Oct 5, 2017 - Cross-linking of the different subunits of the retinal. cGMP-phosphodiesterase (PDE) with its activator. GaGTPrS (a subunit of the retinal ...
Vol . 267, No. 28, Issue of October 5, pp. 19948-19953,1992 Printed in U.S.A.
THEJOURNALOF BIOLOGICAL CHEMISTRY
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction between cGMP-phosphodiesterase and Transducin a-Subunit in Retinal Rods A CROSS-LINKING STUDY* (Received for publication, May 6, 1992)
Armel Clerc, Patrice Catty, and Nelly Bennett$ From the UnitiAssociee NO520 d u Centre National de la Recherche Scientifique, Laboratoire de Biophysique Moleculaire et Cellulaire, Centre d’Etudes Nucliaires deGrenoble, BP 85X, 38041 Grenoble Cedex, France
Cross-linking of the different subunits of the retinal cGMP-phosphodiesterase (PDE)withits activator GaGTPrS (a subunit of the retinal G-protein transducin with GTPrS (guanosine 5’-0-(3-thiotriphosphate) bound) has been investigated using purified proteins, with a N-hydroxysuccinimide homobifunctionalcrosslinker, bis(sulfosuccinimidy1)suberate (BS3) and its cleavable analog 3,3’-dithiobis(sulfosuccinimidylpropionate) (DTSSP). Interaction of purified G-protein and PDE is achieved in the presence of lecithin vesicles, at protein concentrations sufficient for full PDE activation. Protein subunits linked with DTSSP areseparated by cleavage of the disulfide bridge and identified by electrophoresis. Complexes of PDEa (PDEB) with 1 and 2 molecules of activator GaGTPrS are observed, providing direct evidence for an interaction or at least a close proximity between 2 molecules of activator Gar and each of the catalytic PDE subunits in the activated state of PDE. The results also reveal symmetrical roles of PDEa and PDEB, with the existence of one site for PDEr and one site for Ga on each catalytic subunit.
PDE) from excited rhodopsin and at the same time from the GPy subunits. Wehave previously reported (Bennettand Clerc, 1989) that, consistent with the existence of two PDEr subunits per PDEaP, 2 molecules of activator GaGTP are involved in the activation of 1 molecule of PDE: the first activator molecule binds with higher affinity inducing partial activation of PDE, and the second one binds with lower affinity, inducing full activation. The structure of the activated states of PDE is still, however, the subject of discussions. In a recent work (Clerc and Bennett, 1992),we studied the specific attachment of GaGTPyS to thedisk membrane in the presence of PDE, and/or purified PDEy; the results suggest that under the conditions used, active PDE is almost totally in the form of a membrane-bound undissociated (Ga),PDEa@yycomplex, in agreement with Sitaramayya et al. (1986). On the other hand, Yamazaki et al. (1983, 1990) describe solubilization of PDEy subunit upon activation of PDE in frog rods, and Wensel and Stryer(1986,1990) propose that activation of bovine PDE is also achieved by dissociation of GaPDEy from PDEaP. The existence of at least one site of interaction of Ga on the PDEy subunit has been demonstrated by Deterre et al. (1986) by isolation of a GaPDEy complex by ion exchange chromatography; by Fung andGrisAbsorption of aphoton by the photosensitive pigment wold-Prenner (1989) who used coimmunoprecipitation of purhodopsin in retinal rods triggers an enzymatic cascade which rified PDEy with Ga; and by Morrison et al. (1989) who used ends by closing of cGMP-activated channels in the plasma synthetic peptides corresponding to various regions of PDEr. membrane (reviewedby Pugh and Lamb, 1990). The two There is no direct evidence yet for the existence of an interproteins involved in this cascade are peripheral membrane action between G a and the catalytic PDEa or @ subunits. proteins situated on the disk membrane: a GTP-binding pro- However, an indirect argument has been produced by Kroll tein (transducin, or G),’ composed of three subunits a (40 et al. (1989) who describe inhibition of trypsin-activated PDE kDa), @ (37 kDa), and y (8 kDa), and a cGMP-phosphodies- (PDEaP) by GaGDP. High molecular mass complexes of 180 terase (PDE),composed of two catalytic subunits a (88 kDa) and /3 (84 kDa), and inhibitory y subunits (10 kDa) with a and 210 kDa cross-reacting with both anti-G and anti-PDE stoichiometry of 2 y per a@(Deterre et al., 1988; Fung et al., antisera were described by Hingorani et al. (1988), and may 1990). Photoexcited rhodopsin catalyses the exchange of represent complexes of Ga or GP with PDE; themethod used bound GDP for GTP on the Ga subunit; this induces disso- did not allow, however, unambiguous determination of the G ciation of GaGTP (Ga with GTP bound, activator of the and PDE (PDEa, PDEP, and/or PDEy?) subunits involved in the complex. In the present study, we have used purified preparations of * The costs of publication of this article were defrayed in part by bovine GandPDEto investigate the sites of interaction the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 between Ga and PDE subunits by use of a cross-linking solely to indicate this fact. reagent. Nonambiguous identification of the cross-linked sub$ T o whom correspondence shouldbeaddressed:BMC/DBMS, units is performed by reduction of a cleavable analog of the C.E.N.-G. 85X, 38041 Grenoble Cedex, France. The abbreviations used are: G, GTP-binding protein of retinal cross-linker followed by gel analysis. rods(transducin);Ga,Goy,subunits of G; GaGDP,GaGTP, MATERIALS AND METHODS’ GaGTPyS, Ga with GDP, GTP, or GTPrSbound; GGDP,GaGDP. G& (complex of three subunits); G G T ~ ,GGTP~S,GaGTP,or Portions of this paper (including “Materials and Methods,” part GrvGTPrS + Gpr (Ga separated fromGpy); PDE, cGMP phosphodiesterase; PDEa, PDEP, catalytic subunits of PDE; PDEr, inhibi- of “Results,” andFig. 1)are presented in miniprint at the endof this tory subunit of PDE; GTPyS, guanosine 5’-0-(3-thiotriphosphate); paper. Miniprint iseasily read with the aidof a standard magnifying SDS, sodium dodecyl sulfate;DTSSP, 3,3’-dithiobis(sulfosuccini- glass. Full size photocopies are included in the microfilm edition of the Journal that isavailable from Waverly Press. midylpropionate); BS3, bis(sulfosuccinimidy1) suberate.
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RESULTS
Optimization of Experimental Conditions for Cross-linking GaGTPyS to PDE2 Cross-linked Products Obtained by Incubation of GCDp, GcTp+, and/or PDE with BS3 Cross-linked products of PDE alone or of GcDp (complex of GaGDP and GPy) and GCTP~S (GaGTPyS + GPy,dissociated) in the presence or absence of PDE obtained with BS3 are shown in Fig. 2. The experiments were carried out in the presence of lecithin vesicles as membrane support instead of discmembranes; in the presence of GcT~,s, PDE isfully activated under the conditions used, as described inthe “Miniprint” section. The concentration of BS3 and duration of incubation were varied, and thesamples shown correspond to the best yield of cross-linking. Two additional bands PI (105 & 5 kDa) and P2 (122 f 5 kDa) are observed in the PDE sample (lane b). Cross-linked products of GCDP(lane d ) and GCT~,S (lane f ) are similar although they appear in slightly different proportions. In both samples, apparently the same three additional bands GI (46 k 5 kDa), GI (76 f 5 kDa), and GS (95 f 5 kDa) are observed. When PDE is mixedwith inactive G (PDE + GCDp, lane c), the additional bands are those observed in sample b (PDE alone) plus those observed in sample d (GCDp alone), in the same proportions. However, when PDE is mixed with active G (PDE + GCTP,~, lane e ) , the complexes formedare different from those observed with PDE alone and GCTP~S alone, (i) theamount of P1 is reduced and P2is absent and (ii) two additional complexes, PGI (120 & 5 kDa in the experiment shown) and PG2(210 k 15 kDa), the latter appearing as a double band, are observed. PGI is not always observed, and when it is, it is always in lower amounts than PG2. PG1 and PG2 are also observed when a preparation enriched in GacTp,~ (lane i ) is used instead of GGTP,S(GaGTPyS + GPy, dissociated), but not with a preparation enriched in Goy (lane g). PG, and PG2, which are observed only in the presence of PDE and GCTP,S(or PDE and GaCTpyS), may therefore possiblybecomplexes of GaGTPyS andPDE. In order to test thishypothesis, we have performed the same experiments with the cleavable analog of BS3, DTSSP.
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FIG.3. Cross-linked products obtained in the presence of DTSSP (4 mM, 2 min incubation). a, PDE. b, PDE + GCTP~S. c,
e, GCDp Control, no DTSSP. Same protein PDE + GCDp. d, GCTP~S. concentrations as in Fig. 2. Samples are treated for SDS-gel electrophoresis (10% acrylamide) under nonreducing conditions (no @-mercaptoethanol) to avoid cleavageof the disulfide bond of DTSSP.
obtained in Fig. 2 with BS3. The main differences are lower amounts of P2 (absent in the experiment shown), PG1 and PG2, suggesting a lower reactivity of DTSSP, and slightly different apparent molecular massof some of the complexes: GI appearing close to 100 kDa and PGl to 150 kDa (as also observed in some experiments with BS3,not shown).It should be noted that thestructure of the 2 cross-linker molecules is not absolutely identical since the two median -CH2 in BS3 are replaced by a disulfide bridge in DTSSP (see “Materials and Methods”). Increasing the concentration of DTSSP or the duration of incubation mainly results in increasing the amount of aggregates. To identify unambiguouslythe subunits which composethe complexes, all the additional bands have been electrodialyzed and analyzed as described under “Materials and Methods.” One aliquot of the eluate is analyzed on gel to check the presence (and purity) of the complex and to estimate its concentration. In the remainder of the eluate, the disulfide bridge in the DTSSP molecule is cleaved in the presence of reducing agents and the proteins which were linked in the complexes are identified by gel electrophoresis. Complexesof G subunits in the absence of PDE and PDE subunits in the absence of G have also been analyzed, since understanding their composition isa necessary control for understanding the Cross-linked Products Obtained by Incubation of CCD, composition of complexes of Ga and PDE subunits. GGTP,~, and/or PDE with DTSSP Analysis of Complexes of GCTP~Sand G C DSubunits ~ in the Results from cross-linking experiments in the presence of DTSSP are shown in Fig. 3. They are very similar to those Absence of PDE-After electroelution and reduction of DTSSP, GI is found to be composed ofGO and Gy, while G2 and Gt contain both Ga and GP in similar amounts (data not a b c d e f g h i j shown). We have further studied these complexes usingprep““rr Is.”” = arations enriched in GacTp,s or GPy. A complex of the same 200. 1Po1 molecular weight as GI is observed when a preparation of / 116. -m enriched GPy is incubated with DTSSP (Fig. 4A, I, band a ) . P D W- P1 After reduction (Fig.4B), this complex is shownto be exclu. ‘w ‘ 6 2 sively composed ofGB and Gy; it is not observed with prep45. - G1 ”. arations enriched in GaGTPyS. Complexes of the same moaI lecular weight as G2 and GS are observed with enriched Ga 31 preparations (Fig. 4, 11, bands b and c) and also, although FIG. 2. Cross-linked products obtained with BS3. a, controls scarcely visible inthe experiment shown, with enriched GPy without BS3 (PDE, PDE + G, or G); the protein above PDEa@is a contaminant in the PDE preparation. b-j, BS3 2 mM, 50-s incubation. preparations; a low intensity band of about 120 kDa is obProtein concentrations during incubation: 4 p~ PDE, 25 p~ G in the served with both Ga (band d ) and GPy. Higher molecular presence of 5 mg/ml lecithin. Amount of PDE andG per lane: 24 and weight complexes have not been analyzed. After reduction (Fig. 4 B ) , the complexes b,c, and dobtained with GaGTPyS 60 pg, respectively. b, PDE. c and d, G ~ o p2 PDE. e and f, GGTPTS & PDE. g and h, enriched GPy 2 PDE. i and j , enriched GaGTPyS f preparations are shown to only contain Ga. Given their PDE. Additional bands: PI (105 & 5 kDa) and Pp(122 & 5 kDa)(lanes molecular weight, complex b (76 kDa, corresponding to G2) b, c, g, and to a lesser extent PI in lanes e and i ) ; GI (46 kDa) (lanes is likely to be a Ga dimer and complex d (120 kDa) a trimer, c-h); Gp (76 kDa)(lanes c-j); Gs (95kDa) (lanes d, f, and;); PGI (120 kDa) and PG, (210 kDa)(lanes e and i). Asterisk (64-66 kDa), con- while it is not clear whether complex c (100 kDa, correspondtaminant protein. Proteins are denatured in the presence of @-mer- ing to G3) is a dimer or a trimer whose abnormal (too slow or too fast) migration could be related to an additional internal captoethanol; SDS gel, 10% acrylamide.
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Cross-linking of Transducin to Phosphodiesterase
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