phe-350 (Navon and Fung, 1987;
Hingorani *et al.*, 1988; Mazzoni and Hamm, 1989; Hingorani
and Ho, 1990, Mazzoni *et al.*, 1991). The cleavage of these
termini does not impair the ability of Tα* to interact with
PDE in the absence of membranes (Navon and Fung, 1987;
Mazzoni and Hamm, 1989) (Fig. 3B).

The effects of the cleavages on Tα* that we observed (Fig. 3) could be simply explained by assuming that each Tα* terminus itself would be anchored to the membrane. Interestingly, it has been hypothesized that, in the tertiary structure, N and C termini are close to each other (Hingorani and Ho, 1987; Mazzoni et al., 1991). It is noteworthy that, in the Tα-GDP conformation, these Tα termini are engaged in the interaction with Tβγ and with photoexcited rhodopsin (Hamm et al., 1987; Navon and Fung, 1988). No posttranslational modification has been reported on the Tα-subunit, except for the potential ADP-ribosylation of the Cys-347. We found that this ADP-ribosylation does not impair the ability of a Tα* to activate PDE. This result was also found by Ramdas et al. (1991). The anchoring capacity of the Tα* termini resides therefore probably solely in the hydrophobic or charged nature of the residues 1-19 and 310-346. These domains, which are unable to bind to the membranes the Tα* molecule alone, could complement the anchoring of the complex by the PDEβ C terminus. This simple model could explain why Tα*.PDE* is more attached to the membrane than PDE alone (Fig. 1). Then the involvement of the Tα* termini in PDE activation would simply result indirectly from the stabilization of the Tα*.PDE* complex in its membrane-bound form (Fig. 3). Alternative models might however be proposed, which could state that the PDE activated by Tα* would have a different structural conformation than the PDE stripped of PDEγ, especially in its membrane-anchoring site. This would imply that the Tα* termini are essential for the proper conformation of the protein. They could directly participate in the Tα*.PDE activating site and would indirectly modify the PDE anchoring domains. We tend however to favor our simpler model, which is moreover consistent with the current knowledge on the interaction between the adenylate cyclase and its stimulatory G-protein (Gα), where it has been shown that Gαα forms a complex with the catalyst (Pfeuffer, 1977; Morris et al., 1990). It seems also true that both Gαα termini are close to each other (Osawa et al., 1990). Moreover Journot et al. (1991) showed that the membrane association of the activated Gαα involves a C-terminal region, which is not in the last 20 amino acids, but in the charged Val-367 to Phe-376 domain (Asp-368, Glu-370, Arg-373, and Arg-374). In Tα*, it corresponds to peptide Thr-323 to Phe-332 which is indeed included in the above-discussed 310-346 region and which also bears charged residues (Asp-324, Gln-326, and Lys-329).

Activation Processes of PDE by Tα* Are Modulated by the
Presence of Membranes—It is clear from the present work
that the activation process of the PDE by Tα* in situ, i.e.
PDE being membrane-bound, is different from the process
studied when ROS membranes are absent or present at low

<figure><img src="image_6.png" /></figure>

FIG. 5. Tentative scheme of the (Tα\*)₂·PDEαβγ₂ complex, its internal interactions, and its binding sites to the membrane.

<footnote n="4">⁴ A. Sitaramayya *et al.* (1986) already reached the same conclusion from differences in kinetic parameters of trypsin- and light-activated PDE. Yet, we never could reproduce their results.</footnote>
concentration (<0.1 mg of Rh/ml).

Without any membrane, $T\alpha^*$ interacts with PDE$\gamma$ and the PDE$\gamma \cdot T\alpha^*$ complex dissociates from PDE$\alpha\beta$ (Yamazaki et al., 1983; Deterre et al., 1986; Fung and Griswold-Prenner, 1989; Yamazaki et al., 1990). In these conditions the activation constant of $T\alpha^*$ for PDE is weak (>2 $\mu$M, see for instance Fig. 3 in Bennett and Clerc, 1989). This explains why the PDE$\gamma$ subunit is more exchangeable in the presence of $T\alpha^*$ than in its absence (Wensel and Stryer, 1990). The activated PDE is in a partially or totally stripped form, PDE$\alpha\beta\gamma$ or PDE$\alpha\beta$, and is therefore easily inhibitable by addition of excess of PDE$\gamma$, as is the trypsin-activated PDE (Hurley and Stryer, 1982; Sitaramayya et al., 1986; Brown and Stryer, 1989; Wensel and Stryer, 1990). In the presence of membranes and $T\alpha^*$ sufficient to totally bind PDE ([membranes] > 0.01 mg of Rh/ml, see Fig. 1; [$T\alpha^*$] > 2 $\mu$M, see Fig. 4), $T\alpha^* \cdot PDE^*$ seems insensitive to the addition of an excess of PDE$\gamma$ (Bennett and Clerc, 1992). An intermediate situation is encountered, when the concentration of membranes is not high enough to bind all the native PDE (<2 mg of Rh/ml, see Fig. 1) and when $T\alpha^*$ is not saturating (<1 $\mu$M as in Wensel and Stryer (1986) and Bennett and Clerc (1989), see Fig. 4). Then, an equilibrium between the two above situations exists: the membrane-bound ($T\alpha^*$)$_2 \cdot PDE\alpha\beta\gamma_2$ complex is partially dissociated into soluble PDE$\gamma \cdot T\alpha^*$ and stripped PDE (PDE$\alpha\beta$ or PDE$\alpha\beta\gamma$). This can explain why, under those conditions, activated PDE can still be inhibited by an excess of PDE$\gamma$, but with a lower efficiency than for the trypsin-activated PDE (Wensel and Stryer, 1986; Sitaramayya et al., 1986; Brown and Stryer, 1989).

**Probable Existence of a Direct Interaction between $T\alpha^*$ and PDE$\alpha\beta$**—In $T\alpha^* \cdot PDE^*$, the only well documented physical link concerns $T\alpha^*$ and PDE$\gamma$ (Deterre et al., 1986, Fung and Griswold-Prenner, 1989). The cohesion of the complex would then rely on two high affinities of PDE$\gamma$, for PDE$\alpha\beta$ on the one hand, for $T\alpha^*$ on the other. The dissociation constant between PDE$\gamma$ and PDE$\alpha\beta$ has been evaluated to be in the nanomolar range (Hurley and Stryer, 1982; Wensel and Stryer, 1986; Bennett and Clerc, 1989). In the activated complex, this inhibitory link has to be weakened to allow for PDE activation. In the membrane-bound $T\alpha^* \cdot PDE^*$ complex, no release of PDE$\gamma \cdot T\alpha^*$ was observed (Fig. 2), contrary to observations in the absence of membranes (Deterre et al., 1986). This suggests the existence of another link between $T\alpha^*$ and $PDE^*$, namely a direct interaction between $T\alpha^*$ and PDE$\alpha\beta$, as supported by the result of the experiment reported in Fig. 4. This would also be in accordance with the results obtained from reticulation studies of the $T\alpha^*$-activated PDE<fnref n="5" /> (Hingorani et al., 1988). The cartoon in Fig. 5 summarizes our present hypothesis concerning the interactions between subunits and with the membrane in the ($T\alpha^*$)$_2 \cdot PDE\alpha\beta\gamma_2$ complex.

**Concluding Remark**—It appears clearly from the present study that the characteristics of the activation of PDE by $T\alpha^*$ depend not only on the presence of membranes but also on the exact amount of membrane-bound $T\alpha^* \cdot PDE^*$ complexes formed in the given experimental conditions. Previously reported studies were done in the presence of membranes at low concentrations (never exceeding 0.1 mg of Rh/ml, Bennett and Clerc (1989) and Wensel and Stryer (1986)), conditions in which the membrane binding of PDE was increased when increasing $T\alpha^*$ concentration, as evidenced in Fig. 4. It is noteworthy that the apparent activation constant obtained in those studies (ED$_{50}$ close to 1 $\mu$M, except for the

first $T\alpha^*$ site in Bennett and Clerc, 1989) is very close to the ED$_{50}$ of $T\alpha^*$ action for binding the PDE to the membrane (see Fig. 4). This merely means that, in this assay, the activation of the membrane-bound enzyme is limited by the membrane binding step. Further experiments are required to determine the relevant activation constant of PDE by $T\alpha^*$ when they are, as *in vivo*, in this membrane-bound ($T\alpha^*$)$_2 \cdot PDE\alpha\beta\gamma_2$ complex.

**Acknowledgments**—We thank Marc Chabre, Joël Bockaert, Laurent Journot, Yves Audigier, Nelly Bennett, and Armel Clerc for fruitful discussions. We are grateful to M. Chabre and Harel Weinstein for critical reading of the manuscript.

## REFERENCES

Anant, J. S., Ong, O. C., Xie, H., Clarke, S., O'Brien, P. J., and Fung, B. K.-K. (1992) *J. Biol. Chem.* **267**, 687–690

Baehr, W., Devlin, M. J., and Applebury, M. L. (1979) *J. Biol. Chem.* **254**, 11669–11677

Bennett, N., and Clerc, A. (1989) *Biochemistry* **28**, 7418–7424

Blum, H., Beier, H., and Gross, H. J. (1987) *Electrophoresis* **8**, 93–99

Broadford, M. M. (1976) *Anal. Biochem.* **72**, 248–254

Brown, R. L., and Stryer, L. (1989) *Proc. Natl. Acad. Sci. U. S. A.* **86**, 4922–4926

Bruckert, F., Vuong, T. M., and Chabre, M. (1988) *Eur. Biophys. J.* **16**, 207–218

Caretta, A., and Stein P. J. (1986) *Biochemistry* **25**, 2335–2341

Catty, P., and Deterre, P. (1991) *Eur. J. Biochem.* **199**, 263–269

Chabre, M., and Deterre, P. (1989) *Eur. J. Biochem.* **179**, 255–266

Clerc, A., and Bennett, N. (1992) *J. Biol. Chem.* **267**, 6620–6627

Deterre, P., Bigay, J., Robert, M., Pfister, C., Kühn, H., and Chabre, M. (1986) *Proteins Struct. Funct. Genet.* **1**, 188–193

Deterre, P., Bigay, J., Forquet, F., Robert, M., and Chabre, M. (1988) *Proc. Natl. Acad. Sci. U. S. A.* **85**, 2424–2428

Fesenko, E. E., Kolesnikov, S. S., and Lyubarsky, A. L. (1985) *Nature* **313**, 310–313

Fung, B. K.-K., and Griswold-Prenner, I. (1989) *Biochemistry* **28**, 3133–3137

Fung, B. K.-K., and Nash, C. R. (1983) *J. Biol. Chem.* **258**, 10503–10510

Gillespie, P. G., Prusti, R. K., Apel, E. D., and Beavo, J. A. (1989) *J. Biol. Chem.* **264**, 12187–12193

Gray-Keller, M. P., Biernbaum, M. S., and Bownds, M. D. (1990) *J. Biol. Chem.* **265**, 15323–15332

Hamm, H. E., Deretic, D., Hofmann, K. P., Schleicher, A., and Kohl, B. (1987) *J. Biol. Chem.* **262**, 10831–10838

Hingorani, V. N., and Ho, Y. K. (1987) *FEBS Lett.* **220**, 15–22

Hingorani, V. N., and Ho, Y. K. (1990) *J. Biol. Chem.* **265**, 19923–19927

Hingorani, V. N., Tobias, D. T., Henderson, J. T., and Ho, Y. K. (1988) *J. Biol. Chem.* **263**, 6916–6926

Hurley, J. B., and Stryer, L. (1982) *J. Biol. Chem.* **257**, 11094–11099

Journot, L., Pantaloni, C., Poul, M. A., Mazarguil, H. Bockaert, J., and Audigier, Y. (1991) *Proc. Natl. Acad. Sci. U. S. A.* **88**, 10054–10058

Kühn, H. (1981) in *Current Topics in Membrane and Transport* (Miller, W. H., ed) Vol. 15, pp. 171–201, Academic Press, New York

Liebman, P. A., and Sitaramayya, A. (1984) *Adv. Cyclic Nucleotide Protein Phosphorylation Res.* **17**, 215–225

Lipkin, V. M., Khramtsov, N. V., Vasilevskaya, I. A., Atabekova, N. V., Muradov, K. G., Gubanov, V. V., Li, T., Johnston, J. P., Volpp, K. J., and Applebury, M. L. (1990) *J. Biol. Chem.* **265**, 12955–12959

Mazzoni, M. R., and Hamm, H. E. (1989) *Biochemistry* **28**, 9873–9880

Mazzoni, M. R., Malinski, J. A., and Hamm, H. E. (1991) *J. Biol. Chem.* **266**, 14072–14081

Morris, D., McHugh-Sutkowski, E., Moos, M., Jr., Simonds, W. F., Spiegel, A. M., and Seamon, K. B. (1990) *Biochemistry* **29**, 9079–9084

Navon, S. E., and Fung, B. K.-K. (1987) *J. Biol. Chem.* **262**, 15746–15751

Navon, S. E., and Fung, B. K.-K. (1988) *J. Biol. Chem.* **263**, 489–496

Osawa, S., Dhanasekaran, N., Woon, C. W., and Johnson, G. L. (1990) *Cell* **63**, 697–706

Ovchinnikov, Y. 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

Ovchinnikov, Y. 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

Pfeuffer, T. (1977) *J. Biol. Chem.* **252**, 7224–7234

Phillips, W. J., Trukawinski, S., and Cerione, R. A. (1989) *J. Biol. Chem.* **264**, 16679–16688

Ramdas, L., Disher, R. M., and Wensel, T. D. (1991) *Biochemistry* **30**, 11637–11645

Sitaramayya, A., Harkness, J., Parkes, J. H., Gonzalez-Oliva, C., and Liebman, P. A. (1986) *Biochemistry* **25**, 651–655

Stryer, L. (1991) *J. Biol. Chem.* **266**, 10711–10714

Tyminski, P. N., and O'Brien, D. F. (1984) *Biochemistry* **23**, 3986–3993

Van Dop, C., Yamanaka, G., Steinberg, F., Sekura, R. D., Manclark, C. R., Stryer, L., and Bourne, H. R. (1984) *J. Biol. Chem.* **259**, 23–26

Wensel, T. G., and Stryer, L. (1986) *Proteins Struct. Funct. Genet.* **1**, 90–99

Wensel, T. G., and Stryer, L. (1990) *Biochemistry* **29**, 2155–2161

West, R. E., Jr., Moss, J., Vaughan, M., Liu, T., and Liu, T.-Y. (1985) *J. Biol. Chem.* **260**, 14428–14430

Whalen, M. M., and Bitensky, M. W. (1989) *Biochemistry* **J. 259**, 13–19

Yamazaki, A., Stein, P. J., Chernoff, N., and Bitensky, M. W. (1983) *J. Biol. Chem.* **258**, 8188–8194

Yamazaki, A., Hayashi, F., Tatsumi, M., Bitensky, M. W., and George, J. S. (1990) *J. Biol. Chem.* **265**, 11539–11548

<footnote n="5">⁵ A. Clerc, P. Catty, and N. Bennett, manuscript to be published.</footnote>