Signal Transduction in the Visual Cascade Involves Specific Lipid-Protein Interactions

Abstract

In retinal rod photoreceptor cells, transducin (Gt) and cyclic GMP phosphodiesterase (PDE) are peripherally anchored to the cytoplasmic surface of the disk saccules. We have examined the role of specific phospholipids in the interaction of these proteins with native osmotically intact disk vesicles, employing spin-labeled phospholipid analogues (2% of total phospholipids) and bovine serum albumin back-exchange assay. Inactive GDP-bound transducin exclusively reduced the extraction of negatively charged phosphatidylserine. The effect disappeared upon activation of the G-protein with guanosine 5′-O-(3-thiotriphosphate) (GTPγS). PDE affected the extraction of the zwitterionic phosphatidylcholine and, to a smaller extent, of phosphatidylethanolamine. When active GtGTPγS interacted with the PDE to form the active effector, the interaction with phosphatidylcholine was specifically enhanced. Each copy of the G-protein bound 3 ± 1 molecules of phosphatidylserine, whereas the PDE bound a much larger amount (70 ± 10) of a mixture of phosphatidylcholine and ethanolamine. The results are interpreted as a head group-specific and state-dependent interaction of the signaling proteins with the phospholipids of the photoreceptor membrane. In retinal rod photoreceptor cells, transducin (Gt) and cyclic GMP phosphodiesterase (PDE) are peripherally anchored to the cytoplasmic surface of the disk saccules. We have examined the role of specific phospholipids in the interaction of these proteins with native osmotically intact disk vesicles, employing spin-labeled phospholipid analogues (2% of total phospholipids) and bovine serum albumin back-exchange assay. Inactive GDP-bound transducin exclusively reduced the extraction of negatively charged phosphatidylserine. The effect disappeared upon activation of the G-protein with guanosine 5′-O-(3-thiotriphosphate) (GTPγS). PDE affected the extraction of the zwitterionic phosphatidylcholine and, to a smaller extent, of phosphatidylethanolamine. When active GtGTPγS interacted with the PDE to form the active effector, the interaction with phosphatidylcholine was specifically enhanced. Each copy of the G-protein bound 3 ± 1 molecules of phosphatidylserine, whereas the PDE bound a much larger amount (70 ± 10) of a mixture of phosphatidylcholine and ethanolamine. The results are interpreted as a head group-specific and state-dependent interaction of the signaling proteins with the phospholipids of the photoreceptor membrane. The transduction of the light signal in the retinal rod photoreceptor is a well studied model system for G-protein-coupled signal transduction. It involves the sequential activation of rhodopsin, transducin (Gt), 1The abbreviations used are: Gt, transducin; BSA, fatty acid-free bovine serum albumin; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]-propane buffer; Gtα, α-subunit of transducin; Gtβγ, βγ heterodimer subunit of transducin; GTPγS, guanosine 5′-O-(3-thiotriphosphate) bound; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PDE, cGMP-phosphodiesterase; ROS, rod outer segment; SL-PC, 1-palmitoyl-2-(4-doxylpentanoyl) phosphatidylcholine; SL-PE, 1-palmitoyl-2-(4-doxylpentanoyl) phosphatidylethanolamine; SL-PS, 1-palmitoyl-2-(4-doxylpentanoyl) phosphatidylserine; SL-PL, spin-labeled phospholipids. and cGMP phosphodiesterase (PDE) on the cytoplasmic surface of the disk saccules that fill the rod outer segment (ROS). Absorption of light transforms rhodopsin into the activated metarhodopsin II state, which then interacts with Gt to rapidly catalyze the exchange of GDP for GTP in the nucleotide-binding site of the Gtα-subunit. Gtα-GTP dissociates and couples to the effector PDE (1Fung K.K.B. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2500-2504Crossref PubMed Scopus (262) Google Scholar, 2Fung B.K. Hurley J.B. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 152-156Crossref PubMed Scopus (513) Google Scholar). Activation of PDE results in a decrease in the cytosolic cGMP concentration which in turn leads to closure of cGMP-regulated channels in the plasma membrane, hyperpolarization, and neuronal signaling (see Ref. 3Helmreich E.J. Hofmann K.P. Biochim. Biophys. Acta. 1996; 1286: 285-322Crossref PubMed Scopus (128) Google Scholar). Heterotrimeric Gt is peripherally attached to the disk membrane by weak hydrophobic and ionic interactions. Both N-terminal acylation of the Gtα-subunit and C-terminal farnesylation of the Gtγ-subunit are required for membrane association of Gt (4Bigay J. Faurobert E. Franco M. Chabre M. Biochemistry. 1994; 33: 14081-14090Crossref PubMed Scopus (87) Google Scholar). Electrostatic interactions, especially of Gtβγ, further enhance the membrane binding to negatively charged surfaces or to vesicles containing the acidic lipid phosphatidylserine (5Matsuda T. Takao T. Shimonishi Y. Murata M. Asano T. Yoshizawa T. Fukada Y. J. Biol. Chem. 1994; 269: 30358-30363Abstract Full Text PDF PubMed Google Scholar, 6Seitz H.R. Heck M. Hofmann K.P. Alt T. Pellaud J. Seelig A. Biochemistry. 1999; 38: 7950-7960Crossref PubMed Scopus (48) Google Scholar, 7Murray D. McLaughlin S. Honig B. J. Biol. Chem. 2001; 48: 45153-45159Abstract Full Text Full Text PDF Scopus (59) Google Scholar). Inactive transducin in its GDP-bound form is attached as a Gtαβγ heterotrimer to the membrane. Gt couples to activated rhodopsin, triggering GDP release as a first step of catalytic nucleotide exchange. The interaction with the receptor occurs directly from the membrane-bound state of the holoprotein. After activation of transducin, Gtα dissociates from Gtβγ and couples stoichiometrically to the effector PDE. The PDE is a heterotetrameric protein composed of the undissociable αβ complex (8Artemyev N.O. Surendran R. Lee J.C. Hamm H.E. J. Biol. Chem. 1996; 271: 25382-25388Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and two identical γ-subunits (9Fung B.K. Young J.H. Yamane H.K. Griswold Prenner I. Biochemistry. 1990; 29: 2657-2664Crossref PubMed Scopus (81) Google Scholar). Full activation of the enzyme requires the binding of activated (GTP bound) Gt to both of its inhibitory γ-subunits (see Ref. 10Lamb T.D. Pugh Jr., E.N. J. Physiol. (Lond.). 1992; 449: 719-758Crossref Scopus (509) Google Scholar). Like Gt, the PDE complex is weakly associated to the surface of the disk membrane by geranylgeranylation of PDE-β and farnesylation of PDE-α (11Catty P. Deterre P. Eur. J. Biochem. 1991; 199: 263-269Crossref PubMed Scopus (50) Google Scholar, 12Anant J.S. Ong O.C. Xie H.Y. Clarke S. O'Brien P.J. Fung B.K. J. Biol. Chem. 1992; 267: 687-690Abstract Full Text PDF PubMed Google Scholar). The geranylgeranyl moiety was mainly made responsible for membrane attachment, whereas the farnesyl group may play a role in protein-protein interactions. It is known that efficient activation of PDE by active GtαGTP requires the presence of membranes, and protein-membrane interactions co-determine the interaction of Gtα-GTP with the PDE (13Hofmann K.P. Heck M. Lee A.G. Biomembranes II. JAI Press Inc., Greenwich, CT1996: 142-184Google Scholar). PDE activation studies on vesicles of different lipid composition showed that the activity of PDE depended on the nature of the lipid. The highest PDE activation was found on large unilamellar vesicles composed of the unsaturated phospholipid dioleoylphosphatidylcholine (14Melia T.J. Malinski J.A. He F. Wensel T.G. J. Biol. Chem. 2000; 275: 3535-3542Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This study provides direct information on the phospholipid interaction of the signaling proteins in native disk membranes. In particular, we are interested in the dynamics of this protein-lipid interaction during activation of the signal cascade. To characterize this interaction, we incorporate spin-labeled phospholipids into disk membranes, and we probe their accessibility to extraction by bovine serum albumin (BSA) (15Haest C.W. Plasa G. Deuticke B. Biochim. Biophys. Acta. 1981; 649: 701-708Crossref PubMed Scopus (48) Google Scholar, 16Morrot G. Herve P. Zachowski A. Fellmann P. Devaux P.F. Biochemistry. 1989; 28: 3456-3462Crossref PubMed Scopus (154) Google Scholar). In a previous study, employing this approach, we have shown that the interaction of rhodopsin with phosphatidylserine (PS) is dependent on its state of the activation, in agreement with earlier results (17Crain R.C. Marinetti G.V. O'Brien D.F. Biochemistry. 1978; 17: 4186-4192Crossref PubMed Scopus (55) Google Scholar) obtained by chemical labeling of PS in which rhodopsin was found to protect some of the PS of the membrane. During the lifetime of the active metarhodopsin II conformation, one molecule of PS became released by rhodopsin (18Hessel E. Müller P. Herrmann A. Hofmann K.P. J. Biol. Chem. 2001; 276: 2538-2543Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We have now measured the extractability by BSA of spin-labeled phospholipid analogues from the disk membrane in the absence or presence of Gt and PDE, both in the dark and under of light In the study, we interactions of Gt and PDE phospholipids. Gt in a of PS, whereas PDE to a large of ± molecules of phosphatidylcholine and spin-labeled phospholipid analogues and as G. Herve P. Zachowski A. Fellmann P. Devaux P.F. Biochemistry. 1989; 28: 3456-3462Crossref PubMed Scopus (154) Google Scholar, M. Devaux P.F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). phospholipids a fatty the and a spin-labeled fatty the with a group the from and of and from and bovine and in the dark to PubMed Scopus Google Scholar). intact disk vesicles by extraction of and proteins as (18Hessel E. Müller P. Herrmann A. Hofmann K.P. J. Biol. Chem. 2001; 276: 2538-2543Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, E. Herrmann A. Müller P. Hofmann K.P. Eur. J. Biochem. 2000; 267: PubMed Scopus Google Scholar) and in 1,3-bis[tris(hydroxymethyl)methylamino]-propane (2% BTP, 1 or ± as in the To the was a with and used The concentration of rhodopsin was To lipid during the we used disk that and used for that with their of cytosolic to J. Physiol. (Lond.). Scopus Google and in a the results as to PubMed Scopus Google containing and in J. Physiol. (Lond.). Scopus Google Scholar) and in (see showed the and PDE from bovine as M. Hofmann K.P. Biochemistry. PubMed Scopus Google Scholar) and in 1 and amount of and Gt and PDE was the 1980; PubMed Scopus Google Scholar, M. A. Hofmann K.P. 2000; PubMed Google Scholar). The to the as of the used for the rhodopsin with Gt 1 and by After of the was in of to the membrane To the amount of protein bound to the membrane or in the the of or was by of of The in to a under and with the of to a of labeling in the disk membrane rhodopsin, with with the to for The concentration was of total phospholipids. of the we found a of spin-labeled phospholipids into disk the of After of into the outer membrane of the decrease of the from the membrane was measured (15Haest C.W. Plasa G. Deuticke B. Biochim. Biophys. Acta. 1981; 649: 701-708Crossref PubMed Scopus (48) Google Scholar, 16Morrot G. Herve P. Zachowski A. Fellmann P. Devaux P.F. Biochemistry. 1989; 28: 3456-3462Crossref PubMed Scopus (154) Google Scholar, E. Müller P. Herrmann A. Hofmann K.P. J. Biol. Chem. 2001; 276: 2538-2543Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). in the from the membrane with fatty acid-free BSA concentration for 1 on to lipid analogues to BSA E. Herrmann A. Müller P. Hofmann K.P. Eur. J. Biochem. 2000; 267: PubMed Scopus Google Scholar). After and for further of on the of PDE, disk vesicles with PDE for in the dark or PDE was to disk the two membrane (see To PDE from the disk the of the spin-labeled disk in of concentration ± for and the of in the and in the membrane measured by as To the of Gt, first to the disk in the transducin was and activation of G-protein was by signal for the effect by the of PDE, transducin, To the extractability by BSA PDE of disk vesicles with and PDE with of BSA concentration and further in the from the and and both the and for amount of in the and was from the of the To reduced was In the presence of was under the are in the as the amount of from the disk membranes, measured in the or from the In both identical results The with of the was to a a are as ± the of Gt and membrane binding of both Gt and PDE on protein and membrane ionic and We used a to membrane binding of both proteins under the of the 1980; PubMed Scopus Google Scholar, M. Hofmann K.P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). the membrane concentration of the Gt and PDE bound to the membrane in the dark and activation of Gt by in a of Gt from the whereas Gt activation in the presence of PDE membrane binding of PDE In in the a of Gtα was on the membranes, with membrane binding of the active complex J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, P. F. Deterre P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). BSA of and characterize the interaction of Gt and PDE with spin-labeled phospholipid analogues we measured the of analogues from disk in the absence or presence of the (18Hessel E. Müller P. Herrmann A. Hofmann K.P. J. Biol. Chem. 2001; 276: 2538-2543Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, E. Herrmann A. Müller P. Hofmann K.P. Eur. J. Biochem. 2000; 267: PubMed Scopus Google Scholar) of the of into the outer membrane extraction of in the outer with BSA and of and of in the lipid analogues which in the and in the lipid by mixture with membranes, analogues into the outer membrane a U. G. Müller P. Herrmann A. Biophys. J. Full Text PDF PubMed Scopus Google Scholar). the amount of which is by a of the outer and of the membrane. The of for back-exchange with BSA the of We have E. Herrmann A. Müller P. Hofmann K.P. Eur. J. Biochem. 2000; 267: PubMed Scopus Google Scholar) to the the outer and of the of the is a of the phospholipid It that in the of PS, results (17Crain R.C. Marinetti G.V. O'Brien D.F. Biochemistry. 1978; 17: 4186-4192Crossref PubMed Scopus (55) Google Scholar, 1980; PubMed Scopus Google Scholar, J. Biol. 1981; PubMed Scopus Google Scholar). direct was for PC, that these directly PE, chemical of the native lipid found with the E. Herrmann A. Müller P. Hofmann K.P. Eur. J. Biochem. 2000; 267: PubMed Scopus Google Scholar, G. Biochemistry. PubMed Scopus Google Scholar). The analogues a with a of The of spin-labeled phosphatidylcholine and and a of spin-labeled phosphatidylserine of the in the outer This is the as was under different (see (18Hessel E. Müller P. Herrmann A. Hofmann K.P. J. Biol. Chem. 2001; 276: 2538-2543Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, E. Herrmann A. Müller P. Hofmann K.P. Eur. J. Biochem. 2000; 267: PubMed Scopus Google Scholar). the BSA of the interaction of Gt with SL-PL, Gt was to the of the two the extraction of by BSA for was in and with that of The of Gt activation on lipid interaction was in a in which was with The was in the dark to the amount of activated rhodopsin as as under these Gt is activated by a of in the D. Hofmann K.P. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). the of that in the BSA extraction in the presence of or active upon of In the presence of Gt, ± of was to This was upon activation of Gt with GTPγS, that is the that specifically the extraction of much effect was measured for or with we found a of the effect was with The extraction of was with different of The membrane-bound amount of with Gt concentration results obtained in the absence and presence of 1 (see PDE the BSA of and the of PDE on BSA extraction of SL-PL, disk with PDE PDE, in the dark to membrane-bound and PDE. and their was as In PDE was to the disk both membrane 3 the amount of that in the BSA with and of the with PDE. in PDE effect on the extractability of and the of PS In to SL-PS, both and in the presence of PDE and these of and of found in the BSA the effect on extractability was the first was 1 3 both the and the of of and When the PDE was a of was extraction was affected both and became The effect was the first 1 of 1 PDE and of the and of the the BSA a this effect is to that for with PDE. When the extraction was with different of PDE the membrane-bound amount of both and with PDE, whereas of PDE was in the of The amount of and the concentration the of membrane 3 PDE the study of PDE The of PDE the of the PDE with PDE in the was and for the back-exchange in In and the by and with ionic to release PDE from (see of PDE was by of the with the in the release of PDE from the disk ionic in a and of the PDE effect on To that the the extraction of the of was with PDE and the amount of analogues to the of the The of the PDE effect was the containing the lipid with BSA with or PDE of PDE Activation on the of the presence of Gt and PDE their interaction in the active complex affected the extraction of the of and Gt, PDE, and to the membrane with the results of Gt in a reduced extraction of of or The of PDE reduced the of and to a that of that of in the presence of both Gt and PDE, the effect of the proteins is in their the two proteins on the membrane we a of the interaction upon activation of Gt (see this was Gt was activated in the presence of PDE. this reduced extractability of was a of active Gt from the (see and Activation of Gt and PDE in a further decrease of extraction with the membrane binding of PDE (see and the effect was with We have examined the interaction with membrane phospholipids of two proteins in signal the G-protein transducin (Gt) and the effector, a cyclic GMP phosphodiesterase In the osmotically intact disk vesicles, rhodopsin molecule is by PubMed Scopus (29) Google Scholar). the membrane composition 1992; PubMed Scopus (55) Google Scholar) and the of the membrane of PS, PC, and rhodopsin is found in the outer which Gt and PDE BSA back-exchange was to and interaction in native membrane To this the different amount of spin-labeled phospholipid in the presence or absence of the protein was as a with Gt and PDE, or in and in their active or The of the is that the lipid interactions of the two proteins are lipid head group-specific and on the activation state of the We interaction of Gt with the negatively charged phosphatidylserine, and of the PDE with the zwitterionic phosphatidylcholine and phosphatidylethanolamine. The two proteins in the in the amount of The Gt in a of of Gt to rod disk a reduced extraction of spin-labeled PS by This was in the of and SL-PE, a head group-specific lipid interaction of of the total into disk by BSA membrane binding of This was obtained in both the absence and presence of 1 the and of PS in the disk membrane, the effect on total lipid into of Gt heterotrimer is on the α-subunit and on the and membrane association of the protein is to the of both of these into the of the membrane (4Bigay J. Faurobert E. Franco M. Chabre M. Biochemistry. 1994; 33: 14081-14090Crossref PubMed Scopus (87) Google Scholar). the outer to the effect of Gt on for molecules of outer bound to the specific of Gt to a lipid It is known that the unsaturated lipid of the transducin binding (14Melia T.J. Malinski J.A. He F. Wensel T.G. J. Biol. Chem. 2000; 275: 3535-3542Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 2000; PubMed Scopus Google Scholar). In bovine disk membranes, phospholipids a of fatty PS the highest PubMed Scopus Google Scholar). on the of Gtβγ have to the that the negatively charged Gtβγ subunit complex is and a of the site of This may Gtβγ to vesicles of acidic phosphatidylserine (5Matsuda T. Takao T. Shimonishi Y. Murata M. Asano T. Yoshizawa T. Fukada Y. J. Biol. Chem. 1994; 269: 30358-30363Abstract Full Text PDF PubMed Google Scholar, A. A. E. T. J.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and to negatively charged whereas the Gtα-subunit H.R. Heck M. Hofmann K.P. Alt T. Pellaud J. Seelig A. Biochemistry. 1999; 38: 7950-7960Crossref PubMed Scopus (48) Google Scholar, A. A. E. T. J.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The attached protein in a that the farnesyl moiety into the membrane with a negatively charged surface the membrane of transducin D. McLaughlin S. Honig B. J. Biol. Chem. 2001; 48: 45153-45159Abstract Full Text Full Text PDF Scopus (59) Google Scholar). the interaction of Gtβγ with of the membrane surface may to of Gt heterotrimer and its H.R. Heck M. Hofmann K.P. Alt T. Pellaud J. Seelig A. Biochemistry. 1999; 38: 7950-7960Crossref PubMed Scopus (48) Google Scholar) that in the Gt and farnesyl on Gtα and are to one to as one hydrophobic We now that 3 ± 1 molecules of PS this of and hydrophobic interactions. The activation of transducin by leads to Gtα and Gtβγ from the membrane 1980; PubMed Scopus Google which the absence of the effect on PS The PDE a of and of the and membrane-bound native PDE found in rod outer that PDE is associated to the disk membrane to phospholipids J.A. Wensel T.G. Biochemistry. 1992; PubMed Scopus Google Scholar). on the effect of PDE on BSA extraction of SL-PL, and the as for the the PDE interacts with the phospholipids and the PDE was found on the extraction of the negatively charged phospholipid The to BSA extraction of and was shown to in a to the amount of PDE with a effect of and for and SL-PE, In of the native concentration of PDE and the membrane binding under the of of PDE was the native of PDE on the membrane surface was PDE. these extraction of analogues by BSA was reduced by and of total spin-labeled and as with membranes. the known to rhodopsin of of the two and of the PDE, one the that molecules of and of are from BSA extraction molecule of membrane-bound PDE of phospholipid analogues by BSA Gt or PDE to disk of Gt or of total lipid ± ± with the to the ± ± ± to the ± ± with the ± ± to the to the with the in a results membrane which the of BSA to the membranes, extraction of The are of a PDE PS and Gt or we state the the effect from binding of the to the protein for PDE. this the of PDE which was This is with the that with BSA to in the presence of bound PDE to the as for of PDE, The of the and its in the presence of PDE is that the interaction is from the of the that this a of analogues to PDE, and a of the analogues to the membrane The of the is with a direct in which molecule of PDE bound to the membrane surface the We that the lipid analogues the native phospholipids and directly with the PDE. PDE Activation and of exchange of GDP for GTP or in the nucleotide-binding site of Gtα leads to of Gtα and To the PDE two of the activated to the PDE P. J. F. M. Chabre M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). PDE is anchored to the disk membrane by and of the of its and It is well that the of Gt to the PDE is dependent on the presence composition of (14Melia T.J. Malinski J.A. He F. Wensel T.G. J. Biol. Chem. 2000; 275: 3535-3542Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, J.A. Wensel T.G. Biochemistry. 1992; PubMed Scopus Google and studies P. F. Deterre P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, J.A. Wensel T.G. Biochemistry. 1992; PubMed Scopus Google Scholar) have shown that PDE is membrane-bound activated by The interaction Gtα-GTP and a in the light of the membrane, which the activity M. Hofmann K.P. Biochemistry. PubMed Scopus Google Scholar). The from these previous studies that the activation of PDE both requires the presence of the disk membrane and its is and by It was found that the of the complex a of spin-labeled phospholipids to BSA as with the PDE and Gtα the of phospholipid interaction with the complex the of the The complex PS the Gt and a for PC, which may activated PDE to the membrane. to that protein-lipid interaction on the activation state of the proteins the role of membrane and the of lipid in signal transduction. The as to these to the known as which are as The to the PDE to or protein binding to the membrane a in the lipid M. M. Y. M. Y. F. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) have that both Gt and PDE their the disk membrane and the membrane in a state-dependent Gt, into the that of the which Gt to in P. Biol. 2001; PubMed Scopus Google Scholar). activation was under of Gt to with a in the and with the the which the activation J. 2001; PubMed Scopus Google Scholar). by into results on interaction, we may that of the G-protein requires the binding of the protein to different lipid is and in the of Gt Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) that the proteins are to in the of the rod The of with may into the of phospholipid are in these of study a of signal that the two signaling proteins specific of phospholipids to with the membrane. We have found that the amount of lipid that extraction by BSA the required to the of the protein on the membrane. The G-protein is anchored by a molecules of on known a of the negatively charged PS head In the of the PDE effector, membrane association is by a interaction with both and the of PDE is we that its on the membrane is much molecules of and and phospholipid interaction may the of interaction during signal transduction. may that a Gt for the and interaction of Gt with the activated the the of interaction Gt and PDE is on the of M. Hofmann K.P. Biochemistry. PubMed Scopus Google a for The of these interactions to that a to the of the G-protein-coupled transduction in We S. for the of spin-labeled phospholipid analogues and I. for