Inducible Dimerization of RET Reveals a Specific AKT Deregulation in Oncogenic Signaling

Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, a receptor tyrosine kinase, are causally associated with the development of multiple endocrine neoplasia type 2A (MEN2A) syndrome. Such oncogenic RET mutations induce its ligand-independent constitutive activation, but whether it spreads identical signaling to ligand-induced signaling is uncertain. To address this question, we designed a cellular model in which RET can be activated either by its natural ligand, or alternatively, by controlled dimerization of the protein that mimics MEN2A dimerization. We have shown that controlled dimerization leaves proximal RET signaling intact but impacts substantially on the tuning of the distal AKT kinase activation (delayed and sustained). In marked contrast, distal activation of ERK remained unaffected. We further demonstrated that specific temporal adjustment of ligand-induced AKT activation is dependent upon a lipid-based cholesterol-sensitive environment, and this control step is bypassed by MEN2A RET mutants. Therefore, these studies revealed that MEN2A mutations propagate previously unappreciated subtle differences in signaling pathways and unravel a role for lipid rafts in the temporal regulation of AKT activation. Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, a receptor tyrosine kinase, are causally associated with the development of multiple endocrine neoplasia type 2A (MEN2A) syndrome. Such oncogenic RET mutations induce its ligand-independent constitutive activation, but whether it spreads identical signaling to ligand-induced signaling is uncertain. To address this question, we designed a cellular model in which RET can be activated either by its natural ligand, or alternatively, by controlled dimerization of the protein that mimics MEN2A dimerization. We have shown that controlled dimerization leaves proximal RET signaling intact but impacts substantially on the tuning of the distal AKT kinase activation (delayed and sustained). In marked contrast, distal activation of ERK remained unaffected. We further demonstrated that specific temporal adjustment of ligand-induced AKT activation is dependent upon a lipid-based cholesterol-sensitive environment, and this control step is bypassed by MEN2A RET mutants. Therefore, these studies revealed that MEN2A mutations propagate previously unappreciated subtle differences in signaling pathways and unravel a role for lipid rafts in the temporal regulation of AKT activation. The RET 4The abbreviations used are:RETrearranged during transfectionDRMsdetergent-resistant membranesERKextracellular signal regulated kinaseGDNFglial cell line-derived neurotrophic factorGFRαglial cell line-derived neurotrophic factor receptor αMBCmethyl-β-cyclodextrineMEN2multiple endocrine neoplasia type 2PI3Kphosphatidylinositol 3 kinasePLCγphospholipase C γShcSrc homology and collagen proteinTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingGFLGDNF family ligandsHAhemagglutininMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideAPAP20187. 4The abbreviations used are:RETrearranged during transfectionDRMsdetergent-resistant membranesERKextracellular signal regulated kinaseGDNFglial cell line-derived neurotrophic factorGFRαglial cell line-derived neurotrophic factor receptor αMBCmethyl-β-cyclodextrineMEN2multiple endocrine neoplasia type 2PI3Kphosphatidylinositol 3 kinasePLCγphospholipase C γShcSrc homology and collagen proteinTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingGFLGDNF family ligandsHAhemagglutininMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideAPAP20187. proto-oncogene is located on chromosome 10q11.2 and encodes a receptor tyrosine kinase with four cadherin-related motifs and a cysteine-rich domain in the extracellular domain (1Airaksinen M.S. Saarma M. Nat. Rev. Neurosci. 2002; 3: 383-394Crossref PubMed Scopus (1445) Google Scholar). It associates with ligand-specific co-receptors known as GFRαs (GDNF, glial-cell-line-derived neurotrophic factor, family receptors α), to form functional receptors for the GFL (GDNF family ligands). In the current view, homodimeric GFL binding induces a GFL2-GFRα2-RET2 complex (2Leppanen V.M. Bespalov M.M. Runeberg-Roos P. Puurand U. Merits A. Saarma M. Goldman A. EMBO J. 2004; 23: 1452-1462Crossref PubMed Scopus (52) Google Scholar). RET dimerization leads to increased receptor kinase activity and autophosphorylation of cytoplasmic tyrosine residues, which serve as docking sites for Src homology 2 (SH2)- and phospho-tyrosine-binding domain-containing proteins, such as Shc or phospholipase Cγ (3Ichihara M. Murakumo Y. Takahashi M. Cancer Lett. 2004; 204: 197-211Crossref PubMed Scopus (162) Google Scholar). These proteins then recruit additional effector molecules, resulting in the assembly of signaling complexes and the activation of intracellular signaling pathways, including the Ras/extracellular-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. GFL-mediated signaling pathways are involved in the development and maintenance of a broad spectrum of neuronal subpopulations (1Airaksinen M.S. Saarma M. Nat. Rev. Neurosci. 2002; 3: 383-394Crossref PubMed Scopus (1445) Google Scholar). rearranged during transfection detergent-resistant membranes extracellular signal regulated kinase glial cell line-derived neurotrophic factor glial cell line-derived neurotrophic factor receptor α methyl-β-cyclodextrine multiple endocrine neoplasia type 2 phosphatidylinositol 3 kinase phospholipase C γ Src homology and collagen protein terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling GDNF family ligands hemagglutinin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide AP20187. rearranged during transfection detergent-resistant membranes extracellular signal regulated kinase glial cell line-derived neurotrophic factor glial cell line-derived neurotrophic factor receptor α methyl-β-cyclodextrine multiple endocrine neoplasia type 2 phosphatidylinositol 3 kinase phospholipase C γ Src homology and collagen protein terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling GDNF family ligands hemagglutinin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide AP20187. Recently, membrane microdomains, or lipid rafts, have been shown to profoundly influence the functional impact of GDNF-stimulated RET downstream signaling (4Tansey M.G. Baloh R.H. Milbrandt J. Johnson Jr., E.M. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 5Paratcha G. Ledda F. Baars L. Coulpier M. Besset V. Anders J. Scott R. Ibanez C.F. Neuron. 2001; 29: 171-184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Lipid rafts are suggested to be lateral microdomains in membranes of living cells, enriched in sphingolipids, cholesterol, and specific membrane proteins. They are characterized by higher order and by having a lower buoyant density than bulk plasma membranes (6Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5149) Google Scholar, 7Sprong H. van der Sluijs P. van Meer G. Nat. Rev. Mol. Cell. Biol. 2001; 2: 504-513Crossref PubMed Scopus (476) Google Scholar). Although uncertainties about the precise molecular nature of rafts remain (8Edidin M. Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 257-283Crossref PubMed Scopus (1134) Google Scholar, 9Munro S. Cell. 2003; 115: 377-388Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar, 10Sharma P. Varma R. Sarasij R.C. Ira Gousset K. Krishnamoorthy G. Rao M. Mayor S. Cell. 2004; 116: 577-589Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 11Kusumi A. Koyama-Honda I. Suzuki K. Traffic. 2004; 5: 213-230Crossref PubMed Scopus (333) Google Scholar), compelling evidence indicates that lipid rafts can coalesce into larger and more stable structures where proteins can segregate to perform functions (12Golub T. Wacha S. Caroni P. Curr. Opin. Neurobiol. 2004; 14: 542-550Crossref PubMed Scopus (135) Google Scholar, 13Rajendran L. Simons K. J. Cell Sci. 2005; 118: 1099-1102Crossref PubMed Scopus (468) Google Scholar). With regard to GDNF-stimulated RET signaling, it has been shown that at steady state, GFRα1, but not RET, is a resident of lipid raft-related detergent-resistant membranes (DRMs). GDNF stimulation was demonstrated to trigger RET recruitment and activation into DRMs. This was strongly correlated with downstream signaling, cell survival, and differentiation (4Tansey M.G. Baloh R.H. Milbrandt J. Johnson Jr., E.M. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Furthermore, Paratcha et al. (5Paratcha G. Ledda F. Baars L. Coulpier M. Besset V. Anders J. Scott R. Ibanez C.F. Neuron. 2001; 29: 171-184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) showed that RET association with DRMs may influence the nature of the intracellular signaling. Together, these studies demonstrate a connection between RET association with DRMs and RET signaling and support a role for lipid rafts in controlling GDNF-stimulated RET signaling. Mutations in the RET proto-oncogene have been identified as causative for human papillary thyroid carcinoma, multiple endocrine neoplasia (MEN) type 2A and 2B, and familial medullary thyroid carcinoma (14Hansford J.R. Mulligan L.M. J. Med. Genet. 2000; 37: 817-827Crossref PubMed Scopus (208) Google Scholar). MEN2A is an autosomal dominant cancer syndrome, characterized by medullary thyroid carcinoma, pheochromocytoma, and hyperplasia of the parathyroid gland. MEN2A mutations were identified in the cysteine-rich region, and ∼90% of those mutations affect codon 634 (most frequently a cysteine to arginine change) (15Eng C. J. Clin. Oncol. 1999; 17: 380-393Crossref PubMed Google Scholar). They result in a constitutive activation of RET through formation of covalently linked dimers of the receptor, independent of GFL (16Asai N. Iwashita T. Matsuyama M. Takahashi M. Mol. Cell. Biol. 1995; 15: 1613-1619Crossref PubMed Google Scholar, 17Santoro M. Carlomagno F. Romano A. Bottaro D.P. Dathan N.A. Grieco M. Fusco A. Vecchio G. Matoskova B. Kraus M.H. Di Fiore P.P. Science. 1995; 267: 381-383Crossref PubMed Scopus (796) Google Scholar). Dimerization occurs early during the maturation process and results in additional activation of incompletely glycosylated intracellular RET precursors (18Cosma M.P. Cardone M. Carlomagno F. Colantuoni V. Mol. Cell. Biol. 1998; 18: 3321-3329Crossref PubMed Scopus (51) Google Scholar). Whether MEN2A signaling is identical to GFL-triggered signaling is to activation of the constitutive MEN2A protein be in cells, a and with the signaling pathways. the current model to MEN2A RET the nature of MEN2A signaling. To address this we have a protein that can be activated in the either with the natural GDNF or with a T. M. Jr., F. M. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The ligand-independent RET dimerization by MEN2A mutants. This activation of RET in AKT activation but not ERK activation. Furthermore, we demonstrated that a lipid-based cholesterol-sensitive the tuning of AKT activation, a role of lipid we showed that MEN2A this control Therefore, these studies revealed complex in oncogenic signaling by MEN2A RET mutants. and hemagglutinin and human receptor were and were Cell was A. P. M. M. L. I. M. 15: PubMed Scopus Google Scholar). or were by D. G. G. A. G. Vecchio G. Fusco A. M. J. Clin. 2000; PubMed Scopus Google Scholar). human and were and and were was by The domain was the into the sites of the The A. P. M. M. L. I. M. 15: PubMed Scopus Google Scholar) was by to the codon and into the sites of the The by Ibanez M. C. N. Ibanez C.F. Mol. Cell. Neurosci. 1998; PubMed Scopus (162) Google Scholar) was into the sites of the H. 18: PubMed Scopus Google Scholar). Cell and cells, GFRα1, were in with and were with and with the of to were in with cell by RET MEN2A C. M. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) were in with MEN2A were further with GFRα1, and stable were with and and of were in to in and for to were for the of with either GDNF or in and at for in and at were to and the as previously C. C. D. S. EMBO J. 2000; PubMed Scopus Google Scholar). of the was a were with and at 3 of in a of of and were through a and with for at They were then with of in and to The were by a of by of and in and for at in a at The was into the of the for the that of were by and and as were in and for at with were then in and to DRMs. To RET protein were or not with and in for and further with for the of for Cell and were at in in with GDNF or the 3 2 were and an was for of of in was to and were for at The was then with of with was at The is to the of were on at in and with GDNF for in the with for was to the cell and of activation of RET is controlled by the It is on the of a protein RET linked to an domain to be to in the of to RET activation and were in that can be activated with GDNF or the of and in The RET to the incompletely glycosylated protein in the form in and to the glycosylated protein at the plasma membrane I. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the form of was on the cellular the form was not and was to to an of with an of or GDNF for a activation of RET proteins, as with an specific to in to into the and then to and activated the This is to is with MEN2A RET in which the and the of RET are the additional activation of the form of RET, a in tyrosine of cellular proteins was either GDNF or stimulation lower It that the of the to the cytoplasmic of RET not RET activation and signaling a of tyrosine was were with type RET of and with GDNF these results that of RET can to its activation the for They suggested that activation of the intracellular protein not to the of tyrosine RET to an we whether activation of RET by or GDNF can affect were with either GDNF or and the was GDNF during the of of and GDNF-stimulated in an and as shown on of of a these In contrast, to that the to be These results the of specific cellular that RET activation leads to of intracellular signaling pathways. RET on AKT whether activation of RET by or GDNF can into or signaling pathways. We the of RET autophosphorylation specific to and that a of these was by GDNF or with the of in these results that or GDNF stimulation leads to activation of RET tyrosine It is of that RET in remained 2 of stimulation and that it for results were with type RET of that the is not involved in the of RET in and are binding sites for and The molecular are in by RET (3Ichihara M. Murakumo Y. Takahashi M. Cancer Lett. 2004; 204: 197-211Crossref PubMed Scopus (162) Google Scholar). that Shc and were in a GDNF or stimulation and that of those of These results that RET activation by or GDNF not influence proximal RET signaling, autophosphorylation of RET and the resulting of the molecular Shc and We the activation of downstream pathways by at ERK and AKT GDNF a of ERK and AKT Therefore, and signaling pathways can be with the of RET in with ERK to a and in marked contrast, the of AKT was and for and of AKT was on and which AKT D.P. Sci. 2004; 29: Full Text Full Text PDF PubMed Scopus Google Scholar). It be that not affect AKT activation of AKT be GDNF of RET of in the of These results that RET activation into regulated AKT activation, ERK activation. of RET to AKT GDNF has been to signal of RET M. Scott R. Ibanez C.F. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, D. A. J. M. M. U. Saarma M. Lett. 1999; PubMed Scopus Google Scholar), it was to whether signaling to AKT and ERK in We of and have been to of AKT and ERK GDNF stimulation of these we whether of were involved in the of To address this were with and to RET maturation and to the plasma membrane T. U. C. C. C. J. PubMed Scopus Google Scholar). The of the protein maturation was was not to of RET proteins for with the the of the plasma of RET the of the form of RET these stimulation of induce of the of Shc was that activated of RET can recruit molecular these activated of RET to AKT and ERK activation. These with the that additional activation of of RET not the tyrosine strongly that of RET not to the signaling pathways RET with RET associates with the raft-related and this association has been correlated with the control of downstream signaling (4Tansey M.G. Baloh R.H. Milbrandt J. Johnson Jr., E.M. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 5Paratcha G. Ledda F. Baars L. Coulpier M. Besset V. Anders J. Scott R. Ibanez C.F. Neuron. 2001; 29: 171-184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). to an into the for the regulation of AKT activation, we whether stimulation induce the association of RET with DRMs. GDNF stimulation an association of of with DRMs and receptor were used as and of proteins. Although RET with be activated by more than of was in This not to a of RET of DRMs recruitment and activation RET remained associated with DRMs for the of the stimulation in and These results suggested that RET association with DRMs is than association and that it can be by In marked to these RET association with DRMs was not upon RET proteins a MEN2A at C. M. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) were not in the These results demonstrated a in the association the of of RET with DRMs between and RET activation. RET with DRMs with of results the that regulation of but not is linked to association of This was in association of proteins with DRMs has been shown to be to the R. T. C. B. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of for with stimulation not affect RET and its protein it the association of RET with DRMs by indicates a in the association of RET with DRMs GDNF-stimulated AKT was a 2 the of the stimulation and these AKT was by of the cells, a of the on cellular membranes J. S. D. L. M.P. M. Sci. U. S. A. 2003; PubMed Scopus Google Scholar) and a of RET with DRMs activated by the AKT 2 of GDNF stimulation in the of it remained stimulation This that RET association with DRMs not with a AKT but with a AKT these a between association of RET and a specific regulation of AKT GDNF we of the MEN2A that not with DRMs MEN2A at has been shown to AKT in C. M. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). MEN2A can to GDNF in the of B. R. I. T. D. G. I. B. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar), we that stimulation of RET may to its association with DRMs and impact on AKT MEN2A RET were with GFRα1, and stable were in the of GFRα1, GDNF of MEN2A RET not induce an association of RET with DRMs In contrast, that GDNF stimulation of induces association of MEN2A RET with DRMs constitutive with in the form of the MEN2A RET plasma associates with DRMs. this association a of AKT a role for DRMs in the regulation of AKT activation. The of AKT between and in independent and to GDNF cellular AKT can a in cell D.P. Sci. 2004; 29: Full Text Full Text PDF PubMed Scopus Google Scholar), we the of the in AKT activation by the with the B. R. I. T. D. G. I. B. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). that were more to than cells, as revealed by a GDNF stimulation a in the in AKT activation is of these strongly a role for DRMs in the specific regulation of AKT In suggested that the intracellular signaling by MEN2A RET a lipid raft-related control that results in of AKT activation. MEN2A mutations of RET tyrosine kinase receptor result in constitutive activation of RET ligand-independent dimerization. Whether such mutations identical signaling to ligand-induced signaling was previously The revealed more complex of MEN2A oncogenic signaling than previously and a linked to a lipid that the temporal activation of Dimerization of RET an of AKT MEN2A signaling to unravel of RET signaling pathways is by the activation of RET mutants. MEN2A protein activation be in cells, a with the signaling pathways. we that MEN2A activation with an the of RET signaling pathways involved in of evidence were through these studies that the of a binding to the C of RET not RET activation and This RET activation model showed that proximal RET signaling, RET autophosphorylation and of RET such as and is whether the receptor is activated with the natural GDNF or the Therefore, the complex was not for RET activation. This is in with the studies of et al. A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), demonstrated a autophosphorylation of type RET and a MEN2A RET model revealed that ligand-independent activation of RET into an tuning of the the remained these that ligand-independent dimerization of RET leads to an regulation of specific signaling pathways. for Lipid in the of the of AKT the of on the cellular regulated by AKT D.P. Sci. 2004; 29: Full Text Full Text PDF PubMed Scopus Google Scholar), the molecular that the of AKT activation is of The RET used these studies is in which the autophosphorylation tyrosine is in the activation of and pathways V. Scott Ibanez C.F. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In the current view, tyrosine AKT and ERK activation through the protein Shc or and It is to that the regulation of AKT activation occurs downstream of recruitment proximal RET signaling and ERK activation are whether RET is activated in a or ligand-independent the that a lipid is involved in this regulation of Although lipid rafts remain membrane (8Edidin M. Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 257-283Crossref PubMed Scopus (1134) Google Scholar, 9Munro S. Cell. 2003; 115: 377-388Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar), that a lipid-based cholesterol-sensitive a role in the temporal regulation of AKT activation. the regulation of AKT activation by GDNF showed to in membrane Although can influence of the membrane J. S. D. L. M.P. M. Sci. U. S. A. 2003; PubMed Scopus Google Scholar), it AKT activation, a the cell showed a in the association of RET with DRMs GDNF or Although GDNF an Although the association is a to association H. H. T. J. J. Mol. Biol. 2003; PubMed Scopus Google Scholar, H. Biophys. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), it can be as a of a lipid raft-related of this is to and with specific AKT support for a with a results are in with a that of to DRMs not RET (4Tansey M.G. Baloh R.H. Milbrandt J. Johnson Jr., E.M. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). of to DRMs was with the of a of of a that not with DRMs. these AKT and ERK with This with the of AKT and on ERK we with the ligand-independent activation of signaling stimulation were not and be with the AKT activation we at The for ERK activation It of the cellular used in the as with a cell the association of RET with lipid rafts influence AKT The at rafts may of signaling that AKT the phosphoinositide lipid has been to in lipid rafts P. EMBO J. 2001; PubMed Scopus Google Scholar, K. A. A. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of by the that is to the of the signaling an association with lipid rafts the for RET to trigger the resulting in AKT activation. tyrosine or lipid the of AKT We have the of and lipid in the specific regulation of AKT but evidence for its recruitment in DRMs Lipid rafts have been as an to B. J. Cell Sci. 2003; 116: PubMed Scopus Google Scholar). Therefore, it is that RET activated by GDNF not the intracellular as MEN2A mutations activated intracellular of receptors can have in controlling the nature of the activation C. Nat. Rev. Mol. Cell Biol. 2005; PubMed Scopus Google Scholar). it is to that a a process of RET to the tuning regulation of AKT activation. these Dimerization of RET a to of MEN2A of evidence that model of dimerization of RET is to MEN2A signaling. in RET was activated by of the protein in the of and to we in by MEN2A RET activation of RET with in of In marked contrast, not activation of RET with Such a is not in the of the J. Cell Biol. PubMed Scopus Google Scholar), as of cellular stimulation to a of in this this of may not be to into the we as in of receptor tyrosine such as the factor receptor Fiore P.P. R. A. J. Cell. Full Text PDF PubMed Scopus Google L. I. Science. PubMed Scopus Google Scholar), can to activated by the controlled dimerization of RET that AKT activity C. M. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) an tuning of its activation. this activity as MEN2A were to with DRMs through an with AKT was strongly Such a is of the more to It has been that a of MEN2A RET may with DRMs M. A. S. C. Carlomagno M.S. L. V. Cell. 2004; PubMed Scopus Google Scholar). We may not have such an association of the are with a of Whether the AKT activation to of has been by the with the used or these on to cell a of this further of MEN2A to the model to MEN2A RET has been with the nature of MEN2A signaling. results have in this in the has been whether the intracellular activated RET to the oncogenic of 2A proteins. We have evidence that the RET is to the Shc but to trigger further activation of AKT or Although an of the used be these results are with a that an activated RET may not to S. A. G. C. Fusco A. T. M. M. 1998; 17: PubMed Scopus Google Scholar). support for MEN2A signaling the plasma In revealed a complex in which MEN2A signaling is not identical to ligand-induced signaling but differences in the signaling In the studies revealed that a lipid the tuning of AKT activation, a control step that bypassed by MEN2A mutants. The are to the molecular that the of AKT activation and to connection with a lipid-based cholesterol-sensitive We are to for the used in We M. for C. Ibanez for and F. for