Parallels and Distinctions in FGFR, VEGFR, and ... - ACS Publications

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Parallels and Distinctions in FGFR, VEGFR, and EGFR Mechanisms of Transmembrane Signaling Sarvenaz Sarabipour* Institute for Computational Medicine and Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States

ABSTRACT: Receptor tyrosine kinase (RTK) signal transduction is essential in human skeletal, nervous, and vascular development, in homeostasis, and in disease. RTKs are activated by dimerization in the plasma membrane. The mechanisms of receptor dimerization and activation are multifaceted and complex, and unraveling them remains challenging. Most studies of RTKs have been devoted to crystallographic analysis of their isolated extracellular domain and biochemical analysis of the catalytic domain. However, the past few years have seen direct biophysical studies of (intact) RTK dimerization in native membranes lead to significant progress in our fundamental understanding of the mechanisms of their signal transduction across the plasma membrane. This perspective focuses on recent insights into the mechanisms of fibroblast growth factor receptor and vascular endothelial growth factor receptor transmembrane signaling, derived from studies of wild-type and mutant RTKs in a number of environments, including plasma membrane-derived vesicles. These insights reveal distinct steps in and factors of RTK signaling across the plasma membrane that can guide the drug discovery process for RTK targeting therapeutics.

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FGF, VEGF, AND ERBB RECEPTORS AS EXEMPLAR RTKS

cascades such as RAS-MAPK-ERK, JAK-STAT, and PI3KAKT pathways.2−4 FGF, VEGF, and ErbB receptors are members of the RTK family playing key roles in animal neonatal development (pattern formation during embryogenesis),5 adult physiology (bone growth, skeletogenesis, and vasculogenesis),6−8 and pathophysiology (chodrodysplasias, craniosynostosis, angiogenesis, and tumor growth in cancers)9−12 of multicellular eukaryotes. The receptors and their cognate ligands initiate signaling cascades that are responsible for critical cellular functions, including cell migration, differentiation, and proliferation. The initiated signaling pathways are critical, as a large number of gene knockouts are embryonically lethal in mice, with the animals exhibiting sever phenotypes such as growth retardation (FGFR1−/−13), abnormal lung development (FGFR2−/−14), bone overgrowth (FGFR3−/−15,16), impaired limb and organ development among other phenotypes (fgfs−/− reviewed in refs 6 and 17), impaired vascular network formation (VEGF−/− and VEGFR2−/− reviewed in ref 3), or heart, lung, and eye defects and abnormal development of the

Receptor tyrosine kinases (RTKs) make up the second most abundant superfamily of integral membrane proteins of metazoan cells. RTKs are comprised of a single-pass transmembrane domain and multiple flexible soluble domains: extracellular domain, intracellular juxtamembrane domain, tyrosine kinase domain, and C-terminal tail. The glycosylated extracellular domain is comprised of multiple subdomains, some of which are docking sites for multiple ligands (Figure 1). Alternative splicing results in numerous splice isoforms of each member of an RTK family, yielding more than 50 FGFR isoforms. The membrane-bound isoforms laterally dimerize in the plasma membrane. Dimerization stabilizes unliganded (inactive or mildly active) and ligand-bound (highly active) RTK complexes. Dimerization is necessary for closeness, contact, and correct juxtapositioning of the intracellular catalytic domains of receptor monomers to form active (transphosphorylating) signaling complexes.1 Upon phosphorylation of key catalytic domain residues in the dimer, intracellular docking proteins interact with the activated tyrosine kinase domains to initiate intracellular signaling © XXXX American Chemical Society

Received: April 30, 2017

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DOI: 10.1021/acs.biochem.7b00399 Biochemistry XXXX, XXX, XXX−XXX

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Figure 1. Mechanistic models of dimerization and activation for wild-type (A) FGFR1, FGFR2, and FGFR327 and (B) VEGFR228 compared to (C) ErbB1 (EGFR).36,37 Receptors exist as monomers in equilibrium with dimers. At low expression levels, FGFR1, FGFR2, FGFR3, VEGFR2, and EGFR predominantly exist as monomers. The number of unliganded RTK dimer species increases in a concentration-dependent manner.27,28 Upon binding of ligands to monomers or preformed dimers, the dimer undergoes a structural change that results in a change in distance between the tyrosine kinase domains responsible for catalytic activation of the receptors. This yields maximal phosphorylation of the receptors in a dimer. Ligand binding has different structural consequences for each family of RTKs. The change in distance between the C-termini of the TMDs is similar for FGFR family members upon binding of fgf1 and fgf2.27 Using FRET reporters (YFP and mCherry), a tightly packed (closed) conformation is measured upon fgf2 binding compared to fgf1 binding (open conformation) to FGFR1, FGFR2, FGFR3.27 FGFR3 JMD contacts exist and stabilize the unliganded FGFR3 dimers.76 Binding of VEGF-A121a, -165a, and -165b, VEGF-C, and VEGF-D to VEGFR2, however, results in an increase in the TMD C-terminal distance,28 demonstrating diverse mechanisms of activation for two close families of RTKs. EGFR monomers adopt a closed (tethered) autoinhibited conformation with domain IV locked by contact with domain II.37,56 Upon binding of two EGF molecules to domain I and domain III, the receptor undergoes a conformational change to yield an extended extracellular domain conformation.63 Homotypic contacts in domain IV comprise a dimerization interface and stabilize the unliganded dimer.37 Homotypic contacts in domains II and IV stabilize the ligand (EGF or TGFα)-bound EGFR dimers.37,77 G-XXX-G-like motifs at the N- and C-termini of the EGFR TMD are predicted by MD simulations to contribute to unliganded and ligand-bound dimer conformations.36,37 EGFR JM domains stabilize the dimer by forming an antiparallel helical dimer that facilitates asymmetric kinase contacts and phosphorylation.78 Human EGFR is an ECD dimer that is capable of binding to a single EGF;79 however, the transmembrane conformation of the EGFR dimer bound to only one EGF has not yet been elucidated. This figure excludes the formation of higher-order oligomers and negative cooperativity in ligand binding,80 also postulated to be important for EGFR function, and focuses on dimerization thermodynamics and transmembrane domain conformations.

nervous system (ErbB2−/− or ErbB4−/− reviewed in ref 18). The receptors are well-known prognostic indicators of a wide spectrum of human disorders as their point mutations and overexpression are linked to skeletal syndromes, vascular anomalies, and many forms of cancer.19−23 As a result, a number of currently approved drugs targeting membrane proteins target the ligands, extracellular domain, or tyrosine kinase domain of FGFRs in gastric, endometrial, and lung cancers, ErbBs in lung and breast cancers, VEGFRs in solid tumors, angiogenic sprouting of blood vessel, and lymphangio-

genesis with high binding efficiency but poor patient outcomes.24,25 Despite extensive efforts in basic RTK research and in therapeutic design, however, the modes of RTK action and mechanisms of interactions remain poorly understood. This review covers recent insights into structural and thermodynamic determinants of interactions of two RTK families, FGFRs and VEGFRs, that are important and whose mutations are pathologic. It further examines the roles that various receptor domains and pathogenic mutations play in normal and B


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ROLE OF RTK DOMAINS IN DIMERIZATION AND ACTIVATION Dual Role of the Extracellular Domain (ECD). The ECD Is an Autoinhibitory Domain in the Absence of Ligands. Given that FGFRs, ErbBs, and VEGFRs exist as low-activity preformed dimers in the plasma membrane, the extracellular domain of these receptors tightly regulates receptor activity by inhibiting maximal dimerization and activation in the absence of ligands. Recent work using quantitative imaging fluorescence resonance energy transfer (QI-FRET) microscopy, comparing dimer stabilities of truncated TM and EC+TM domains of FGFR1, FGFR2, FGFR3, and VEGFR2, demonstrates that the free energy of receptor dimerization (ΔG) in mammalian plasma membranes is less favorable when the EC domain is present compared to the values with only the TM domain.27,28 Hence, the ECD acts to destabilize unliganded dimers as ΔGECTM − ΔGTM = ΔΔGEC ∼ 1.5−2 kcal mol−1, where ΔΔGEC is the contribution of the ECD to dimerization.27,28,53 In the absence of ligands, FGFR D1 plays a small role in autoinhibition of receptor activation and signaling.54 The presence of VEGFR2 D4−D7 reduces the VEGF binding affinity by 10-fold.55 EGFR monomers assume an autoinhibited conformation in the absence of EGF with domain IV locked in contact with domain II (Figure 1).56 The EGFR EC domain is postulated to inhibit receptor dimerization in the absence of ligands, as deletion of ECD results in ligand-independent receptor activation.36,37,57 In the absence of ligands, all the studied EC domains inhibit receptor dimerization; they destabilize RTK dimers by shifting the dimerization equilibrium toward dissociation to monomers.27,28 Thus, the extracellular domain exerts an inhibitory role in interactions of members of three RTK families in the absence of ligands. Ligand Binding to ECD Stabilizes RTK Dimers. The EC domains of FGFR and VEGFR families are comprised of multiple immunoglobulin (Ig)-like subdomains (D1, D2, etc.) and linker regions connecting the subdomains. D2, D3, and the D2−D3 linker constitute the fgf1−18 binding sites of FGFRs.58,59 VEGFs and homologues (VEGF-A, -B, -C, -D, -E, -F, PlGF, and splice isoforms) bind to D2, D3, and the D2− D3 linker of VEGFRs.60 The loss of the D2−D3 linker of VEGFR2 results in a 1000-fold decrease in VEGF binding affinity.61 There are at least seven different EGFR binding ligands: EGF, TGFα, AR, BTC, EPN, EPR, and HB-EGF.62 EGF and TGF-α binding engages EGFR (L domains) D1 and D3.63,64 Ligand binding to RTKs results in an increased level of receptor activation as it increases the thermodynamic stability of RTK dimers (lowers receptor−receptor dissociation rates).27,28,41,65 For all families of receptors discussed here, ligand binding stabilizes a thermodynamic state distinct from the preformed dimer state (the dimer conformationally selects this state).27,28,37 In the absence of ligands, this highly active state is autoinhibited because of (1) steric hindrance between receptor ECDs blocking ligand-independent receptor dimerization, inhibiting the full-length receptor from achieving the optimal active conformation. Steric hindrance between ECDs prevents the ECD and TMD from forming correct contacts and hence from exploring key active conformations, and (2) intramolecular interactions within extracellular and/or juxtamembrane domains inhibiting correct conformational rearrangements of preformed dimers (in the absence of ligands).66 Ligand-induced conformational changes are an additional mechanism of receptor dimerization and dimer stabilization

dysregulated transmembrane signal transduction, emphasizing results of RTK studies in native membrane environments.

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STRUCTURAL AND THERMODYNAMIC DETERMINANTS OF RTK INTERACTIONS IN THE ABSENCE AND PRESENCE OF LIGANDS

Homodimerization Often Leads to Activation. While RTKs have long been studied in the presence of ligands, ligand binding is not always essential for receptor dimerization and activation as mutant FGFRs and VEGFRs form stable dimers and undergo phosphorylation in the absence of ligands.26−30 Furthermore, at high local receptor concentrations, even unliganded (preformed) wild-type ErbB1, FGFR1−3, and VEGFR2 exist in a monomer−dimer equilibrium in the plasma membrane.27,28,31 Consistent with this, with the exception of IGF-1R that forms constitutive but inactive dimers,32 basal activation has been observed for wild-type MET, TIE2, TRK-A, FGFR1, FGFR2, FGFR3, VEGFR2, and VEGFR3, in the absence of ligands.27,28,32−35 Similarly, ErbB family members, in particular the best studied member of this family (EGFR), dimerize and are phosphorylated in the absence of ligands.31,36−38 Crystal structures of monomeric and the ligand-bound EGFR extracellular domain reveal a large conformational change upon binding of EGF.39 Hence, the preformed EGFR dimers may also serve as intermediate active complexes, priming the receptor for ligand binding by easing the conformational change upon binding of EGF. VEGFR3 monomers form low-activity dimers in the absence of VEGFs, as basal activation is measured on the surface of endothelial cells.40 Unliganded dimerization is not merely receptor aggregation at high concentrations as members of the RTK superfamily (even different members of the same RTK family) form unliganded dimers with distinct propensities (free energies of dimerization or ΔG).27,28 Low-activity preformed dimers prime the receptors, ensuring a swift response to ligand binding.41 Heterodimerization as a Complementary Interaction Mechanism. Heterodimerization in the absence and presence of ligands is a common feature of multiple RTK families and serves to diversify RTK-mediated signaling pathways.42,43 ErbB family members form heterodimers.42 ErbB2/ErbB3 heterodimerization is a means of activating ErbB2 (as this receptor does not bind to any known ligands)44 and ErbB family activation diversity.42 The NMR structure of the ErbB1/ErbB2 transmembrane dimer is distinct from that of the ErbB1 transmembrane homodimer,36,45 and the ErbB homo- and heterodimer signaling output varies with different partners from the same family.42 FGFR1/FGFR2, FGFR2/FGFR3, and FGFR1/FGFR3 heterodimers form in the absence and presence of fgfs at strengths (dimerization propensities) similar to those of homodimers. 46,47 VEGFR1/VEGFR2 and VEGFR2/VEGFR3 heterodimers also form in the absence of VEGFs and in response to VEGF-A and VEGF-C.48−50 RTKs are overexpressed in many cancers, including breast cancer, gastric cancer, multiple myeloma, and glioblastomas;11,23,51,52 hence, the preformed RTK homo- and heterodimers may be the key mediators of a number of downstream effects of such RTK overexpression. C


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in non-native environments. An emergent technique, quantitative imaging FRET (QI-FRET),84,86 has revealed that VEGFR2 and FGFR1, FGFR2, FGFR3 TM domains strongly selfassociate in the nativelike lipid bilayers (PMVs).27,28,88 TM domains drive ligand-independent interactions of full-length RTKs27,28 previously believed to be monomeric on the basis of studies of their isolated ECD in solution.40 Upon binding of ligands, TM−TM interactions further critically impact thermodynamic and conformational states for the full-length receptors in the plasma membrane (Figure 1).27,28 Specific TMD interactions have been studied by highresolution solution NMR spectroscopy of the isolated TMDs, i.e., NMR studies of human ErbB4,89 FGFR3,90 ErbB1,91 and ErbB2.92 Only two transmembrane domain structures have been determined for FGFRs and VEGFRs to date, the wildtype FGFR3 TMD in DPC/SDS micelles90 and the wild-type VEGFR2 TMD also in DPC micelles.93 Although these structures provide valuable atomistic insights into a particular TM domain dimer conformation, solution NMR structures of isolated transmembrane domains in micelles may not capture the complete mechanism of RTK TM domain-mediated interactions in the context of the full-length receptor. Any Dimer Will Not Do: TMDs Undergo Distinct Conformational Switches in Response to Ligand Binding. Studies of wild-type and mutant RTKs in their nativelike lipid environments have provided key insights into the role of the TM domain in full-length receptor dimer thermodynamics (dimerization propensity) and structure. FGFR1, FGFR2, FGFR3, and VEGFR2 exist in monomer−dimer equilibria, and ligands mediate receptor conformational selectivity regulating their transmembrane signaling (Figure 1).27,28 This additional mechanism of regulation, TMD conformational switch, is captured by experimental studies using the QI-FRET technique.27,28,86 These studies used truncated TMD, large truncated (ECD+TMD), and full-length VEGFR2 and FGFR3 in the presence of ligands. The results showed that upon dimerization, specific interfaces are formed in the extracellular, transmembrane, and intracellular domains.27,28 These interfaces mediate distinct signaling modes. The findings demonstrate that it is not necessarily correct to infer structural and functional properties of the full-length receptor from isolated structures. QI-FRET studies of wild-type and mutant FGFR3 and VEGFR2 EC+TM constructs further showed that TMD αhelices in the dimer utilize specific amino acid contacts that define distinct specific conformations (dimeric states) dictated upstream by different ligands (Figure 1). Multiple distinct interfaces have been observed for FGFR3 and VEGFR2 upon binding of their ligands, fgf1 and fgf2, VEGF-A isoforms, and VEGF-C, -D, -E,27,28 or in the presence of pathogenic mutations (examples of studied mutations are FGFR3 A391E, VEGFR2 C482R, FGFR2 C342R, and FGFR3 C228R).27,28,95 These specific TM−TM contacts are critical for the correct conformation of the receptors to yield maximal catalytic activation.27,28,96 NMR studies reveal atomistic details of possible TM−TM contacts typically only available experimentally by a few techniques.97 Using the FGFR3 wild-type TMD NMR structure, an extended heptad interface, involving L777, G380, and A391 among other residues,90 was identified to be involved in the fgf2-mediated interactions (Figure 2).27 In plasma membrane-derived vesicles, a number of FGFR3 mutations, A391E mapping to TMD and C228R mapping to ECD Ig-like D2, emulate this FGFR3 TMD dimer

and are a common feature of RTK dimers that significantly contributes to the multifold increase in the level of receptor phosphorylation (Figure 1).27,28,32 The ECD Subdomains Engage in Homotypic Contacts To Stabilize RTK Dimers. Despite the inhibitory role of the EC domain as a whole, ECD−ECD subdomain contacts remain key determinants of receptor activation. The ECD does not merely serve as a ligand docking site. Homotypic contacts of various subdomains are necessary for formation of correct and potent (catalytically active/signaling competent) dimer conformations (catalytic domain orientations) in the absence and presence of ligands.28,67,68 As noted above, ligand binding stabilizes RTK−RTK contacts within a dimer and critically contributes to an increase in the level of receptor transphosphorylation.27,28,32 Homotypic contacts form between membrane proximal regions (D4, D5, and D7) of VEGFR2 monomers in a dimer.67 D4, D5, and D7 contacts are similarly formed in VEGFR3 dimers as observed in the ECD crystal structure.40,69 Furthermore, D5 and/or D7 mutations impair receptor activation in the absence and presence of VEGF-C.40 Homotypic interface regions, the EF loop of D4, the L/ IxRϕxxxD/ExG motif in D5 (key residues being Thr446 and Lys516), and D7 (key residues being Arg726 and Asp731), are conserved in all VEGFRs.69 Complete loss of, or key point mutations in, VEGFR-2 D7 results in receptor oligomerization and loss of efficient receptor dimerization and activation in the absence and presence of VEGF despite VEGF binding to the receptor.28,69,70 Loss of D4 results in inhibition of ligand binding to the D2−D3 binding pocket on VEGFR228 and inhibition of receptor activation in the absence and presence of VEGFs.68 Hence, D4−D7 contacts are not dispensable for correct dimerization and for maximal activation of VEGFRs. Both D4 and the D4−D5 linker of VEGFR1 enhance ligandmediated receptor dimerization, supporting a role in VEGFR1 dimerization.71 The full-length VEGFR1 ECD crystal structure further highlights distinct homotypic contacts in D4, D5, and D7.72 As noted earlier, ligand binding to FGFR1, FGFR2, FGFR3, and VEGFR2 ECD initiates conformational changes that propagate to the transmembrane and intracellular domains (Figure 1).27,28 In the VEGFR1, VEGFR2, VEGFR3 dimers, ECD contacts are a conserved feature and play key roles in unliganded and ligand-bound receptor dimerization, activation, and positioning of kinase domains. Hence, homotypic contacts in receptor dimers can play multiple roles in altering receptor dimerization and in inhibition of receptor activation: disruption of such contacts prevents proper preformed dimer formation (as shown by VEGFR2 D7 mutations28,70) and/or prevents efficient ligand binding (as shown by VEGFR2 D4 mutation28). As highlighted by the crystal structures and predicted by MD simulations, homotypic contacts in subdomains I, II, and IV are involved in ErbB ligand-independent and ligand-dependent dimerization.37,73 Similarly, a homotypic contact site in the extracellular D2−D3 linker of FGFRs is involved in receptor dimerization.74,75 Homotypic contacts between RTK extracellular subdomains are critical for correct formation of unliganded and ligand-bound dimers and their optimal activation.28,67,68,74,75 The Transmembrane Domain Stabilizes RTK Dimers. A number of biophysical techniques, TOXCAT studies in bacterial membranes,81,82 FRET studies in simplified synthetic lipid bilayers,27,83−86 and NMR studies in micelles and bicelles,87 have demonstrated that RTK TM domains, including FGFRs, VEGFRs, and ErbBs, have a propensity to self-associate D


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contact as well as a second conformation with a C-terminal contact.36,37 The EGFR TMD sequence has N-terminal and Cterminal G-xxx-G motifs (Figure 2). MD simulations predict EGFR TMD interactions at the N-terminus to be more stable than C-terminal interactions based on longer association times. This could be associated with the higher number of G-xxx-G motifs at the TMD N-terminus than at the C-terminus. MD simulations further postulate that the ECD of EGFR prevents close N-terminal contacts between TMDs in the unliganded full-length EGFR dimer.37 The ligand induces a conformational change in EGFR that results in an increased level of receptor activation. This conformational change is predicted to result in an increase in the intermolecular distance at TMD C-termini in the EGFR dimers.37 Molecular dynamics simulations of HER2 (ErbB2), another receptor of the ErbB family, also predict dimer association at the C-terminus where a G-xxx-G motif is present. Furthermore, a solution NMR structure of HER2 TMD highlights dimer contacts at the N-terminus where a Sxxx-G motif is present.99 Small-xxx-Small or G-xxx-G motifs are present in ErbB1, ErbB2, and ErbB4 and, as NMR structures show, are involved in the TM−TM helix packing interface (Figure 2).89,91 Mutating the extended, experimentally verified, FGFR3 G-xxx-G motif or the experimentally identified FGFR3 NMR interface90 results in a distinctly diminished level of receptor activation in the presence of fgf1 and fgf2.27 Mutating the unliganded (NMR interface93) or proposed ligand-bound VEGFR TMD interface also affects receptor phosphorylation.28 Mutating all possible N-terminal G-xxx-G motifs of the EGFR TMD (T648I/G649I/G652I/A653I) significantly inhibits receptor activation36 (Figure 2), while disrupting only two small amino acids of the Small-xxx-Small motif on both N- and C-termini yields an ∼50% reduction in the level of EGFinduced (ligand-bound) EGFR phosphorylation.36 A number of distinct integral membrane protein TMD conformations are stabilized by the long recognized Small-xxx-Small motif of integral transmembrane proteins.100−102 Binding of different ligands can stabilize distinct active conformations as directly demonstrated for the fgf1-bound and fgf2-bound FGFR1, FGFR2 and FGFR3 homodimer conformations at saturating fgf concentrations.27 Binding of different EGF family ligands to EGFR may also induce distinct receptor conformations.103 Binding of saturating concentrations of VEGF-A121a, VEGFA165a, VEGF-A165b, VEGF-C, VEGF-D, or VEGF-E to CHO cell PMVs, only over expressing VEGFR2, however induces a single VEGFR2 conformation.28 The Intracellular Domain (ICD) Further Stabilizes RTK Dimers. The ICDs of a number of RTKs, FGFR2, FGFR3, and VEGFR2, stabilize the full-length unliganded receptor dimer conformation in the absence of ligands.27,28 ICD contacts are required to further facilitate intermolecular phosphorylation of the tyrosine kinase domains. In the absence of ligands, the Cterminal (most intracellular) tail of VEGFR2 negatively regulates receptor activity by sterically blocking the tyrosine kinase activation loop.104 The juxtamembrane domains (JMDs) of both EGFR and FGFR3 form contacts that further stabilize the receptor dimer.27,78 JMD stabilizes the FGFR3 contacts in the absence of ligands.76 Studies of human ErbB1 and ErbB4 show that JMD regulates ErbB1 receptor autoinhibition in the absence of ligands.78 Mutagenesis studies have demonstrated that the ErbB1 JMD plays a key role in receptor activation in the presence of ligands.78,105 Pathogenic Modes of RTK Dimerization and Activation. Increased Levels of Receptor and Ligand Expression.

Figure 2. FGFR3, VEGFR2, and ErbB1 solution NMR structures in a micellar environment show key contacts in the dimer. Complementing this insight, mutagenesis experiments have examined these interfaces in native cellular lipid bilayers. One or more of the highlighted residues are involved in the full-length (intact) receptor TMD dimer interface. (A) Key interface residues are highlighted on the NMR FGFR3 heptad interface (PDB entry 2LZL)90 engaged during binding of fgf2 to FGFR3.27 (B) The extended Small-xxx-Small motif (where Small is any residue with small side chains, i.e., glycine, alanine, or serine) interface identified by mutagenesis (structure not determined) is involved in fgf1 binding27 and highlighted here on the only available structure.90 (C) Key interface residues are highlighted on the NMR structure of the unliganded wild-type VEGFR2 TMD (PDB entry 2M59).93 (D) Key residues that likely are at the interface of the ligandbound VEGFR2 TMD dimer28 (structure not determined) are highlighted here on the only available structure.93 (E) N-Terminally engaged, computationally stable NMR structure of wild-type human EGFR. The Small-xxx-Small motif (where Small is glycine, alanine, or serine) is identified by mutating resides at the interface of the wildtype EGFR structure (PDB entry 2M20).36,37 Full-length EGFR activation is not significantly affected by G649I/A653I mutagenesis. Mutating two additional residues (T648I/G649I/G652I/A653I), however, abolishes a larger interface, dramatically reducing receptor phosphorylation levels.36 (F) A second NMR structure (PDB entry 2M0B) for the wild-type EGFR TM domain reveals an interface involving another Small-xxx-Small motif at the receptor TMD Cterminus. This TMD dimer conformation is predicted by MD simulations to be the unliganded, low-activity full-length EGFR dimer conformation.94 The amino acid numbering in all cases includes the signal peptide residues.

conformation.27,95 A second conformation uses one or more of the extended N-terminal G-xxx-G motifs, where G is a residue with a small side chain, i.e., glycine, alanine, or serine, common to integral membrane proteins (Figure 2).27 This interface is responsible for the fgf1-mediated dimer structure (Figure 1).27 EGFR also dimerizes in the absence of ligands.31,41 The EGFR monomers adopt a tethered conformation characterized by a wide separation of domains I and III and the tether interaction between domain IV and the dimerization arm of domain II.98 The EGFR dimer conformation in the absence of ligands, however, remains elusive. Ligand binding to the EGF receptor results in an extracellular rearrangement (ECD conformational change)36 (Figure 1). Upon binding of EGF, the EGFR dimer adopts an extended extracellular conformation.37 Molecular dynamics simulations of the EGFR TMD predict that the TMD dimer also adopts two different conformations, an N-terminal E


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Figure 3. QI-FRET experiments86 in nativelike88 plasma membrane-derived vesicles have directly demonstrated that an increase in the dimer fraction and structural changes can both regulate RTK catalytic domain transphosphorylation. (A) Increase in the dimer fraction. In the absence of ligands, the sequence-specific dimerization propensity and an increase in total (YFP-tagged and mCherry-tagged) receptor concentrations drive dimer formation. The dimeric fraction increases by 80% when full-length FGFR2 (filled black squares) concentration increases by 1000-fold.27 The full-length FGFR3 (filled light green squares) dimeric fraction, however, increases by 50% over the same concentration range (10−10000 receptors μm−2).27 At low concentrations, these dimers are active at low levels.27 Ligand binding further stabilizes the receptor dimers, and at saturating ligand concentrations, shown for binding of fgf1 to EC+TM FGFR3 (solid purple circles) here, all receptors are dimeric independent of receptor concentration (constitutive dimerization).27 Pathogenic mutations increase the receptor dimeric fraction by increasing the receptor dimerization propensity in a sequence-specific manner. Different pathogenic mutations can increase the receptor dimerization propensity by different degrees; C228R (filled red diamonds), associated with colorectal cancer, increases the level of FGFR3 dimerization more than A391E does (empty dark green circles), associated as germline mutation with Crouzon syndrome and as somatic mutation with bladder cancer.76,95 The C342R mutation results in constitutive dimerization of FGFR2 (full-length receptor data as empty blue diamonds, EC+TM data as filled blue circles).95 (B) Structural changes. Ligand binding or mutations can induce a conformational change in receptor dimers (distinct from the wild-type dimer conformation) that maximizes receptor activation. The QI-FRET method allows measurements of this change as the interfluorophore distance of a FRET pair of YFP and mCherry.84,86 This distance determines the measured intrinsic FRET (Ĕ or I-FRET, which is the FRET efficiency of a dimer) via the equation Ĕ = 1/[1 + (d/R0)6], where R0 is the Förster radius. A number of the studied FGFR2, FGFR3, and VEGFR2 mutations mimic the most active ligandbound conformation. The distributions of I-FRET values are plotted for constitutive dimers of FGFR2+fgf1, FGFR2+fgf2, and FGFR2 C342R. The similarity of the conformations of the mutant receptor dimers and the fgf2-bound wild-type dimers and their distinction from fgf1-bound WT FGFR2 dimers are shown by the overlap of C342R and WT+fgf2 histograms of measured intrinsic FRET where at excess fgf1 or fgf2 concentrations all WT FGFR2 molecules are constitutively dimerized.27,95

(2) An increase in the level of ligand expression and ultimately excessive ligand availability (receptor saturation) results in dramatically higher levels of stable dimers (constitutive dimerization) (Figure 3A). An increase or decrease in ligand expression levels results in altered receptor homo- and heterodimer formation in the plasma membrane, and hence altered cellular phosphorylation levels of the receptors and altered intracellular signaling and trafficking. (3) The ligandbound dimers possess a different conformation that is characterized by distinct intrinsic FRET values (interfluorophore distances) in QI-FRET experiments.84,86 This conformation is optimal for juxtapositioning of the tyrosine kinase domains for efficient receptor activation (Figure 3B). Receptor or ligand expression levels are elevated in many cancers and associated with their poor prognosis;106−109 hence, overexpressed unliganded and ligand-bound wild-type RTK dimers

RTK dimerization and activity are regulated by their distinct sequence-specific receptor dimerization propensities and dimer conformations in the absence and presence of ligands (as experimentally shown for FGFRs and VEGFR227,28). Additional contributors to dysregulation of wild-type and mutant RTK dimerization in the plasma membrane are altered receptor monomer and/or ligand levels as (1) an increase in the level of wild-type FGFR, VEGFR, and EGFR monomer expression results in formation of higher numbers of receptor dimers (by the law of mass action) in the absence and presence of ligands (Figure 3A).27,28 A 1000-fold increase in the level of wild-type receptor expression results in 50% and 80% increases in wildtype FGFR3 and FGFR2 dimeric fractions, respectively, but in the absence of ligands, dimerization reaches 100% only at extremely high receptor concentrations (Figure 3A). Despite this, the wild-type dimers are stable in a suboptimal conformation, resulting in low levels of receptor activation. F


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Figure 4. Pathogenic mutations can mimic ligand-bound wild-type receptor conformations. QI-FRET was used as a structural monitoring technique to show that: (A) The A391E mutation in FGFR3 renders the receptor dimer as a tightly packed conformation distinct from the wild-type receptor dimer conformation. Wild-type FGFR3 adapts this conformation only when bound to fgf2. The A391E FGFR3 dimer, however, adapts this closed conformation in the absence and presence of both fgf1 and fgf2.27 (B) The C482R mutation mapped to VEGFR2 stirs the receptor to mimic the most active VEGFR2 conformation (the wild-type VEGF-bound conformation) in the absence and presence of VEGFs.28

proportional to dimeric fraction and conformational changes in a dimer; hence, an increased level of dimerization of the mutant receptors can have significant effects on receptor phosphorylation and downstream signaling in particular at low receptor concentrations [highest (dimeric fraction of MUT)/ (dimeric fraction of WT) ratio]. (2) Mutant RTKs can adopt the conformation with the highest activity (most potent ligandbound wild-type structure) without the need for ligands. Thermodynamic stability (which regulates monomer−dimer concentrations) and conformational changes (which regulate correct tyrosine kinase juxtapositioning within a dimer/ complex geometry) (Figure 3B) both contribute to mutant RTK activation and downstream signaling. Point mutations such as FGFR1 C178S and FGFR3 G380R, mapping to the extracellular or transmembrane domain of the receptor, increase the ligand-independent receptor homo- or heterodimerization propensity (dimer stability) and so alter dimer transphosphorylation in the absence of ligands.26,27,46 Extracellular and transmembrane domain mutations can instead or additionally induce a conformational change in the receptor dimer, altering transphosphorylation in a dimer [A391E in the FGFR3 TMD,27 C228R in FGFR3 D2,95 and C342R in FGFR2 D395 (Figure 4)].

embedded in the cellular plasma membrane are postulated to play important roles in pathological conditions. Mutations Dysregulate RTK Interactions. FGFR and ErbB family mutations are recognized point mutations of multiple disorders.110,111 A majority of FGFR1, FGFR2 and FGFR3 mutations occur in the ligand binding pocket (D2, D3, and D2−D3 linker), the transmembrane domain, and the catalytic tyrosine kinase domain. Mutations may affect the unliganded and/or ligand-bound RTK dimeric fraction and/or conformation. The relative increase in the dimeric fraction of mutant receptors depends on the concentration of monomers as well and most prominently affects RTK dimerization and activation at low receptor concentrations (as shown for FGFR3 A391E and C228R in Figure 3).27,28 (1) Most of the studied extracellular and transmembrane domain pathogenic mutations increase unliganded receptor dimer stability (i.e., dimerization propensity).27,28,95 As shown in Figure 3A, which consists of dimerization curves for the FGFR3 wild type, A391E, and C228R, the (dimeric fraction of MUT)/(dimeric fraction of WT) ratio at a single concentration is highest in the lowreceptor concentration range [e.g., (dimeric fraction of MUT)/ (dimeric fraction of WT) at 100 receptors μm−2 compared to 100000 receptors μm−2]. Receptor activation is directly G


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Biochemistry

Figure 5. Mechanisms of normal and pathological RTK dimerization and activation. (A) RTK overexpression in cancers can lead to an increase in the level of unliganded dimer formation (examples are FGFR1, -2, and -3 and VEGFR227,28). (B) RTK family members heterodimerize in the absence of ligands (FGFR1, FGFR2, and FGFR3 heterodimization shown here;46 VEGFR1, -2, and -3 and ErbB1, -2, and -3 also form intrafamilial heterodimers in the absence and presence of ligands42,48). (C) Ligand binding to RTK monomers or preformed dimers further stabilizes the receptor dimer. Additionally, ligand binding to the receptor results in a conformational change leading to an increased level of receptor transphosphorylation in a dimer. The degree of conformational change and activation can depend on the type of ligand (for example, binding of fgf1 and fgf2 to FGFR1, FGFR2, and FGFR327,28). The degree of the conformational change, the added ligand-induced stability, and the presence of coreceptors and/or cofactors such as heparin sulfate proteoglycans130 regulate the RTK phosphorylation threshold. (D) Missense mutations in the transmembrane or extracellular domain of RTKs in most cases result in increased dimerization propensity and activation (an example is unliganded FGFR3 G380R26). (E) Missense mutations in the transmembrane or extracellular domain of RTKs in the absence of ligands in many cases result in higher dimer stabilities and the optimal conformation of the receptor dimer (examples are FGFR3 A391E27 and Y373C117). (F) Missense mutations in various extracellular subdomains of RTKs can result in constitutive dimerization and activation and a structural change in the receptor dimer that mimics the most active receptor dimer conformation (for example, FGFR3 C228R,95 FGFR2 C342R,95 and VEGFR2 C482R28).

receptor.28 R250Q affects binding of fgf to FGFR1.112 Point mutations at a site other than the ligand binding pocket in the extracellular domain of the receptor may result in impaired receptor homo- and/or heterodimerization by disruption of homotypic contacts between receptors in a dimer.28,40,68,70 Pathogenic Transmembrane and Extracellular Domain Mutations That Mimic Known Ligand-Mediated Mechanisms of Action. The Ala391Glu mutation mapped to FGFR3 and the Cys482Arg mutation mapped to VEGFR2 affect both the receptor dimer stability and conformation in the absence and presence of ligands. The A391E mutation increases the FGFR3 dimer stability by 1.8 kcal mol−1 in the plasma membrane.76 It further induces a conformational change in the dimer as measured by a 7 Å shift in distance between the fluorescent reporters fused to the TMD C-termini in the dimer.76 Glutamate side chains and other highly polar residues commonly cause noncovalent association of transmembrane helices through hydrogen bonding and salt bridge formation.123

Altered Mutant Receptor Expression. Point mutations of RTKs can decrease or increase receptor expression levels. FGFR1 Y99C, Y228D, and I239T mutants impair the tertiary folding of the FGFR1 extracellular domain, resulting in incomplete glycosylation and a reduced level of cell surface expression, and ER retention of misfolded mutant FGFRs.112 Mutations at the Ligand Binding Pocket. Mutations may also affect ligand binding to the receptor only by enhancing the ligand binding affinity. Examples are Pro250Arg in FGFR3 IIIc and Pro252Arg in FGFR1 IIIc, which enhance the binding affinity of fgf for FGFRs,113 and Pro253Arg and Asp321Ala associated with Pfeiffer syndrome and Ser252Trp associated with Apert syndrome, which enhance the binding of FGFR2 IIIc to multiple fgfs.113,114 Mutations at the ligand binding sites may also lead to enhanced ligand binding or an alteration of the ligand specificity or complete abolition of ligand binding (the latter postulated for mapping of C342R to D3 of FGFR295). D4 → D5 mutation in VEGFR2 abolishes ligand binding to the H


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Biochemistry

Table 1. Pathogenic Mutations Mapping to RTK Extracellular or Transmembrane Domains Often Perturb the Thermodynamics (dimerization propensity) and Structure (conformation) of RTK Dimers26−28,95,117 perturbation receptor tyrosine kinase

pathogenic mutation

thermodynamic (dimer stability)

structural (dimer conformation)

associated disorder

FGFR1 FGFR2

Cys178Ser95 Cys342Arg95

√ √

√ √

FGFR3

Cys228Arg95 Arg248Cys117 Ser249Cys117 Tyr373Cys117 Gly380Arg26 Ala391Glu76

√ √ × × √ √

√ √ √ √ × √

VEGFR2

Cys482Arg28

√

√

Kallman syndrome115 Pfeiffer syndrome (Pf) Jackson-Weiss syndrome (JW) Crouzon syndrome (Crz)116 colorectal cancer111 type 1 thanatophoric dysplasia (TD1)118 type 1 thanatophoric dysplasia (TD1)118 type 1 thanatophoric dysplasia (TD1)118 achondroplasia (ACH)119 Crouzon syndrome (Crz)120 bladder cancer121 infantile hemangioma122

FGFR1 (C178S)-mediated inter-FGFR1 disulfide bond, however, stabilizes an unliganded FGFR1 dimer conformation closely resembling the unliganded wild-type FGFR1 dimer structure. In the case of VEGFR2, however, there are no disulfide bonds known to be mediated by pathogenic cysteine mutations.28 However, HER2 (human ErbB2) ECD mutations C311R and C334S stabilize HER2 dimers by formation of intermolecular disulfide bonds.127 While the FGFR2 C342Y mutation increases the level of FGFR2 activation96 and ErbB2 C334S is active,127 ErbB2 C311R inhibits receptor activation.127 These findings highlight the complexity and diversity of mechanisms by which RTK mutations affect receptor dimerization and activation. An increase in the dimer fraction by disulfide bond formation is often coupled with a receptor dimer conformation (Table 1) that may be active or inactive, yielding distinct outcomes for phosphorylation of the intracellular domain. Cysteine mutations do not always result in the formation of constitutive RTK dimers (C178S in D2 of FGFR1 is one studied example), and the extent (strength) of disulfide bridge formation is different among RTK families and even for receptors from the same family.95 The ICD homotypic contacts result in added stability for the receptor dimer stabilized by disulfide bonds and stabilize the full-length FGFR dimer (as shown for FGFR3 C228R) (Figure 3A).95 When new disulfide bonds make FGFR mutant dimers more stable than the wildtype dimers (FGFR2 C278F and FGFR2 C342R/S/Y/W/F), the dimers become hyperactive (Figure 5E,F).96,128 Hence, formation of extracellular disulfide bonds by pathogenic point mutations directly impacts pathogenesis by regulating dimerization, activation, and downstream signaling of RTKs.129 Juxtamembrane Domain Mutations. Point mutations in the (intracellular) juxtamembrane domain of receptors may directly contribute to pathogenesis by stabilizing or destabilizing signaling competent receptor dimers. As a result, these mutations may increase or decrease the level of receptor activation by driving the catalytic activity or causing a loss of catalytic activity. R470L in FGFR1, A441T in FGFR3, and V674I and E709K/E709G in EGFR are associated with a number of human disorders and may fall into this mechanism. Tyrosine Kinase Domain Mutations Alter Receptor Activation. Increased catalytic activity is a hallmark of most mutant RTKs. Point mutations in the tyrosine kinase domain or intracellular tail may result in enhanced or maximally enhanced (constitutive) RTK activation. Examples are K650E mapping to the FGFR3 TKD that is associated with thanatophoric dysplasia

The side chain of Glu664 in the Neu (rat ErbB2) receptor TM domain is protonated and participates in hydrogen bonding.124 Hydrogen bonds are proposed to be important mediators of TMD dimer stability. Hydrogen bonds are postulated to form in the membrane and contribute to TM−TM stabilization in the case of A391E125 as modeling by CHI126 predicts formation of two intermolecular hydrogen bonds by the A391E mutation stabilizing the FGFR3 TMD dimer.83 The H-bond formed between the glutamic acid at residue 391 and side chains of backbone amino acids stabilizes the FGFR3 dimer in the absence of fgfs regardless of the type of bound fgf (Figure 4).27 Pathogenic mutations FGFR3 A391E and VEGFR2 C342R yield the highest phosphorylation level of FGFR3 and VEGFR2, respectively, by altering the receptor dimer so that it resembles the most active ligand-bound wild-type receptor conformation (Figure 4). Disulfide Bond Formation by Mutant RTKs. Pathogenic FGFR germline cysteine mutations are linked to skeletal dysplasias (thanatophoric dysplasia and achondroplasia), craniosynostosis syndromes (Crouzon, Pfeiffer, Jackson− Weiss, and other disorders of short bones of the skull, hand, and foot), and Kallmann syndrome. Somatic cysteine mutations linked to cancers [bladder (FGFR3), colorectal (FGFR2 and FGFR3), and breast (FGFR1 and FGFR2)] are found as germline mutations in individuals with skeletal disorders of dysplasias, chondroplasias, and craniosynostosis syndromes. Inter-receptor disulfide bonds form when cysteine mutations are substituted (added or removed) at key sites on the receptor ECD or TMD. Disulfide bond formation by mutant FGFR3 (C228R, R248C, S249C, Y373C), FGFR2 (C342R), or FGFR1 (C178S) results in increased dimer stability and a conformational change and has distinct effects; not every disulfide bond contributes to RTK dimer stability to the same degree.95,117 Loss of one of the six IgD cysteines from the FGFR ECD yields inter-FGFR disulfide bonds forming between remaining (unpaired) cysteines at the expense of the intradomain disulfide bond in that IgD. Cysteine mutations (in particular cysteine loss mutations) in D2 and D3 of FGFRs are associated with perturbed dimer structure, additional dimer stability, and possibly a perturbed/distorted ligand binding pocket that results in disrupted ligand binding and ligand-independent overactivation of the FGFR dimer (Figure 5E,F). Thus, FGFR3 (C228R, R248C, S249C, Y373C) and FGFR2 (C342R) mutations stabilized an unliganded FGFR dimer conformation distinct from the wild-type FGFR dimer conformation. The I


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Biochemistry II131 and L858R mapping to ErbB1, identified in patients with lung cancer.29 These mutation may be involved in stabilizing the active conformation(s) of the activation loop.132,133 FGFR1 K618N, A671P, and Q680X mutations of the intracellular domain impair tyrosine kinase activity.112 Structural and Thermodynamic Coupling between RTK Domains across the Plasma Membrane and Implications for Development of RTK Targeting Agents. RTK kinase domains are valuable drug targets in multiple diseases.24 RTKs are conveniently druggable, and as the past few decades have shown, TKIs are excellent kinase inhibitors.134 Inhibitors of kinases, however, may activate or inhibit other downstream pathways via nonspecific activation of a number of kinases. Thus, the early logic of targeting the ATP binding pocket of RTKs may not be the best strategy for fully and specifically inhibiting VEGFR or FGFR signaling. Small molecules often can bind and interact with multiple downstream protein kinases135 and are less specific than antibodies and small proteins that can target various subdomains of the receptor extracellular domain. We do not know in which ways drugs that are identified by high-throughput screens and characterized for their function work on intact receptor complexes in the native plasma membrane and how these agents affect transmembrane action. A clear understanding of the agents that interfere with RTKs thermodynamically and conformationally will pave the way for a better understanding of RTK families and better drug development programs with more exclusive high-throughput screens and more specific approaches. These could be further explored using computational methods such as long time-scale molecular dynamics to improve our understanding of the mode of action of the drug on receptors.

modes to induce elevated activation and signaling. Both elevated wild-type receptor levels and the existence of mutant receptors alter normal signaling by means of altered dimerization and phosphorylation threshold. RTK interactions are regulated by ligand binding specificity, key domain interfaces, relative domain contributions, and the role of pathogenic mutations in the context of the full-length receptors. Drug discovery efforts so far have mostly relied on highthroughput screening for function using compound libraries. This process has a low efficiency rate, and many of the identified molecules may have poor druglike properties, low affinity, and/or low efficacy. Moreover, high-throughput functional screening of these agents cannot distinguish between receptors that have a high dimerization propensity and receptors that undergo a conformational change. A better understanding of RTK modes of action in the absence and presence of ligands and mutations is required. This would be an understanding of (1) how ligand binding alters the dimerization propensity, conformation, and function of a receptor dimer complex and (2) what is the the nature of the extracellular− intracellular coupling via the transmembrane domain via which ligands and drugs act. Knowledge of molecular determinants, thermodynamics, and conformational dynamics that contribute to signaling is key for more informed prospective drug design. This could facilitate drug design by guiding optimization of lead candidates. We need to understand mechanisms using methods beyond binding and functional assays to screen for thermodynamic and conformational modulators. Fluorescence microscopy methods such as QI-FRET and computational methods such as MD simulations, which allow studies of nearly fulllength receptors, hold promise. These methods can use spatial structures obtained by NMR and X-ray crystallography to estimate dynamic parameters of interaction to report conformational changes in the nativelike lipid membranes.27,28,86,95 The two methods can reveal differences in conformation and thermodynamics between wild-type and mutant RTKs27,95,117 and the interplay between RTKs and lipids,136,137 specifically how distinct lipid species aberrantly activate different RTKs or inhibit particular RTK dimeric states. 37,138 These are challenging to unravel using NMR and X-ray crystallographic techniques, including cysteine mutations that are difficult to probe using crystallography and challenging to probe in nonreduced environments where cysteine-mediated disulfide bonds remain intact. Computational methods such as molecular docking and molecular dynamics can investigate novel compound scaffolds and potential receptor binding pockets. It is also important to determine how binding of one agent would change the affinity of the ligands for the ligand binding site. It is also not clear if these agents target preformed dimers or ligand-bound dimers, thereby altering RTK thermodynamics. Clinical translation remains a challenge, with very few nontraditional drugs being approved to date. Mechanistic insights from normal regulation and pathogenic dysregulation of RTK dimerization thermodynamics and conformational changes can lead the way to the design of more effective therapeutic interventions.

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CONCLUDING REMARKS Recent developments have revealed that VEGFRs, ErbBs, and FGFRs dimerize in the absence of ligands and are active to a small extent at low (physiological) concentrations. The TMD plays a key role in RTK activation by forming distinct contacts that propagate the ECD conformational changes to the ICD. Additional contacts in the ECD subdomains and the ICD significantly contribute to stabilization of both unliganded (lowactivity) and ligand-bound (high-activity) dimer conformations. Furthermore, there is thermodynamic and conformational synergy between RTK TM and IC domains as well as EC and TM domains; they do not act independently of one another. Dimerization is mediated and stabilized by contacts in the ECD, TMD, and ICD regulating distinct self-assembly and structural propensities. Distinct conformational changes in the ECD are coupled to juxtapositioning of the ICD for activation. Specific ECD conformational changes are coupled to activating conformational changes in the ICD. The existence of less active RTK dimers suggests a tight conformational coupling between the extra- and intracellular domains to prevent kinase activity in the absence of ligands. Dimerization in the absence and presence of ligands brings the tyrosine kinase domains close to each other but is not adequate for maximal cross-phosphorylation of TK domains. Thus, recent direct experimental evidence further affirms that RTK dimerization is necessary but not sufficient for full receptor activation (efficient phosphorylation and hence signaling). Distinct FGFR and VEGFR dimer conformations correspond to distinct activation states (in the absence of ligand or in the presence of different ligands). Mutations in FGFR2, FGFR3, and VEGFR2 mimic these

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AUTHOR INFORMATION

Corresponding Author

*E-mal: [email protected]. ORCID

Sarvenaz Sarabipour: 0000-0001-5097-5509 J


Perspective

Biochemistry Notes

mutations associated with craniosynostosis and skeletal dysplasia syndromes. Oncogene 26, 7158−7162. (13) Deng, C. X., Wynshawboris, A., Shen, M. M., Daugherty, C., Ornitz, D. M., and Leder, P. (1994) MURINE FGFR-1 IS REQUIRED FOR EARLY POSTIMPLANTATION GROWTH AND AXIAL ORGANIZATION. Genes Dev. 8, 3045−3057. (14) Peters, K., Werner, S., Liao, X., Wert, S., Whitsett, J., and Williams, L. (1994) Targeted expression of a dominant-negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung. EMBO J. 13, 3296−3301. (15) Deng, C. X., WynshawBoris, A., Zhou, F., Kuo, A., and Leder, P. (1996) Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell 84, 911−921. (16) Wen, X., Li, X. G., Tang, Y. B., Tang, J. Z., Zhou, S., Xie, Y. L., Guo, J. Y., Yang, J., Du, X. L., Su, N., and Chen, L. (2016) Chondrocyte FGFR3 Regulates Bone Mass by Inhibiting Osteogenesis. J. Biol. Chem. 291, 24912−24921. (17) Itoh, N., and Ornitz, D. M. (2011) Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease. J. Biochem. 149, 121−130. (18) Sibilia, M., Kroismayr, R., Lichtenberger, B. M., Natarajan, A., Hecking, M., and Holcmann, M. (2007) The epidermal growth factor receptor: from development to tumorigenesis. Differentiation 75, 770− 787. (19) Brooks, A. N., Kilgour, E., and Smith, P. D. (2012) Molecular Pathways: Fibroblast Growth Factor Signaling: A New Therapeutic Opportunity in Cancer. Clin. Cancer Res. 18, 1855−1862. (20) Roskoski, R. (2014) ErbB/HER protein-tyrosine kinases: Structures and small molecule inhibitors. Pharmacol. Res. 87, 42−59. (21) Yan, J. D., Liu, Y. R., Zhang, Z. Y., Liu, G. Y., Xu, J. H., Liu, L. Y., and Hu, Y. M. (2015) Expression and prognostic significance of VEGFR-2 in breast cancer. Pathol., Res. Pract. 211, 539−543. (22) Blume-Jensen, P., and Hunter, T. (2001) Oncogenic kinase signalling. Nature 411, 355−365. (23) Barker, F. G., Simmons, M. L., Chang, S. M., Prados, M. D., Larson, D. A., Sneed, P. K., Wara, W. M., Berger, M. S., Chen, P. C., Israel, M. A., and Aldape, K. D. (2001) EGFR overexpression and radiation response in glioblastoma multiforme. Int. J. Radiat. Oncol., Biol., Phys. 51, 410−418. (24) Zwick, E., Bange, J., and Ullrich, A. (2002) Receptor tyrosine kinases as targets for anticancer drugs. Trends Mol. Med. 8, 17−23. (25) Katoh, M., and Nakagama, H. (2014) FGF Receptors: Cancer Biology and Therapeutics. Med. Res. Rev. 34, 280−300. (26) Sarabipour, S., and Hristova, K. (2016) Effect of the achondroplasia mutation on FGFR3 dimerization and FGFR3 structural response to fgf1 and fgf2: A quantitative FRET study in osmotically derived plasma membrane vesicles. Biochim. Biophys. Acta, Biomembr. 1858, 1436−1442. (27) Sarabipour, S., and Hristova, K. (2016) Mechanism of FGF receptor dimerization and activation. Nat. Commun. 7, 10262. (28) Sarabipour, S., Ballmer-Hofer, K., and Hristova, K. (2016) VEGFR-2 conformational switch in response to ligand binding. eLife 5, e13876. (29) Wang, Z. H., Longo, P. A., Tarrant, M. K., Kim, K., Head, S., Leahy, D. J., and Cole, P. A. (2011) Mechanistic insights into the activation of oncogenic forms of EGF receptor. Nat. Struct. Mol. Biol. 18, 1388−U1105. (30) Chen, F., Sarabipour, S., and Hristova, K. (2013) Multiple Consequences of a Single Amino Acid Pathogenic RTK Mutation: The A391E Mutation in FGFR3. PLoS One 8, e56521. (31) Nagy, P., Claus, J., Jovin, T. M., and Arndt-Jovin, D. J. (2010) Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis. Proc. Natl. Acad. Sci. U. S. A. 107, 16524−16529. (32) Kavran, J. M., McCabe, J. M., Byrne, P. O., Connacher, M. K., Wang, Z. H., Ramek, A., Sarabipour, S., Shan, Y. B., Shaw, D. E., Hristova, K., Cole, P. A., and Leahy, D. J. (2014) How IGF-1 Activates its Receptor. eLife 3, 54.

The author declares no competing financial interest.

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ACKNOWLEDGMENTS I thank Drs. Michael Edidin, Daniel Leahy, and Patrick Byrne for many inspiring discussions and for reading the manuscript prior to publication.

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ABBREVIATIONS RTK, receptor tyrosine kinase; FGFR, fibroblast growth factor receptor; ErbB, erythroblastosis oncogene B; VEGFR, vascular endothelial growth factor; IR, insulin receptor; IGF-1R, insulinlike growth factor receptor; TMD, transmembrane domain; ECD, extracellular domain; ICD, intracellular domain; JMD, juxtamembrane domain; YFP, yellow fluorescent protein; mCherry, monomeric cherry; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor 2; EGF, epidermal growth factor; TGFα, transforming growth factor α; AR, amphiregulin; BTC, betacellulin; EPN, epigen; EPR, epiregulin; HF-EGF, heparin binding EGF-like growth factor; ER, endoplasmic reticulum; TKI, tyrosine kinase inhibitor; MD, molecular dynamics; WT, wild type; MUT, mutant; CS, craniosynostosis syndromes; ACH, achondroplasia; TD, thanatophoric dysplasia; Crz, Crouzon syndrome; CHO, Chinese hamster ovary; PMVs, plasma membrane-derived vesicles; NMR, nuclear magnetic resonance imaging; FRET, fluorescence resonance energy transfer; EM, electron microscopy; PDB, Protein Data Bank.

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