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Insights & Perspectives Hypotheses

Activation of transmembrane cell-surface receptors via a common mechanism? The ‘‘rotation model’’

Ichiro N. Maruyama

It has long been thought that transmembrane cell-surface receptors, such as typically consist of an extracellular kinases and cytokine receptors, among others, are activated by domain (ECD) and an intracellular ligand binding through ligand-induced dimerization of the receptors. However, domain (ICD) separated by a single transmembrane domain (TMD), with there is growing evidence that prior to ligand binding, various transmembrane the exception of bacterial receptors receptors have a preformed, yet inactive, dimeric structure on the cell surface. such as the aspartate receptor (Tar) Various studies also demonstrate that during transmembrane signaling, ligand and the receptor (Tsr), which binding to the extracellular domain of receptor dimers induces a rotation of have another TMD at their amino transmembrane domains, followed by rearrangement and/or activation of termini. Ligand binding to their ECDs often regulates kinases that are either intracellular domains. The paper here describes transmembrane cell-surface integrated into the receptor ICD, or receptors that are known or proposed to exist in dimeric form prior toligand binding, physically associated with the ICD. and discusses how these preformed dimers are activated by ligand binding. Apart from receptors that initiate signaling pathways inside cells via Keywords: tyrosine , there are .cytokine; dimerization; ligand binding; preformed dimer; transmembrane receptors in bacteria, fungi, and plants signaling; that phosphorylate histidine residues upon ligand binding. Furthermore, natriuretic peptide receptors, which are receptor-type guanylyl cyclases, Introduction cell membranes to the cytoplasm, and produce cGMP upon peptide binding. include receptor tyrosine kinases There are also receptors that recruit Transmembrane, cell-surface receptors (RTKs) and cytokine receptors among adaptor/effector proteins through transmit extracellular signals across many others. Cell-surface receptors protein-protein interactions upon ligand binding. There are two major, mutually DOI 10.1002/bies.201500041 exclusive concepts to explain activation of transmembrane, cell-surface receptors. Ligand binding induces Okinawa Institute of Science and Technology GCY-14, receptor-type guanylyl cyclase-14; Graduate University, Onna, Okinawa, Japan GHR, growth hormone receptor; ICD, intracellular either (i) dimerization of receptors, or domain; IGF1R, -like growth factor-1 recep- (ii) rearrangement of constitutively pre- Corresponding author: tor; IL-6R, 6 receptor; IL-12R, inter- formed dimeric receptors. The former Ichiro N. Maruyama leukin 12 receptor; IR, ; IRR, insulin E-mail: [email protected] receptor-related receptor; JAK, ; mechanism, known as ligand-induced LepR, leptin receptor; NPRA, natriuretic peptide receptor dimerization, was first pro- Abbreviations: receptor A; p75NTR, neurotrophin receptor; PRLR, posed for the BRET, bioluminescence resonance energy trans- prolactin receptor; RTK, ; receptor (EGFR; also called ErbB1 or ; ECD, extracellular domain; EGFR, epidermal STAT, signal transducers and activators of tran- ; Eph, receptor; scription; Tar, bacterial aspartate receptor; TLR, HER1) almost three decades ago [1–3]. In EpoR, receptor; ErbB, originally toll-like receptor; TMD, transmembrane domain; this “dimerization model,” the receptor named because of the homology to the erythro- TNFR, tumor necrosis factor receptor; TpoR, is thought to exist in monomeric form blastoma viral gene product, v-erbB; FGFR, receptor; Trk, tropomyosin- fibroblast growth factor receptor; FnIII, fibronectin related kinase receptor; Tsr, bacterial serine on the cell surface prior to ligand type III; FRET, Forster€ resonance energy transfer; receptor. binding, which induces receptor

Bioessays 37: 959–967, ß 2015 The Author. BioEssays published by WILEY Periodicals, Inc. This is an www.bioessays-journal.com 959 open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. I. N. Maruyama Insights & Perspectives.....

dimerization. According to this model, A variety of receptors kinase activity and in selective trans- ICDs of dimerized receptors are brought exist in dimeric form prior autophosphorylation of tyrosine resi- into close proximity to allow receptor to ligand binding dues. Some of these sites are involved in trans-autophosphorylation, subsequent maintaining active conformations of the tyrosine kinase activation, and initia- Receptor tyrosine kinases kinases, while others become docking tion of downstream signaling path- sites for various adaptor/effector scaf- ways [4, 5]. This mechanism has been The human RTK superfamily consists of fold proteins and . All RTKs, proposed for activation of many other 58 proteins grouped into 20 sub- except for the IR family, are expressed cell-surface receptors, including recep- families [8]. RTKs are integral mem- as single protomers. IR family members, tor tyrosine kinases and cytokine recep- brane proteins with a single TMD, and comprising IR, IGF1R, and IRR, are also tors. In contrast, the insulin receptor their N-terminal ECDs are generally expressed as single subunits, but they Hypotheses (IR), insulin-like growth factor-1 composed of various structural modules undergo processing into two a and two receptor (IGF1R), and insulin receptor- with multiple, intrachain, disulfide b polypeptide chains that are assembled related receptor (IRR) have covalent, bonds, and numerous N-linked into a heterotetramer, or an (ab)2 preformed, dimeric structures linked by glycosylation sites. Their ICDs have homodimer, stabilized by disulfide disulfide bridges [6, 7]. Like- tyrosine kinase domains flanked by bonds. wise, there is growing evidence that intracellular, juxtamembrane regions, prior to ligand binding, various trans- and C-terminal tails, which differ in size membrane receptors exist in preformed, and tyrosine content among family EGFR (ErbB) family yet inactive, dimeric form on the cell members. Ligand binding to the ECDs surface (Table 1). results in elevation of their tyrosine The ErbB receptor family consists of EGFR, ErbB2 (also known as Neu/ HER2), ErbB3 (HER3), and ErbB4 (HER4), and the receptors play crucial Table 1. Transmembrane cell-surface receptors that exist or are proposed to exist roles in cell growth, differentiation, in dimeric form prior to ligand binding survival, and migration. A number of studies demonstrate that prior to ligand References Rotation angle and reference binding, ErbB receptors exist in dimeric Receptor tyrosine kinases form on the cell surface (see [9], and EGFR [10–21] [140˚: 10] ErbB2 [13, 18, 80] references therein]. Chemical cross- ErbB3 [18, 21] linking showed that >80% of EGFR ErbB4 [18] molecules were dimeric in the absence EphA1 [40, 41] of bound ligand [10]. Forster€ resonance EphA2 [38, 39, 41] [60˚: 41] energy transfer (FRET) [11–14] and fluo- EphA3 [37] rescence correlation spectroscopic anal- FGFR3 [43] IGF1R [32] yses [13, 15, 16] further demonstrated that IR [32] preformed EGFR and ErbB2 dimers are IRR [33] present at physiological expression lev- MET [42] els on surfaces of living cells. Single- TrkA [28] molecule observations using total inter- TrkB [29] nal reflection fluorescence microscopy VEGFR2 [180˚: 99] with oblique illumination also supports Cytokine receptors EpoR [50, 51] [100˚: 91, 92] the existence of receptor dimers [17]. GHR [48, 49] [40˚: 93, 94; 45˚: 95] Fluorescent protein fragment comple- IL-6R [60] mentation indicates that all the members IL-12R [58] of the ErbB family exist in dimeric LepR [55–57] NTR form [18]. This is consistent with results p75 [30] of reversible firefly luciferase fragment PRLR [52] TNFR [62] complementation analysis, showing that TpoR [100˚: 53] 100% of EGFR and ErbB3 receptor Other cell-surface receptors molecules exist as dimers [19–21], since EnvZ [69] luciferase activity did not increase after GCY-14 [65] addition of EGF to the cell culture. LINGO-1 [66] Depending on methods used, dimer- NPRA [64] [40˚: 96] Tar [67] [50˚: 74] to-monomer ratios vary from 40 to TLR9 [63] 100%. Considering the inefficiency of Tsr [68] chemical cross-linking [22] and of fluo- rescent protein folding [23–25], these Receptors in which TMDs have been proposed to rotate during signaling are ratios are likely to be underestimated. indicated with bold letters, with or without rotation angles and reference(s). However, when EGFR mutants with

960 Bioessays 37: 959–967, ß 2015 The Author. BioEssays published by WILEY Periodicals, Inc...... Insights & Perspectives I. N. Maruyama

cysteine substitutions at different loca- extracellular a-subunits that contain (signal transducers and activators of tions in the TMD were expressed in a ligand-binding domains and two trans- transcription) proteins. A series of land- murine pre-B lymphocyte line, Ba/F3, membrane b-subunits that possess mark publications gave rise to the Hypotheses disulfide cross-linking of the receptors intracellular kinase domains [32, 33]. textbook view that ligand binding was observed only in the presence of There is also evidence for the existence initiates cytokine receptor dimerization, EGF [26]. This result is inconsistent with of a disulfide-linked (ab)2 hybrid which then leads to activation of a the previous result in which similar EGFR dimeric receptor (IR::IGF1R), which is tyrosine kinase associated with the or ErbB2 constructs with a cysteine composed of an IR ab hemireceptor and receptor [46, 47]. However, at least nine substitution spontaneously formed an IGF1R ab hemireceptor [34, 35]. Eph distinct cytokine receptors have been dimers in the absence of bound EGF, RTKs mediate contact-dependent, cell- shown or proposed to exist in preformed when expressed in mouse fibroblast B82 cell communication by interacting with dimeric form (Table 1). cells or monkey fibroblast-like COS-7 surface-associated ligands (ephrin) on The growth hormone receptor (GHR) cells, respectively [10, 27]. All disulfide neighboring cells [36]. EphA3, which is is required for postnatal growth, as well cross-linkings of EGFR extracellular jux- essential for cell guidance during as for lipid and carbohydrate metabo- tamembrane regions induced autophos- embryogenesis, clusters as a result of lism. It dimerizes in the endoplasmic phorylation to various extents, EphA3-EphA3 interactions, which are reticulum before reaching the cell sur- depending upon where had independent of ligand binding [37]. face [48, 49]. Ligand-independent oligo- been replaced. These phosphorylated Similarly, EphA2 also constitutively merization of the cell-surface receptors were internalized and forms dimers without bound ligand [38, erythropoietin receptor (EpoR), which degraded in the absence of bound EGF, 39]. Consistently, EphA1 and EphA2 is crucial for production of mature red and their efficiency is likely to be cell TMDs spontaneously form homodimers blood cells, has also been observed by type-dependent. Therefore, it would be in lipid [40, 41]. The MET receptor for immunofluorescence co-patching [50]. necessary to observe the spontaneous /scatter factor, Consistently, the crystal structure of the dimerization of the cysteine-replaced which is essential during embryonic EpoR ECD is homodimeric in the absence EGFR mutants expressed in Ba/F3 by development and plays an important of bound ligand [51]. The prolactin inhibiting their endocytosis. role during metastasis and tissue receptor (PRLR) mediates effects of regeneration, has been shown to exist prolactin, which stimulates growth and as a dimer, based on photobleaching differentiation of mammary epithelium Neurotrophin receptors experiments using single-molecule and initiation and maintenance of lacta- fluorescence microscopy [42]. TMDs of tion. Co-immunoprecipitation assays There is also evidence that many non- fibroblast growth factor receptor 3 were used to confirm its ligand- ErbB family RTKs exist as dimers in the (FGFR3), which is a negative regulator independent dimerization. In this dime- absence of bound ligand. Chemical of bone growth, and which is critically rization process, the TMDs play a cross-linking and firefly luciferase com- important for skeletal development, significant role [52]. The thrombopoietin plementation analyses demonstrate interact as dimers and this interaction receptor (TpoR) regulates the prolifera- that the neurotrophin receptors, TrkA persists if their ECDs are present [43]. tion of multipotent, hematopoietic bone and TrkB, which bind nerve growth marrow stem cells, their differentiation factor and brain-derived neurotrophic into mature megakaryocytes, and pro- factor, respectively, exist in dimeric Cytokine receptors duction of platelets in response to form [28, 29]. Luciferase activity did thrombopoietin binding. Using a combi- not increase with addition of ligand to There are more than 30 Class I cytokine nation of cysteine cross-linking, the cell cultures, indicating that 100% receptors [44] and at least 12 Class II alanine-scanningmutagenesis,andcom- of these receptors have preformed cytokine receptors [45] that activate putationalsimulations,itwasshownthat dimeric structures. p75NTR is a member JAK1 (), JAK2, JAK3, or TpoR TMDs dimerize strongly in mem- of the tumor necrosis factor (TNF) TYK2 (). Class I and branes in the absence of bound receptor (TNFR) superfamily. It does Class II receptors are distinguished by ligand [53]. not have kinase activity and binds all the position of class-specific cysteine The leptin receptor (LepR) plays a neurotrophins with low affinity. It exists residues, and by the presence of a central role in control of body weight and as a disulfide-linked dimer owing to a highly conserved “WSXWS” motif in energy homeostasis. LepR shows great highly conserved cysteine in its the carboxyl terminal half of Class I similarity to the interleukin 6 (IL-6) TMD [30]. receptor ECDs. Cytokine receptors com- signaling receptor chain glycoprotein prise two receptor subunits, each of 130 (gp130), the granulocyte colony- which associates with a JAK monomer. stimulating factor receptor, and the IR and Eph families, and others Upon cytokine binding, the receptor leukemia inhibitory factor receptor, and activates the associated JAKs, which in uses JAK2 and STAT3 for its signaling IR and IGF1R, which play critical roles in turn phosphorylate tyrosine residues pathway [54]. In cell membranes, LepR metabolism and cell growth, and IRR, within the receptor ICD. The phosphory- assembles as preformed dimers or which is an extracellular alkaline pH lated tyrosine residues serve as docking oligomers, as evidenced by a high basal sensor [31], are covalent, disulfide- sites for downstream adaptor and effec- signal in the absence of leptin in analysis linked (ab)2 dimers comprising two tor proteins, which include the STAT of differently tagged LepRs by

Bioessays 37: 959–967, ß 2015 The Author. BioEssays published by WILEY Periodicals, Inc. 961 I. N. Maruyama Insights & Perspectives.....

co-immunoprecipitation, biolumines- Natriuretic peptides play key roles in “Rotation model” for cence resonance energy transfer (BRET), cardiovascular homeostasis, and their transmembrane signaling by and FRET [55–57]. IL-12 is a heterodimeric cellular effects are mediated via the Tar and EGFR cytokine composed of two disulfide- transmembrane natriuretic peptide bonded, glycoprotein subunits, and has receptor A (NPRA). NPRA, which consists Bacterial chemotaxis is a model system pleiotropic effects on NK and T cells, of an extracellular ligand-binding for signal transduction, and the chemo- which are mediated through IL-12 recep- domain, a single TMD, a kinase- receptor Tar is one of the best- tors (IL-12Rs). When IL12-Rs were homology domain, and a guanylyl characterized cell-surface receptors. A expressed in COS cells, they expressed cyclase domain, produces cGMP upon proposed “rotation model” for trans- both monomers and disulfide-linked ligandbinding. Crystallographic analysis membrane signaling by the Tar dimer dimers or oligomers on their surfaces in demonstrated that the ECD of NPRA is indicates that ligand binding to the Tar Hypotheses the absence of IL-12, among which only homodimeric in the absence of bound ECDs is likely to restrict rotation of the the IL-12R dimers/oligomers, but not the ligand [64]. Domain-swapping analysis TMDs at specific positions about their monomers, bind IL-12 [58]. Upon IL-6 and site-directed mutagenesis demon- long axes [74]. The model predicts that binding, the IL-6 receptor (IL-6R), which strated that the transmembrane guanylyl Tar molecules with and without bound is essential for regenerative and anti- cyclase GCY-14, which senses extracel- aspartate have similar structures, since bacterial effects of IL-6 [59], activates the lular alkalinity in the nematode, bound aspartate stabilizes the most gp130homodimer,leadingtoinitiationof Caenorhabditis elegans, also has a homo- stable structure of the apo-receptor. JAK/STAT signaling. Co-immunoprecipi- dimeric structure [65]. Histidine residues Indeed, crystal structural analysis dem- tation experiments with two differently in the GCY-14 ECD are essential for onstrated that the membrane proximal tagged IL-6R variants expressed in COS-7 extracellular alkalinity sensing. The region of the Tar ECD with bound ̊ cells showed that an IL-6R dimer exists in transmembrane protein LINGO-1 is a aspartate translates 1A downward or the plasma membrane in the absence of negative regulator in the nervous system, toward the cytoplasm compared to its IL-6 [60]. mainly affecting axonal regeneration, position without bound ligand [75]. A ̊ TNF is a cytokine for immune neuronal survival, oligodendrocyte dif- similar subtle (1A) movement of the responses and inflammation, and is a ferentiation, and myelination. Co- TMD was also detected by electron para- homotrimer with a molecular mass of immunoprecipitation and BRET satura- magnetic resonance spectroscopy anal- 52 kDa. TNF binds with high affinity to tion analyses have shown that LINGO-1 is ysis of spin-labeled receptors, with and two type-I transmembrane receptors: homodimeric on the cell surface [66]. It without bound aspartate [76]. These TNFR1, which is activated by both has long been known that bacterial results can be interpreted to indicate soluble TNF and transmembrane TNF, chemoreceptors, such as Tar and Tsr, that binding of aspartate further stabil- and TNFR2, which is activated mainly exist in dimeric form on the cell sur- izes the most stable structure of apo-Tar by transmembrane TNF. Upon TNF face [67, 68]. The same is true for the in the absence of bound aspartate, binding to TNFR, the adaptor molecule Escherichia coli extracellular osmolarity although the “piston model” [75, 76] TRADD binds to the death domain of the sensor EnvZ [69]. among others [77, 78] has also been receptor. TRADD acts as a platform proposed. Furthermore, the “rotation adaptor that can recruit downstream model” also predicts that TMD of attrac- proteins [61]. Chemical cross-linking of How are preformed tant-bound and repellent-bound forms  TNFR molecules expressed on the cell dimeric receptors differ rotationally by an angle of 50˚. surface demonstrated that the receptors These restricted rotations of the TMDs spontaneously form a homotrimeric activated by ligand may in turn restrict rotation of the HAMP structure prior to ligand binding [62]. binding? domains in the cytoplasm in order to regulate activity of the histidine kinase For increasing numbers of cell-surface CheA,whichphysicallyinteractswithTar Other cell-surface receptors receptor dimers, like IR and IGF1R, with help of the adaptor, CheW. This activation cannot be explained by the model is consistent with recent results Toll-like receptors (TLRs) recognize ligand-induced dimerization model. showing axial helix rotations of the Tsr structural and sequence variations Early studies demonstrated that a chi- cytoplasmic domains during transmem- between host and microbial nucleic acids meric receptor with the IR ECD and the brane signaling [79]. in immune cells. TLR9 is activated by EGFR ICD was activated by insulin, and A similar “rotation model” has also DNA that is rich in unmethylated CpG that EGF activated a chimera with the been proposed for activation of the motifs, such as microbial DNA, in the EGFR ECD and the IR ICD [70–72]. EGFR, in which EGF binding to its endosome. This results in production of Furthermore, a chimeric receptor, con- flexible ECDs induces conformational inflammatory cytokines and interferons sisting of the ligand-binding ECD of changes of the domains to form a stable that lead to adaptive immunity. FRET bacterial Tar and the IR ICD, is activated dimeric structure. Extracellular confor- analysis of TLR9 in living cells demon- by aspartate, resulting in phosphoryla- mational changes induce a rotation strated the existence of preformed TLR9 tion of the intracellular IR moiety [73]. (140˚ parallel to the plane of the homodimers. TLR9 activation is regu- These studies suggest that diverse cell- plasma membrane) of the TMDs about lated by conformational changes specif- surface receptors may be regulated their long axes, which in turn dissociate ically induced by foreign DNA [63]. through similar molecular mechanisms. the inactive, symmetric kinase dimer in

962 Bioessays 37: 959–967, ß 2015 The Author. BioEssays published by WILEY Periodicals, Inc...... Insights & Perspectives I. N. Maruyama

the cytosol, followed by rearranging of transmembrane domains for activation activation of dimeric EpoR by erythro- dimeric kinase domains to form active of the kinases. Indeed, cysteine residues poietin binding is achieved by rotation- asymmetric structures [9, 10, 18]. Con- artificially introduced into the extracel- ally reorienting the receptor TMD and Hypotheses sistently, computational analysis of the lular juxtamembrane region of IGF1R connected cytosolic domains, through conformation space of the ErbB2 TMD formeddisulfidebridgesintheabsenceof random mutagenesis of the TMD, which homodimer has supported a molecular bound IGF1, and the cross-linked recep- was followed by cysteine-scanning mechanism for rotation-coupled recep- tors were autophosphorylated as effi- mutagenesis of the receptor juxtamem- tor activation, in which the two stable ciently as in the presence of bound brane and TMDs [91]. Analysis of conformations of the TMD correspond to ligand. These results indicate that the chimeric receptors of the EpoR, in which the active and inactive states of the juxtamembraneregions (hence the trans- its ECD was replaced with a dimeric, receptor [80]. membrane domains) exist in close coiled coil, has also demonstrated three enough proximity to spontaneously form rotationally related conformations disulfide bridges in theabsence of ligand. (active, inactive, and partially active) IR family may also be activated This is consistent with the dimeric of EpoR TMD dimers [92]. When the by its TMD rotation structure of unactivated IGF1R kinase engineered EpoR fusion protein was domains, determined by crystallogra- constrained in seven possible orienta- Crystal structures of IR ECDs without phy, in which two monomers are tions, three dimeric TMD orientations ligand [81, 82] and a fragment of the IR arranged such that their ATP binding corresponding to fully active, partially ECD bound to insulin [83] have been clefts face each other. The ordered N- active, and inactive receptors were determined. In the absence of ligand, terminus of one monomer approaches identified by measuring activity of the ECDs form a symmetric, antiparallel the proximal part of the activation loop, JAK2, STAT, and MAP kinases in the dimer shaped like a folded-over “L.” the ATP binding pocket, and the catalytic cytosol. Average molecular structures The C-terminal half of the ECD consists loop of the other monomer [88]. for active and inactive orientations of three contiguous, fibronectin type III Another model recently proposed for differ by a rotation of 100˚. Ligand- (FnIII) domains, which are followed by IR activation is based on results in which induced rotations of EpoR TMDs may the TMD. Bioinformatics analysis indi- IR TMD peptides supplied extracellularly, induce flexibility of the receptor’s ICDs, cates that only a slight “rotation” of the stimulated a dose-dependent increase in and may rearrange the JAK2 kinase last two FnIII domains is required to IR tyrosine phosphorylation in living cells. dimer for its autophosphorylation. align the proposed binding sites of IR to This result was interpreted as indicating insulin [7, 84]. This subtle “rotation” of that TMD peptides specifically interact the extracellular juxtamembrane region with an inactive form of IR TMD dimers, TMD rotations during signaling and TMD during signaling is compatible resultingindissociationoftheTMDdimer by GHR, TpoR, NPRA, Eph, and with results of a small-angle X-ray to activate the receptor [89]. As discussed VEGFR scattering study of IGF1 binding to the above, the TMD peptides interact with an soluble IGF1R ECD, wherein very little inactive form of IR TMD dimers, and may Within the dimeric GHR, subunit rota- change in the radius of gyration was induce or allow a rotation of the IR TMD tions (40˚ clockwise) have been sug- observed in the ECD upon binding of about its long axis for rearrangement and gested as the activation mechanism, IGF1 [85]. activation of the IR kinase dimers. using FRET, BRET, and co- An alternative model has recently immunoprecipitation [93, 94]. Once GH been proposed based on FRET and is removed from the hormone-bound mutagenesis studies, in which the IGF1R Homodimeric EpoR TMDs receptor complex, consistently, coun-  ECD maintains an auto-inhibited state rotate during signaling ter-clockwise rotations of 45˚ of the withtheTMDsheld apart.Ligandbinding two subunits relative to each other has releases the constraint, allowing associ- Crystallographic structural analysis of been observed in atomistic molecular ation of TMDs and kinase domains for the EpoR ECD in the presence and dynamics simulation [95]. Three differ- trans-autophosphorylation [86]. Dele- absence of bound ligand suggest that ent, rotationally related conformations of tion of the extracellular N-terminal L1 the receptor may exist in dimeric form TpoR TMD dimers, possibly correspond- domain of IGF1R resulted in constitutive prior to ligand binding [51]. From a ing to specific states (active, inactive, and activity of IGF1R. This is reminiscent of subsequent fluorescent study based on partially active) of the full-length the EGFRvIII mutant, in which an dimerization-induced complementation receptor have been discovered with a extracellular, N-terminal ligand-binding of designed fragments of the murine combination of cysteine cross-linking, domain is deleted [87]. EGFRvIII exists in dihydrofolate reductase, an alanine-scanningmutagenesis,andcom- dimeric form and is constitutively active allosteric mechanism of EpoR activation putational simulations [53]. The active in the absence of bound ligand. In both was proposed in which ligand-induced interfacebetweendimerizedTMDsdiffers EGFR and IGF1R, the extracellular reorganization of the dimer brings the fromtheinactive interfacebya rotationof ligand-binding domains seem to play a intracellular domains into closer prox- 100˚. Similarly, a transmembrane rota- role in keeping the intracellular kinase imity, allowing associated JAK2s to tion of 40˚ that leads to constitutive inactive prior to ligand binding. Deletion come into contact and autophosphor- activation of NPRA has been elucidated of their ligand-binding domains may ylate [90]. However, the mechanism is by sequentially replacing nine residues induce or allow a rotation of their now explained differently in which with cysteine and by introducing one to

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five alanine residues into the receptor helices in activated constructs were conformational changes of the ECDs transmembrane a-helix [96]. rotated by 180˚ relative to the interface induce a rotation of the TMDs, which Structural analysis of EphA2 TMD of the wild-type conformation [99]. makestheICDslessstableandrearranges dimers in lipid bicelles using solution the domains. TMD rotations occur NMR found that there are two states, left- together with changes in interhelical handed, parallel-packed, and right- TMD rotations as a common crossing angles and distances, as handed, dimeric structures, suggesting mechanism underlying observed in an NMR study of the EphA2 a rotation-coupled (60˚, average) activa- transmembrane signaling by TMD [41]. In addition to TMD rotations, tion mechanism during EphA2 signal- cell-surface receptors? indeed, such interhelical crossing angles ing [41]. Indeed, site-directed and distances are also crucial for activa- mutagenesis of TMD of full-length EphA2 As described above, transmembrane tion of GHR and EGFR [100, 101]. During Hypotheses suggests that the TMD domains interact signaling by a variety of cell-surface transmembrane signaling mediated by in two different ways, corresponding to receptors may be regulated by bound cell-surface receptors, TMD rotations inactive and active receptor states, ligandthrough a common mechanism, in would be energetically favorable in respectively, as a mechanism underlying which ligand binding to receptor ECDs comparison to TMD’s lateral movement EphA2 signal transduction [97]. When induces their TMD rotation, thereby againstthelipidbilayer barrier,proposed EphA1 TMD dimers in lipid bilayers were regulating ICD activity (the “rotation by the ligand-induced dimerization analyzed with a multiscale approach, model,” Fig. 1). Conformational changes model. The “rotation model” may also combining coarse-grain and atomistic of ECD dimers induced by ligand binding explain not only outside-in but also molecular dynamics simulations, it was are likely to induce a rotation of the inside-outsignalingmechanisms.During found that the interaction of transmem- receptor TMDs, resulting in rearrange- inside-out signaling, conformational brane helices in EphA1 dimers may be ment of the ICDs. As observed in EGFR changes of receptor ICDs induced by intrinsically flexible enough to accom- [9, 10], structures of receptor ECDs and cytoplasmic factor(s) may stabilize ICD modate two states involving helix rota- ICDs are flexible and less flexible, flexibility,andmay induce TMDrotations tionsabouttheirlongaxes[98].Similarly, respectively, prior to ligand binding. opposite in direction to those of outside- NMR revealed that vascular endothelial Ligand binding is likely to stabilize the in signaling, as observed in atomistic growth factor receptor 2 (VEGFR2) TMD flexible ECDs. The resulting molecular dynamics simulation of GHR [95]. This counter rotation of TMDs may induce flexibility of ECDs, and may release ligand from the domains. To test the model, it is necessary to analyze structures of full-length receptors in the presence and absence of bound ligand, since the receptor extracellular juxtamembrane regions, TMD and ICD, seem to play crucial roles in dimer formation [10, 18]. Further- more, the dimeric receptor structures are very unstable outside of the mem- brane. Therefore, structures should be determined in the membrane, intact or artificial. Cryo-electron tomogra- phy [102] may be suited for experiments in cases in which large conformational changes, like those observed in EGFR, are expected. Various conformational structures of ECD dimers with a rela- tively stable ICD dimer may be observed prior to ligand binding. In the presence of bound ligand, in contrast, a relatively stable structure of ECD dimers with various conformational variables of ICD dimers may be observed. Figure 1. “Rotation model” of transmembrane signaling mediated by cell-surface receptors. Prior to ligand binding, receptors exist in dimeric form on the cell surface. The ICD dimer has a relatively stable structure while the ligand-binding ECD dimer has a rotationally flexible Conclusions and outlook structure. Ligand binding stabilizes the flexible ECDs and induces conformational changes of the domains. This extracellular conformational change in turn induces or allows a rotation of the TMDs, which rearranges the ICDs, making them flexible for activation and/or interaction It has traditionally been thought that with other cytoplasmic proteins. Rotation of TMDs occurs together with changes in transmembrane, cell-surface receptors interhelical crossing angles and distances. are activated by ligand-induced

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