Receptor tyrosine kinase transmembrane domains

The transmembrane (TM) domains of receptor tyrosine kinases (RTKs) play an active role in signaling. They contribute to the stability of full-length receptor dimers and to maintaining a signaling-competent dimeric receptor conformation. In an exciting new development, two structures of RTK TM domains have been solved, a break-through achievement in the field. Here we review these structures, and we discuss recent studies of RTK TM domain dimerization energetics, possible synergies between domains, and the effects of pathogenic RTK TM mutations on structure and dimerization.     

Receptor tyrosine kinase substrates: src homology domains and signal transduction.

Among the intracellular milieu of proteins are molecules with defined biochemical functions that serve as substrates for ligand-activated tyrosine kinase receptors. It seems likely that some of these substrate molecules are elements of a critical signaling pathway used by growth factors to control cell proliferation and subverted by oncogenes to deregulate this process. Although the process of cell growth and division is relatively slow compared with other hormonally regulated responses, homeostasis in a human being requires approximately 20 x 10(6) cell divisions per second for the renewal of various cell populations. This review summarizes the present understanding of tyrosine kinase substrates that seem likely to have key roles in the signal transduction pathway that regulates cell proliferation. This includes structural features of these molecules, the influence of tyrosine phosphorylation on their functions, the biological roles of these proteins, and the capacity of these substrates to associate with activated receptor tyrosine kinases.     

Rotational coupling of the transmembrane and kinase domains of the Neu receptor tyrosine kinase

Ligand binding to receptor tyrosine kinases (RTKs) regulates receptor dimerization and activation of the kinase domain. To examine the role of the transmembrane domain in regulation of RTK activation, we have exploited a simplified transmembrane motif, [VVVEVVV](n), previously shown to activate the Neu receptor. Here we demonstrate rotational linkage of the transmembrane domain with the kinase domain, as evidenced by a periodic activation of Neu as the dimerization motif is shifted across the transmembrane domain. These results indicate that activation requires a specific orientation of the kinase domains with respect to each other. Results obtained with platelet-derived growth factor receptor-beta suggest that this rotational linkage of the transmembrane domain to the kinase domain may be a general feature of RTKs. These observations suggest that activating mutations in RTK transmembrane and juxtamembrane domains will be limited to those residues that position the kinase domains in an allowed rotational conformation.     

Direct transmembrane clustering and cytoplasmic dimerization of focal adhesion kinase initiates its tyrosine phosphorylation

We investigated mechanisms for inducing focal adhesion kinase (FAK) tyrosine phosphorylation and their ability to trigger MAP kinase signaling using transmembrane chimeras that localize FAK and its mutants to the plasma membrane. We tested whether tyrosine phosphorylation was triggered by FAK transmembrane aggregation using antibodies against the chimeric extracellular domain. Experimental clustering of chimeras containing integrin beta cytoplasmic domains or FAK induced FAK tyrosine phosphorylation and trans-phosphorylation of endogenous FAK, as well as strong ERK activation. Next, we examined whether lower-order molecular proximity, namely dimerization, could regulate FAK tyrosine phosphorylation. We found that even relatively low-affinity FAK dimerization (K(d)=3.9 x 10(-5) M), in either of two different orientations, could induce FAK tyrosine phosphorylation. However, this cytoplasmic FAK dimerization could not induce MAP kinase activation or trans-phosphorylation of endogenous FAK. We conclude that dimerization of FAK is sufficient to induce its tyrosine phosphorylation, but that higher-order molecular proximity (clustering) at the cell membrane is apparently needed for additional biochemical events. This study identifies a proximity mechanism for regulating the initiation of FAK-mediated biochemical signaling.     

Role of receptor tyrosine kinase transmembrane domains in cell signaling and human pathologies

Receptor tyrosine kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane, and their transmembrane (TM) domains play an important role in the dimerization process. Here we present two models of RTK-mediated signaling, and we discuss the role of the TM domains within the framework of these two models. We summarize findings of single-amino acid mutations in RTK TM domains that induce unregulated signaling and, as a consequence, pathological phenotypes. We review the current knowledge of pathology induction mechanisms due to these mutations, focusing on the structural and thermodynamic basis of pathogenic dimer stabilization.     

A sequence motif in the transmembrane region of growth factor receptors with tyrosine kinase activity mediates dimerization

A mechanism by which ligand binding to the extracellular domain of a growth factor receptor causes activation of its cytoplasmic tyrosine kinase domain is that binding promotes receptor dimerization. Recently we proposed a model in which dimerization of the transmembrane alpha-helices in one member of this family, rat neu, is mediated by the presence of three specific residues. This paper shows that a similar sequence motif is observed in 18 of the 20 transmembrane alpha-helices of the tyrosine kinase family of growth factor receptors. The motif encompasses a five residue segment in which position 0 (P0) requires a small side chain (Gly, Ala, Ser, Thr or Pro), P3 an aliphatic side chain (Ala, Val, Leu or Ile) and P4 only the smallest side chains (Gly or Ala). In addition other features of the transmembrane sequences are reported. It is concluded that the dimerization of transmembrane alpha-helices may be a general mechanism of tyrosine kinase activation in this family of growth factor receptors.     

Receptor tyrosine kinase signaling--a proteomic perspective

The RTKs are one of the most important families mediating transmembrane signaling and they participate and are instrumental in regulating a broad range of physiological activities. Indeed, tyrosine kinases in general, and the processes that they control and/or stimulate, provide a rich source of drug targets, particularly in growth related disorders such as cancer (Zwick et al., 2002; Krause and Van Etten, 2005). However, there remain many questions regarding their activation and downstream signaling and the application of proteomic analyses promises to answer many of them. There have been relatively few detailed studies of this type to date and it will require considerably more of them to better define the pathways with respect to both the major and minor PTMs that, along with the protein-protein interactions, are the means to direct the flow of the signals generated. It will take such approaches to define the specificity that characterize the individual families, even appreciating that to some degree all are capable of activating many, if not all, of the principal pathways. It will also be necessary to understand, in the highly complex networks of intracellular phosphorylation (that contain thousands of sites of modification and clearly have not yet been fully determined in any paradigm), exactly which kinases modify which substrates, and to work out the inter-relationships with other modifications such as O-GlcNAcylation and acetylation. Only then will it be possible to determine which modifications are physiologically significant and which are simply background. Along theway, these studies should continue to provide potential drug targets and perhaps improve the current lackluster biomarker discovery track record.     

Crystal structure of a C-terminal fragment of growth arrest-specific protein Gas6. Receptor tyrosine kinase activation by laminin G-like domains

Receptor tyrosine kinases of the Axl family are activated by Gas6, the product of growth arrest-specific gene 6. Gas6-Axl signaling is implicated in cell survival, adhesion, and migration. The receptor-binding site of Gas6 is located within a C-terminal pair of laminin G-like (LG) domains that do not resemble any other receptor tyrosine kinase ligand. We report the crystal structure at 2.2-A resolution of a Gas6 fragment spanning both LG domains (Gas6-LG). The structure reveals a V-shaped arrangement of LG domains strengthened by an interdomain calcium-binding site. LG2 of Gas6-LG contains two unusual features: an alpha-helix cradled by one edge of the LG beta-sandwich and a conspicuous patch of surface-exposed hydrophobic residues. Mutagenesis of some residues in this patch reduces Gas6-LG binding to the extracellular domain of Axl as well as Axl activation in glioblastoma cells, identifying a component of the receptor-binding site of Gas6.     

A dimerization hierarchy in the transmembrane domains of the HER receptor family

Bitopic membrane proteins offer an opportunity for studying transmembrane domain interactions without the structural complexity inherent to multitopic integral membrane proteins. To date, only homomeric associations have been extensively studied quantitatively. Here we propose to assess the thermodynamics of heteromeric associations, which opens the way to investigating specificity and selectivity. A very interesting system of biological relevance with single transmembrane domains possibly involved in interactions with different partners is the EGFR receptor family. The four members, all tyrosine kinase receptors, are involved in an interaction network that potentially leads to a complete set of homo- and heterodimers, ideally suited to such a study. Furthermore, the transmembrane domains of these receptors have been previously implicated in their function in the past by mutations in the transmembrane domain leading to constitutive activation. We demonstrate, using a fluorescence-based measurement of interaction energies, a hierarchy of transmembrane domain interactions ranging from a noninteractive pair to strong dimerization. We propose a structural model based on the crystal structure of the EGFR dimer, to show how the dimeric structure favors these interactions. The correlation we observe between transmembrane domain and whole receptor interaction hierarchies opens a new perspective, suggesting a role for transmembrane receptor domains in the modulation of receptor signaling.     

Cross-talk between RON receptor tyrosine kinase and other transmembrane receptors

RON is a transmembrane receptor tyrosine kinase that mediates biological activities of Macrophage Stimulating Protein (MSP). MSP is a multifunctional factor regulating cell adhesion, motility, growth and survival. MSP binding to RON causes receptor tyrosine phosphorylation leading to up-regulation of RON catalytic activity and subsequent activation of downstream signaling molecules. Recent studies show that RON is spatially and functionally associated with other transmembrane molecules including adhesion receptors integrins and cadherins, and cytokine and growth factor receptors IL-3 betac, EPOR and MET. For example, MSP-induced cell shape change is mediated via RON-activated IL-3 betac receptor. Activation of integrins causes MSP-independent RON phosphorylation, and the integrin/RON collaboration regulates cell survival. Thus, RON can be activated without MSP by ligand stimulation of RON-associated receptors, and MSP-activated RON can cause ligand-independent activation of RON-associated receptors. As a result of the receptor cross-activation RON-specific pathways become a part of a signal transduction network of other receptors, and conversely signaling pathways activated by other receptors can be used by RON. This receptor collaboration extends the spectrum of cellular responses generated by MSP and by putative ligands of RON-associated receptors. However signaling pathways involved in the receptor cross-talk and underlying activation mechanisms remain to be investigated. The purpose of this review is to summarize data and to discuss a role of cross-talk between RON and other transmembrane receptors.     

Receptor tyrosine kinase signaling and primordial germ cell development

Primordial germ cells (PGCs) give rise to sperms and eggs. Their development is crucial to species propagation and has to be precisely controlled. Studies in several model organisms have identified many genes involved in the specification and guided migration of PGCs. However, the mechanisms governing the behaviors of these unique cells remain to be investigated. Interestingly, PGCs share certain cellular properties with metastasizing cancer cells including proliferation, invasion of other tissues, survival and migration. Recently we have shown that in Drosophila the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development. In later stages, activation of both STAT and Ras, likely by other molecules, is required continuously for PGC migration. The requirement for RTK suggests molecular conservation between flies and mice in PGC development and also suggests that germ cells and cancer cells share certain intracellular signaling strategies.     

Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer

Binding of erythropoietin to the erythropoietin receptor (EpoR) extracellular domain orients the transmembrane (TM) and cytosolic regions of the receptor dimer into an unknown activated conformation. By replacing the EpoR extracellular domain with a dimeric coiled coil, we engineered TM EpoR fusion proteins where the helical TM domains were constrained into seven possible relative orientations. We identify one dimeric TM conformation that imparts full activity to the cytosolic domain of the receptor and signals via JAK2, STAT proteins, and MAP kinase, one partially active orientation that preferentially activates MAP kinase, and one conformation corresponding to the inactive receptor. The active and inactive conformations were independently identified by computational searches for low-energy TM dimeric structures. We propose a specific EpoR-activated interface and suggest its use for structural and signaling studies.     

Spatial structure and pH-dependent conformational diversity of dimeric transmembrane domain of the receptor tyrosine kinase EphA1

Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel alpha-helical bundle, region (544-569)(2), through the N-terminal glycine zipper motif A(550)X(3)G(554)X(3)G(558). Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu(547), whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A(560)X(3)G(564). This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pK(a) value of Glu(547) and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an "extracellular pH sensor," and can have relevant physiological implications.     

Crystal structure of the agrin-responsive immunoglobulin-like domains 1 and 2 of the receptor tyrosine kinase MuSK

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed exclusively in skeletal muscle, where it is required for formation of the neuromuscular junction. MuSK is activated by agrin, a neuron-derived heparan sulfate proteoglycan. Here, we report the crystal structure of the agrin-responsive first and second immunoglobulin-like domains (Ig1 and Ig2) of the MuSK ectodomain at 2.2 A resolution. The structure reveals that MuSK Ig1 and Ig2 are Ig-like domains of the I-set subfamily, which are configured in a linear, semi-rigid arrangement. In addition to the canonical internal disulfide bridge, Ig1 contains a second, solvent-exposed disulfide bridge, which our biochemical data indicate is critical for proper folding of Ig1 and processing of MuSK. Two Ig1-2 molecules form a non-crystallographic dimer that is mediated by a unique hydrophobic patch on the surface of Ig1. Biochemical analyses of MuSK mutants introduced into MuSK(-/-) myotubes demonstrate that residues in this hydrophobic patch are critical for agrin-induced MuSK activation.     

Receptor tyrosine kinase, an autapomorphic character of metazoa: identification in marine sponges

In the present review we summarize sequence data obtained from cloning of sponge receptor tyrosine kinases [RTK]. The cDNA sequences were mainly obtained from the marine sponge Geodia cydonium. RTKs (i) with immunoglobulin [Ig]-like domains in the extracellular region, (ii) of the type of insulin-like receptors, as well as (iii) RTKs with one extracellular speract domain, have been identified. The analyses revealed that the RTK genes are constructed in blocks [domains], suggesting a blockwise evolution. The phylogenetic relationships of the sequences obtained revealed that all sponge sequences fall into one branch of the evolutionary tree, while related sequences from higher Metazoa, human, mouse and rat, including also invertebrate sequences, together form a second branch. It is concluded that the RTK molecules have evolved in sponges prior to the "Cambrian Explosion" and have contributed to the rapid appearance of the higher metazoan phyla and that sponges are, as a taxon, also monophyletic. Due to the fact that protein tyrosine kinases in general and RTKs in particular have only been identified in Metazoa, they are, as a group qualified, to be considered as an autapomorphic character of all metazoan phyla.     

Receptor protein tyrosine phosphatase sigma regulates synapse structure, function and plasticity

The mechanisms that regulate synapse formation and maintenance are incompletely understood. In particular, relatively few inhibitors of synapse formation have been identified. Receptor protein tyrosine phosphatase σ (RPTPσ), a transmembrane tyrosine phosphatase, is widely expressed by neurons in developing and mature mammalian brain, and functions as a receptor for chondroitin sulfate proteoglycans that inhibits axon regeneration following injury. In this study, we address RPTPσ function in the mature brain. We demonstrate increased axon collateral branching in the hippocampus of RPTPσ null mice during normal aging or following chemically induced seizure, indicating that RPTPσ maintains neural circuitry by inhibiting axonal branching. Previous studies demonstrated a role for pre-synaptic RPTPσ promoting synaptic differentiation during development; however, subcellular fractionation revealed enrichment of RPTPσ in post-synaptic densities. We report that neurons lacking RPTPσ have an increased density of pre-synaptic varicosities in vitro and increased dendritic spine density and length in vivo. RPTPσ knockouts exhibit an increased frequency of miniature excitatory post-synaptic currents, and greater paired-pulse facilitation, consistent with increased synapse density but reduced synaptic efficiency. Furthermore, RPTPσ nulls exhibit reduced long-term potentiation and enhanced novel object recognition memory. We conclude that RPTPσ limits synapse number and regulates synapse structure and function in the mature CNS.     

Receptor for bombesin with associated tyrosine kinase activity.

The neuropeptide bombesin is known for its potent mitogenic activity on murine 3T3 fibroblasts and other cells. Recently it has been implicated in the pathogenesis of small cell lung carcinoma, in which it acts through an autocrine loop of growth stimulation. Phosphotyrosine (P-Tyr) antibodies have been successfully used to recognize the autophosphorylated receptors for known growth factors. In Swiss 3T3 fibroblasts, phosphotyrosine antibodies identified a 115,000-Mr cell surface protein (p115) that became phosphorylated on tyrosine as a specific response to bombesin stimulation of quiescent cells. The extent of phosphorylation was dose dependent and correlated with the mitogenic effect induced by bombesin, measured by [3H]thymidine incorporation. Tyrosine phosphorylation of p115 was detectable minutes after the addition of bombesin, and its time course paralleled that described for the binding of bombesin to its receptor. Immunocomplexes of phosphorylated p115 and phosphotyrosine antibodies bound 125I-labeled [Tyr4]bombesin in a specific and saturable manner and displayed an associated tyrosine kinase activity enhanced by bombesin. Furthermore, the 125I-labeled bombesin analog gastrin-releasing peptide, bound to intact live cells, was coprecipitated with p115. These data strongly suggest that p115 participates in the structure and function of the surface receptor for bombesin, a new member of the family of growth factor receptors with associated tyrosine kinase activity.     

Myoglobin and CO: structure, energetics, and disorder

 The physiological significance of steric inhibition of CO binding to heme proteins is a fundamentally quantitative issue, since it has meaning only relative to the enhancement of O₂ binding by the protein. Previously, difficulties in reconciling structural and energetic data have hindered a quantitative assessment of the energetic cost of distorting the bound CO. Recent progress on both fronts suggests that the energetic cost of CO distortion in myoglobin is quite small. However, distortion of the surrounding protein to accommodate the heme-CO complex may contribute significantly to the free energy of CO binding. Polarized IR measurements on single crystals of MbCO not only yield precise structural information on the CO geometry, but also address the limitations of conventional structural refinement by characterizing conformational disorder in the crystalline state. Received, accepted: 23 May 1997