Kinetics in Signal Transduction Pathways Involving Promiscuous Oligomerizing Receptors Can Be Determined by Receptor Specificity: Apoptosis Induction by TRAIL
2012; Elsevier BV; Volume: 11; Issue: 3 Linguagem: Inglês
10.1074/mcp.m111.013730
ISSN1535-9484
AutoresÉva Szegezdi, Almer M. van der Sloot, Devalingam Mahalingam, Lynda O’Leary, Robbert H. Cool, Inés G. Muñoz, Guillermo Montoya, Wim J. Quax, Steven de Jong, Afshin Samali, Luís Serrano,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoHere we show by computer modeling that kinetics and outcome of signal transduction in case of hetero-oligomerizing receptors of a promiscuous ligand largely depend on the relative amounts of its receptors. Promiscuous ligands can trigger the formation of nonproductive receptor complexes, which slows down the formation of active receptor complexes and thus can block signal transduction. Our model predicts that increasing the receptor specificity of the ligand without changing its binding parameters should result in faster receptor activation and enhanced signaling. We experimentally validated this hypothesis using the cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its four membrane-bound receptors as an example. Bypassing ligand-induced receptor hetero-oligomerization by receptor-selective TRAIL variants enhanced the kinetics of receptor activation and augmented apoptosis. Our results suggest that control of signaling pathways by promiscuous ligands could result in apparent slow biological kinetics and blocking signal transmission. By modulating the relative amount of the different receptors for the ligand, signaling processes like apoptosis can be accelerated or decelerated and even inhibited. It also implies that more effective treatments using protein therapeutics could be achieved simply by altering specificity Here we show by computer modeling that kinetics and outcome of signal transduction in case of hetero-oligomerizing receptors of a promiscuous ligand largely depend on the relative amounts of its receptors. Promiscuous ligands can trigger the formation of nonproductive receptor complexes, which slows down the formation of active receptor complexes and thus can block signal transduction. Our model predicts that increasing the receptor specificity of the ligand without changing its binding parameters should result in faster receptor activation and enhanced signaling. We experimentally validated this hypothesis using the cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its four membrane-bound receptors as an example. Bypassing ligand-induced receptor hetero-oligomerization by receptor-selective TRAIL variants enhanced the kinetics of receptor activation and augmented apoptosis. Our results suggest that control of signaling pathways by promiscuous ligands could result in apparent slow biological kinetics and blocking signal transmission. By modulating the relative amount of the different receptors for the ligand, signaling processes like apoptosis can be accelerated or decelerated and even inhibited. It also implies that more effective treatments using protein therapeutics could be achieved simply by altering specificity To enable diverse biological responses, many ligand-receptor systems consist of multiple receptors and/or ligands (1Bodmer J.L. Schneider P. Tschopp J. The molecular architecture of the TNF superfamily.Trends Biochem. Sci. 2002; 27: 19-26Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 2Eswarakumar V.P. Lax I. Schlessinger J. Cellular signaling by fibroblast growth factor receptors.Cytokine Growth Factor Rev. 2005; 16: 139-149Crossref PubMed Scopus (1493) Google Scholar, 3Ferguson K.M. Structure-based view of epidermal growth factor receptor regulation.Annu. Rev. Biophys. 2008; 37: 353-373Crossref PubMed Scopus (241) Google Scholar, 4Shibuya M. Claesson-Welsh L. Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis.Exp. Cell Res. 2006; 312: 549-560Crossref PubMed Scopus (847) Google Scholar). Predicting the biological response in such a multicomponent system can be complex: the presence of the receptors and ligands does not guarantee that the signal is transmitted. The response can be dictated by the final equilibrium distribution of the ligand with the different receptors, but it is equally possible that the biological effect is more dynamically driven by temporal kinetic effects of the ligand interacting with its receptors (5Buchler N.E. Louis M. Molecular titration and ultrasensitivity in regulatory networks.J. Mol. Biol. 2008; 384: 1106-1119Crossref PubMed Scopus (205) Google Scholar). This may be the case in particular when the ligand has a short half-life time. In fact, there is evidence that kinetics could play an important role in the outcome of a signal (6Kiel C. Serrano L. Cell type-specific importance of Ras-c-Raf complex association rate constants for MAPK signaling.Sci. Signal. 2009; 2: ra38Crossref PubMed Scopus (57) Google Scholar). In a simple network, an interaction with similar Kd but having different kon and koff values could respond differently with a short or a long pulse of a ligand. In these multicomponent systems, mathematical modeling is an invaluable tool to understand and predict the outcome of the signaling events (6Kiel C. Serrano L. Cell type-specific importance of Ras-c-Raf complex association rate constants for MAPK signaling.Sci. Signal. 2009; 2: ra38Crossref PubMed Scopus (57) Google Scholar). In principle, excluding well known effects like macromolecular crowding or protein immobilization, one could assume that it should be possible to extrapolate kinetic and equilibrium constants from in vitro to in vivo conditions, provided that the in vitro conditions reflect the in vivo ones. However, this simple assumption could fail if the molecule that triggers a signal can be trapped in nonproductive interactions that equilibrate slowly with the productive complex. This can significantly slow down or even block the progression of a signal. In such cases, one could imagine that increasing the specificity of the ligand toward the productive complex could result in faster kinetics of receptor activation (7Reis C.R. van der Sloot A.M. Natoni A. Szegezdi E. Setroikromo R. Meijer M. Sjollema K. Stricher F. Cool R.H. Samali A. Serrano L. Quax W.J. Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants.Cell Death Dis. 2010; 1: e83Crossref PubMed Scopus (62) Google Scholar). Moreover, if this is the case, it could affect the way many signal transduction pathways are analyzed and simulated because it is conceivable that this scenario could be very common. To explore this possibility, we have selected a relatively well understood signal transduction pathway that involves both a promiscuous ligand and nonproductive - (receptor-ligand) complex formation, the TNF-related apoptosis-inducing ligand (TRAIL)-induced 1The abbreviations used are:TRAILtumor necrosis factor-related apoptosis-inducing ligandDRdeath receptorDcRdecoy receptorFADDFas-associated death domainPLADpreligand assembly domain(s)MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromideDEVDcarbobenzoxy-Asp-Glu-Val-Asp-7AMCamino-4-methylcoumarinCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidXIAPX-linked inhibitor of apoptosis proteinWTwild typeCARPcaspase-8 and -10 associated RING protein. 1The abbreviations used are:TRAILtumor necrosis factor-related apoptosis-inducing ligandDRdeath receptorDcRdecoy receptorFADDFas-associated death domainPLADpreligand assembly domain(s)MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromideDEVDcarbobenzoxy-Asp-Glu-Val-Asp-7AMCamino-4-methylcoumarinCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidXIAPX-linked inhibitor of apoptosis proteinWTwild typeCARPcaspase-8 and -10 associated RING protein. apoptotic pathway (8Ashkenazi A. 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Biol. 2004; 1: 129-138Crossref Scopus (111) Google Scholar). Our simulations suggest that the receptor toward which the ligand has the highest specificity—but without necessarily having increased kinetic binding constants—will be activated much faster than the other receptors and will therefore be the main determinant of the signal transduction pathway activated. To verify whether this prediction holds in cells, we have used a TRAIL variant that selectively binds to DR5 (D269H/E195R) (28van der Sloot A.M. Tur V. Szegezdi E. Mullally M.M. Cool R.H. Samali A. Serrano L. Quax W.J. Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8634-8639Crossref PubMed Scopus (134) Google Scholar). This TRAIL variant has proved to be a very potent inducer of apoptosis in tumor cell lines where the DR5 receptor is active both in vitro (28van der Sloot A.M. Tur V. Szegezdi E. Mullally M.M. Cool R.H. Samali A. Serrano L. Quax W.J. Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8634-8639Crossref PubMed Scopus (134) Google Scholar) and in vivo (29Duiker E.W. de Vries E.G. Mahalingam D. Meersma G.J. Boersma-van Ek W. Hollema H. Lub-de Hooge M.N. van Dam G.M. Cool R.H. Quax W.J. Samali A. van der Zee A.G. de Jong S. Enhanced antitumor efficacy of a DR5-specific TRAIL variant over recombinant human TRAIL in a bioluminescent ovarian cancer xenograft model.Clin. Cancer Res. 2009; 15: 2048-2057Crossref PubMed Scopus (41) Google Scholar). Using this TRAIL variant as a tool, we found that receptor-specific TRAIL variants exhibit much faster receptor activation kinetics, confirming the prediction of the model. We also show that this multireceptor system works in concert to create a temporal control of apoptosis induction and, under physiological conditions, can act as a safeguard against unwarranted induction of apoptosis. Thus, our results indicate that kinetics of signal transduction induced by promiscuous ligands are largely determined by nonproductive interactions and, consequently, by the relative amount of the different receptors sharing the same ligand. Colo205 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mm l-glutamine, 50 units/ml penicillin, 5 mg/ml streptomycin, and 10 mm sodium pyruvate. The cells were treated with recombinant human TRAIL (nontagged, amino acids 114–281 fragment), Wt TRAIL, or DR5-specific TRAIL variant, D269H/E195R, which were produced and purified as described before (28van der Sloot A.M. Tur V. Szegezdi E. Mullally M.M. Cool R.H. Samali A. Serrano L. Quax W.J. Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8634-8639Crossref PubMed Scopus (134) Google Scholar, 30van der Sloot A.M. Mullally M.M. Fernandez-Ballester G. Serrano L. Quax W.J. Stabilization of TRAIL, an all-β-sheet multimeric protein, using computational redesign.Protein Eng. Des. Sel. 2004; 17: 673-680Crossref PubMed Scopus (32) Google Scholar). Analytical gel filtration and nonreducing gel electrophoresis confirmed that D269H/E195R is a trimeric molecule that does not form higher degree aggregates (28van der Sloot A.M. Tur V. Szegezdi E. Mullally M.M. Cool R.H. Samali A. Serrano L. Quax W.J. Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8634-8639Crossref PubMed Scopus (134) Google Scholar). Soluble DR5 receptor, DcR1 and DcR2, and neutralizing antibodies to DcR1 and DcR2 were all from R & D Systems. Other reagents were from Sigma-Aldrich unless otherwise stated. Cell viability was measured by adding 500 μg/ml MTT to control and treated cells and incubated for 3 h at 37 °C. The reaction was stopped, the purple formazan precipitate formed was dissolved in 10% dimethyl formamide and 20% sodium dodecyl sulfate, and the color intensity was measured at 550 nm using a Wallac multilabel counter (32Szegezdi E. Cahill S. Meyer M. O'Dwyer M. Samali A. TRAIL sensitisation by arsenic trioxide is caspase-8 dependent and involves modulation of death receptor components and Akt.Br. J. Cancer. 2006; 94: 398-406Crossref PubMed Scopus (28) Google Scholar). The control value corresponding to untreated cells was taken as 100%, and the viability of treated samples was expressed as a percentage of the control. Externalization of phosphatidylserine on the plasma membrane of apoptotic cells was detected using annexin V-FITC (IQ Corporation, Groningen, The Netherlands) and flow cytometry (FACSCanto II flow cytometer; Becton Dickinson) as previously described (33Holohan C. Szegezdi E. Ritter T. O'Brien T. Samali A. Cytokine-induced β-cell apoptosis is NO-dependent, mitochondria-mediated and inhibited by BCL-XL.J. Cell Mol. Med. 2008; 12: 591-606Crossref PubMed Scopus (48) Google Scholar). Polyacrylamide gel electrophoresis and Western blotting was carried out as previously described (34Szegezdi E. Herbert K.R. Kavanagh E.T. Samali A. Gorman A.M. Nerve growth factor blocks thapsigargin-induced apoptosis at the level of the mitochondrion via regulation of Bim.J. Cell Mol. Med. 2008; 12: 2482-2496Crossref PubMed Scopus (35) Google Scholar). For antigen detection, membranes were incubated with antibodies to actin (1:500; Sigma), caspase-3 and -8 (1:1,000; Cell Signaling Technologies) overnight at 4 °C followed by 2 h of incubation at room temperature with appropriate secondary antibodies (1:5,000; Pierce). Protein bands were visualized using Supersignal Ultra Chemiluminescent Substrate (Pierce) on x-ray film (Agfa). Cells (3 × 105) were resuspended in PBS (25 μl) and were transferred to a microtiter plate and snap-frozen over liquid nitrogen. To initiate the reaction, 50 μm of the caspase substrate DEVD-AMC (Peptide Institute Inc., Osaka, Japan) in assay buffer (100 mm Hepes buffer, 10% sucrose, 0.1% CHAPS, 5 mm DTT, and 0.0001% Igepal 630, pH 7.25) was added to cell lysates. Substrate cleavage leading to the release of free AMC was monitored at 37 °C at 60 s intervals for 25 cycles using a Wallac multilabel counter (excitation, 355 nm; emission, 460 nm). Enzyme activity was expressed as nmol of AMC released/min/mg protein. Binding experiments were performed as previously described (28van der Sloot A.M. Tur V. Szegezdi E. Mullally M.M. Cool R.H. Samali A. Serrano L. Quax W.J. Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8634-8639Crossref PubMed Scopus (134) Google Scholar). The cells were washed twice in PBS containing 1% BSA and then incubated with monoclonal antibodies to DR4, DR5, DcR1, or DcR2 (Enzo Life Sciences) for 40 min. The antibodies have no cross-reactivity among the four TRAIL receptors (shown by the Supplier's data sheet). After two wash steps with PBS/BSA, anti-mouse IgG-FITC (Sigma) secondary antibody was added for 30 min. All of the incubations were carried out on ice. Negative controls contained isotype control antibody. Receptor expression was analyzed on a FACSCalibur or FACSCanto II flow cytometer (Becton Dickinson). Receptor binding by WT TRAIL and D269H/E195R on the cell surface was simulated using a mathematical model describing all of the possible binding events. Both the formation of homotrimeric (e.g. TRAIL-3DR5) and heterotrimeric ligand receptor complexes (e.g. TRAIL-2DR5-DcR2) were allowed, and binding was simulated in a stepwise fashion (Fig. 1). On-rates and off-rates measured for interactions between monomeric DR4, DR5, DcR1 and DcR2 receptors and of TRAIL binding to these receptors were taken from the report of Lee et al. (25Lee H.W. Lee S.H. Lee H.W. Ryu Y.W. Kwon M.H. Kim Y.S. Homomeric and heteromeric interactions of the extracellular domains of death receptors and death decoy receptors.Biochem. Biophys. Res. Commun. 2005; 330: 1205-1212Crossref PubMed Scopus (52) Google Scholar), where monomeric TRAIL receptor constructs have been employed. This allowed us to derive stepwise binding constants and to model heteromeric receptor-ligand interactions (Fig. 1 and supplemental Table 2). Stepwise constants for going from a single ligand-bound receptor, via two ligand-bound receptors, to the complex consisting of three receptors bound by a trimeric ligand were estimated in the following way: the kon reported by Lee et al. was assigned to the first binding event. The first association step is entropically the most unfavorable one because of the loss of rotational degrees of freedom when going from an unbound state to a bound state. To compensate for this entropic penalty in the first step, the Kd for the second and third step was decreased by 2 kcal/mol and attributed completely to an increase in kon (35Tamura A. Privalov P.L. The entropy cost of protein association.J. Mol. Biol. 1997; 273: 1048-1060Crossref PubMed Scopus (109) Google Scholar, 36Ben-Tal N. Honig B. Bagdassarian C.K. Ben-Shaul A. Association entropy in adsorption processes.Biophys. J. 2000; 79: 1180-1187Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The values of 0 to −4 kcal/mol for the entropy penalty were tested; the impact on the final results was negligible. The reported koff was assigned to the first step. Two scenarios were explored: 1) no cooperative binding, i.e. binding of one receptor does not enhance the binding of the next receptor (supplemental Table 2) and 2) cooperative binding, whereby binding to the first receptor enhances the binding of a second receptor of the same type (supplemental Table 2). Cooperativity can be the result of interaction between the intracellular death domains of the DR4 or DR5 receptors, as has been shown for FAS, another death receptor (37Wang L. Yang J.K. Kabaleeswaran V. Rice A.J. Cruz A.C. Park A.Y. Yin Q. Damko E. Jang S.B. Raunser S. Robinson C.V. Siegel R.M. Walz T. Wu H. The Fas-FADD death domain complex structure reveals the basis of DISC assembly and disease mutations.Nat. Struct. Mol. Biol. 2010; 17: 1324-1329Crossref PubMed Scopus (218) Google Scholar). Thus, to model cooperativity, the koff was decreased stepwise 5-fold in each subsequent binding reaction (38Klein J.S. Gnanapragasam P.N. Galimidi R.P. Foglesong C.P. West Jr., A.P. Bjorkman P.J. Examination of the contributions of size and avidity to the neutralization mechanisms of the anti-HIV antibodies b12 and 4E10.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7385-7390Crossref PubMed Scopus (120) Google Scholar, 39Lortat-Jacob H. Chouin E. Cusack S. van Raaij M.J. Kinetic analysis of adenovirus fiber binding to its receptor reveals an avidity mechanism for trimeric receptor-ligand interactions.J. Biol. Chem. 2001; 276: 9009-9015Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Varying this factor between 2 and 10 revealed little impact on the final results. In case of homotrimeric TRAIL-3DR5 and TRAIL-3DR4 complexes, an extra step to an "activated" receptor complex was introduced to mathematically describe the assembly of the intracellular death-inducing signaling complex (consisting of the death receptors, FADD, and caspase-8). As mentioned before, DcR1 does not contain an intracellular domain, and DcR2 only contains a truncated Death domain; therefore no "activation" step was added for decoy receptor containing complexes (supplemental Table 2). The rate constants for the "activation" step that governs the formation of activated homotrimeric death receptor complexes were obtained by increasing the kon and decreasing the koff by a factor of ten relative to the rate constants of the third step (supplemental Table 2). The presence of an activation step was necessary for scenario 1 (no cooperative effects upon binding additional receptor units) to drive TRAIL-3DR4/TRAIL-3DR5 complex formation, but the effect of the actual magnitude of this step was low upon varying this factor between 2 and 10 (relative to the rate constants of the third step). The impact on inclusion of an activation step for scenari
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