Definition of an inhibitory juxtamembrane WW-like domain in the platelet-derived growth factor beta receptor

A variety of tumors contain activating mutations in the cytoplasmic juxtamembrane domain of the type III family of receptor-tyrosine kinases, and some constructed mutations in this domain induce ligand-independent receptor activation. To explore the role of this domain in regulation of receptor activity, we subjected the juxtamembrane domain of the murine platelet-derived growth factor (PDGF) beta receptor to alanine-scanning mutagenesis. The mutant receptors were expressed in Ba/F3 cells and tested for constitutive tyrosine phosphorylation, association with phosphatidylinositol 3'-kinase, and their ability to induce cell survival and proliferation in the absence of interleukin-3. The mutant receptors accumulated to similar levels and appeared to undergo a normal PDGF-induced increase in tyrosine phosphorylation. Alanine substitutions at numerous positions located throughout the juxtamembrane domain caused constitutive receptor activation, as did an alanine insertion in the membrane-proximal segment of the juxtamembrane domain and a six-amino acid deletion in the center of the domain. It is possible to model the PDGF receptor juxtamembrane domain as a short alpha-helix followed by a three-stranded beta-sheet very similar to the known structures of WW domains. Strikingly, the activating mutations clustered in the central portions of the first and second beta strands and along one face of the beta-sheet, whereas the loops connecting the strands were largely devoid of mutationally sensitive positions. These findings provide strong support for the model that the activating mutations in the juxtamembrane region stimulate receptor activity by disrupting an inhibitory WW-like domain.     

Tyrosine 569 in the c-Fms juxtamembrane domain is essential for kinase activity and macrophage colony-stimulating factor-dependent internalization.

The receptor (Fms) for macrophage colony-stimulating factor (M-CSF) is a member of the tyrosine kinase class of growth factor receptors. It maintains survival, stimulates growth, and drives differentiation of the macrophage lineage of hematopoietic cells. Fms accumulates on the cell surface and becomes activated for signal transduction after M-CSF binding and is then internalized via endocytosis for eventual degradation in lysosomes. We have investigated the mechanism of endocytosis as part of the overall signaling process of this receptor and have identified an amino acid segment near the cytoplasmic juxtamembrane region surrounding tyrosine 569 that is important for internalization. Mutation of tyrosine 569 to alanine (Y569A) eliminates ligand-induced rapid endocytosis of receptor molecules. The mutant Fms Y569A also lacks tyrosine kinase activity; however, tyrosine kinase activity is not essential for endocytosis because the kinase inactive receptor Fms K614A does undergo ligand-induced endocytosis, albeit at a reduced rate. Mutation of tyrosine 569 to phenylalanine had no effect on the M-CSF-induced endocytosis of Fms, and a four-amino-acid sequence containing Y-569 could support endocytosis when transferred into the cytoplasmic juxtamembrane region of a glycophorin A construct. These results indicate that tyrosine 569 within the juxtamembrane region of Fms is part of a signal recognition sequence for endocytosis that does not require tyrosine phosphorylation at this site and that this domain also influences the kinase activity of the receptor. These results are consistent with a ligand-dependent step in recognition of the potential cryptic internalization signal.     

Strong oligomerization behavior of PDGFβ ?receptor transmembrane domain and its regulation by the juxtamembrane regions

The platelet-derived growth factor β-receptor (PDGFβR) represents an important subclass of receptor tyrosine kinase (RTK) thought to be activated by ligand-induced dimerization. Interestingly, the receptor is also activated by the bovine papillomavirus E5 oncoprotein, an interaction involving the transmembrane domains of both proteins and resulting in constitutive downstream signalling. This unique mode of activation along with emerging data for other RTKs raises important questions about the role of the PDGFβR transmembrane domain in signalling. To address this, we have investigated the murine PDGFβR transmembrane and juxtamembrane domains. We show for the first time the strong oligomerization behavior of PDGFβR transmembrane domain, forming dimers and trimers in natural membranes and detergents; and that these self-interactions are mediated by a leucine-zipper-like motif. The juxtamembrane regions are found to regulate these helix-helix interactions and select specifically for dimer formation. These data provide evidence that PDGFβR is able to form ligand-independent dimers, supporting similar observations in a number of other RTK's. A point mutant in the PDGFβR juxtamembrane domain previously shown to cause receptor activation was studied and yielded no change in oligomerization or folding, suggesting (in-line with observations of the c-Kit receptor) that it may moderate interactions with other regions of PDGFβR.     

Receptor heterodimerization: essential mechanism for platelet-derived growth factor-induced epidermal growth factor receptor transactivation

Previous studies showed that the epidermal growth factor receptor (EGFR) can be transactivated by platelet-derived growth factor (PDGF) stimulation and that EGFR transactivation is required for PDGF-stimulated cell migration. To investigate the mechanism for cross talk between the PDGF beta receptor (PDGFbetaR) and the EGFR, we stimulated rat aortic vascular smooth muscle cells (VSMC) with 20 ng of PDGF/ml. Transactivation of the EGFR, defined by receptor tyrosine phosphorylation, occurred with the same time course as PDGFbetaR activation. Basal formation of PDGFbetaR-EGFR heterodimers was shown by coimmunoprecipitation studies, and interestingly, disruption of this receptor heterodimer abolished EGFR transactivation. Breakdown of the heterodimer was observed when VSMC were pretreated with antioxidants or with a Src family kinase inhibitor. Disruption of heterodimers decreased ERK1 and ERK2 activation by PDGF. Although PDGF-induced PDGFbetaR activation was abolished after pretreatment with 1 microM AG1295 (a specific PDGF receptor kinase inhibitor), EGFR transactivation was still observed, indicating that PDGFbetaR kinase activity is not required. In conclusion, our data demonstrate that the PDGFbetaR and the EGFR form PDGFbetaR-EGFR heterodimers basally, and we suggest that heterodimers represent a novel signaling complex which plays an important role in PDGF signal transduction.     

Intramolecular regulatory switch in ZAP-70: analogy with receptor tyrosine kinases

ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.     

Autoinhibition of the kit receptor tyrosine kinase by the cytosolic juxtamembrane region

Genetic studies have implicated the cytosolic juxtamembrane region of the Kit receptor tyrosine kinase as an autoinhibitory regulatory domain. Mutations in the juxtamembrane domain are associated with cancers, such as gastrointestinal stromal tumors and mastocytosis, and result in constitutive activation of Kit. Here we elucidate the biochemical mechanism of this regulation. A synthetic peptide encompassing the juxtamembrane region demonstrates cooperative thermal denaturation, suggesting that it folds as an autonomous domain. The juxtamembrane peptide directly interacted with the N-terminal ATP-binding lobe of the kinase domain. A mutation in the juxtamembrane region corresponding to an oncogenic form of Kit or a tyrosine-phosphorylated form of the juxtamembrane peptide disrupted the stability of this domain and its interaction with the N-terminal kinase lobe. Kinetic analysis of the Kit kinase harboring oncogenic mutations in the juxtamembrane region displayed faster activation times than the wild-type kinase. Addition of exogenous wild-type juxtamembrane peptide to active forms of Kit inhibited its kinase activity in trans, whereas the mutant peptide and a phosphorylated form of the wild-type peptide were less effective inhibitors. Lastly, expression of the Kit juxtamembrane peptide in cells which harbor an oncogenic form of Kit inhibited cell growth in a Kit-specific manner. Together, these results show the Kit kinase is autoinhibited through an intramolecular interaction with the juxtamembrane domain, and tyrosine phosphorylation and oncogenic mutations relieved the regulatory function of the juxtamembrane domain.     

Activation of receptor protein-tyrosine kinases from the cytoplasmic compartment

It is widely accepted that receptor protein-tyrosine kinases (RTKs) are activated upon dimerization by binding to their extracellular ligands. However, EGF receptor (EGFR) dimerization per se does not require ligand binding. Instead, its cytoplasmic kinase domains have to form characteristic head-to-tail asymmetric dimers to become active, where one 'activator' domain activates the other 'receiver' domain. The non-catalytic, cytoplasmic regions of RTKs, namely the juxtamembrane and carboxy terminal portions, also regulate kinase activity. For instance, the juxtamembrane region of the RTK MuSK inhibits the kinase domain probably together with a cellular factor(s). These findings suggest that RTKs could be activated by cytoplasmic proteins. Indeed, Dok-7 and cytohesin have recently been identified as such activators of MuSK and EGFR, respectively. Given that failure of Dok-7 signaling causes myasthenia, and inhibition of cytohesin signaling reduces the proliferation of EGFR-dependent cancer cells, cytoplasmic activators of RTKs may provide new therapeutic targets.     

Multiple transmembrane amino acid requirements suggest a highly specific interaction between the bovine papillomavirus E5 oncoprotein and the platelet-derived growth factor beta receptor

The bovine papillomavirus E5 protein activates the cellular platelet-derived growth factor beta receptor (PDGFbetaR) tyrosine kinase in a ligand-independent manner. Evidence suggests that the small transmembrane E5 protein homodimerizes and physically interacts with the transmembrane domain of the PDGFbetaR, thereby inducing constitutive dimerization and activation of this receptor. Amino acids in the receptor previously found to be required for the PDGFbetaR-E5 interaction are a transmembrane Thr513 and a juxtamembrane Lys499. Here, we sought to determine if these are the only two receptor amino acids required for an interaction with the E5 protein. Substitution of large portions of the PDGFbetaR transmembrane domain indicated that additional amino acids in both the amino and carboxyl halves of the receptor transmembrane domain are required for a productive interaction with the E5 protein. Indeed, individual amino acid substitutions in the receptor transmembrane domain identified roles for the extracellular proximal transmembrane residues in the interaction. These data suggest that multiple amino acids within the transmembrane domain of the PDGFbetaR are required for a stable interaction with the E5 protein. These may be involved in direct protein-protein contacts or may support the proper transmembrane alpha-helical conformation for optimal positioning of the primary amino acid requirements.     

The insulin receptor juxtamembrane region contains two independent tyrosine/beta-turn internalization signals

We have investigated the role of tyrosine residues in the insulin receptor cytoplasmic juxtamembrane region (Tyr953 and Tyr960) during endocytosis. Analysis of the secondary structure of the juxtamembrane region by the Chou-Fasman algorithms predicts that both the sequences GPLY953 and NPEY960 form tyrosine-containing beta-turns. Similarly, analysis of model peptides by 1-D and 2-D NMR show that these sequences form beta-turns in solution, whereas replacement of the tyrosine residues with alanine destabilizes the beta-turn. CHO cell lines were prepared expressing mutant receptors in which each tyrosine was mutated to phenylalanine or alanine, and an additional mutant contained alanine at both positions. These mutations had no effect on insulin binding or receptor autophosphorylation. Replacements with phenylalanine had no effect on the rate of [125I]insulin endocytosis, whereas single substitutions with alanine reduced [125I]insulin endocytosis by 40-50%. Replacement of both tyrosines with alanine reduced internalization by 70%. These data suggest that the insulin receptor contains two tyrosine/beta-turns which contribute independently and additively to insulin-stimulated endocytosis.     

Structural basis for autoinhibition of the Ephb2 receptor tyrosine kinase by the unphosphorylated juxtamembrane region

The Eph receptor tyrosine kinase family is regulated by autophosphorylation within the juxtamembrane region and the kinase activation segment. We have solved the X-ray crystal structure to 1.9 A resolution of an autoinhibited, unphosphorylated form of EphB2 comprised of the juxtamembrane region and the kinase domain. The structure, supported by mutagenesis data, reveals that the juxtamembrane segment adopts a helical conformation that distorts the small lobe of the kinase domain, and blocks the activation segment from attaining an activated conformation. Phosphorylation of conserved juxtamembrane tyrosines would relieve this autoinhibition by disturbing the association of the juxtamembrane segment with the kinase domain, while liberating phosphotyrosine sites for binding SH2 domains of target proteins. We propose that the autoinhibitory mechanism employed by EphB2 is a more general device through which receptor tyrosine kinases are controlled.     

Association of the signaling adaptor FRS2 with fibroblast growth factor receptor 1 (Fgfr1) is mediated by alternative splicing of the juxtamembrane domain.

Fibroblast growth factor receptors (FGFRs) are a family of transmembrane tyrosine kinases involved in signaling via interactions with the family of fibroblast growth factors. Alternative splicing of the juxtamembrane region of FGFR1-3 leads to the inclusion or exclusion of two amino acids, valine and threonine, the VT site. The presence or absence of VT (VT+ or VT-, respectively) affects the signaling potential of the receptor. The VT+ receptor isoform is required for Erk2 phosphorylation, a component of the mitogen-activated protein kinase signaling pathway. FRS2 is an adaptor protein that links FGFRs to the mitogen-activated protein kinase signaling pathway. FRS2 interacts with a region of the juxtamembrane domain of FGFR1 that includes the alternatively spliced VT site. We investigated the interaction of FRS2 with murine Fgfr1 juxtamembrane domain. We showed the alternatively spliced VT motif, at the juxtamembrane domain of Fgfr1 is required for FRS2 interaction with Fgfr1. Activation of signaling pathways from FRS2 is likely to be regulated by controlling the Fgfr1/FRS2 interaction through alternative splicing of the VT motif of Fgfr1.     

Tyrosine mutations within the alpha platelet-derived growth factor receptor kinase insert domain abrogate receptor-associated phosphatidylinositol-3 kinase activity without affecting mitogenic or chemotactic signal transduction.

A phosphatidylinositol-3 (PI-3) kinase activity of unknown biological function associates with tyrosine kinase-containing proteins, including a number of growth factor receptors after ligand stimulation. In the beta platelet-derived growth factor (beta PDGF) receptor, phosphorylation of a specific tyrosine residue within the kinase insert domain was required for its interaction with this enzyme. We show that substitutions of phenylalanine for tyrosine residue 731 or 742 within the kinase insert domain of the alpha PDGF receptor do not impair PDGF-induced tyrosine phosphorylation of the receptor or of an in vivo substrate, phospholipase C-gamma. Moreover, phosphatidylinositol turnover in response to ligand stimulation is unaffected. However, both lesions markedly impair receptor association with PI-3 kinase. Antiphosphotyrosine antibody-recoverable PI-3 kinase was also dramatically reduced in PDGF-stimulated cells expressing either mutant receptor. Since neither mutation abolished PDGF-induced mitogenesis or chemotaxis, we conclude that alpha PDGF receptor-associated PI-3 kinase activity is not required for either of these major PDGF signalling functions.     

Compensatory Mutants of the Bovine Papillomavirus E5 Protein and the Platelet-Derived Growth Factor β Receptor Reveal a Complex Direct Transmembrane Interaction

The 44-amino-acid E5 protein of bovine papillomavirus is a dimeric transmembrane protein that exists in a stable complex with the platelet-derived growth factor (PDGF) β receptor, causing receptor activation and cell transformation. The transmembrane domain of the PDGF β receptor is required for complex formation, but it is not known if the two proteins contact one another directly. Here, we studied a PDGF β receptor mutant containing a leucine-to-isoleucine substitution in its transmembrane domain, which prevents complex formation with the wild-type E5 protein in mouse BaF3 cells and inhibits receptor activation by the E5 protein. We selected E5 mutants containing either a small deletion or multiple substitution mutations that restored binding to the mutant PDGF β receptor, resulting in receptor activation and growth factor independence. These E5 mutants displayed lower activity with PDGF β receptor mutants containing other transmembrane substitutions in the vicinity of the original mutation, and one of them cooperated with a receptor mutant containing a distal mutation in the juxtamembrane domain. These results provide strong genetic evidence that the transmembrane domains of the E5 protein and the PDGF β receptor contact one another directly. They also demonstrate that different mutations in the E5 protein allow it to tolerate the same mutation in the PDGF β receptor transmembrane domain and that a mutation in the E5 protein can allow it to tolerate different mutations in the PDGF β receptor. Thus, the rules governing direct interactions between transmembrane helices are complex and not restricted to local interactions.     

The juxtamembrane region of MuSK has a critical role in agrin-mediated signaling

MuSK is a receptor tyrosine kinase expressed selectively in skeletal muscle and localized to neuromuscular synapses. Agrin activates MuSK and stimulates phosphorylation and clustering of acetylcholine receptors (AChRs) at synaptic sites. We expressed wild-type or mutant MuSK in MuSK(-/-) myotubes and identified tyrosine residues in the MuSK cytoplasmic domain that are necessary for agrin-stimulated phosphorylation and clustering of AChRs. The activation loop tyrosines and the single juxtamembrane tyrosine were found to be essential for agrin-stimulated phosphorylation and clustering of AChRs. Further, we show that the juxtamembrane tyrosine, contained within an NPXY motif, is phosphorylated in vivo by agrin stimulation. We constructed chimeras containing extracellular and transmembrane domains from MuSK and cytoplasmic sequences from TrkA and found that inclusion of 13 amino acids from the MuSK juxtamembrane region, including the NPXY motif, is sufficient to convert a phosphorylated but inactive MuSK-TrkA chimera into a phosphorylated active chimera. These data suggest that phosphorylation of the MuSK NPXY site leads to recruitment of a phosphotyrosine-binding domain-containing protein that functions to stimulate phosphorylation and clustering of AChRs.     

In vitro phosphorylation and hormone binding activation of the synthetic wild type human estradiol receptor.

A tyrosine kinase purified from calf uterus activates the hormone binding of endogenous estradiol receptor (ER) predephosphorylated and preinactivated by a nuclear phosphotyrosine phosphatase. The kinase also activates and phosphorylates the human estradiol receptor HEO synthesized in vitro, which differs from the wild type receptor HEGO because a glycine is replaced by a valine at position 400. Moreover, the kinase activates and phosphorylates a deletion mutant of HEO which consists almost exclusively of the hormone binding domain. Using HEGO and HEO in parallel and measuring both binding activation and phosphorylation of ER we now observe that the wild type receptor is a good kinase substrate, slightly better than HEO. Furthermore, HEGO like the calf uterus receptor in the presence of estradiol, stimulates the kinase. From present findings it appears that ER and uterus tyrosine kinase are functionally associated and that this association is abolished by glycine to valine substitution at position 400 of ER.     

Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.     

Crystal structure of the MuSK tyrosine kinase: insights into receptor autoregulation

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed selectively in skeletal muscle. During neuromuscular synapse formation, agrin released from motor neurons stimulates MuSK autophosphorylation in the kinase activation loop and in the juxtamembrane region, leading to clustering of acetylcholine receptors. We have determined the crystal structure of the cytoplasmic domain of unphosphorylated MuSK at 2.05 A resolution. The structure reveals an autoinhibited kinase domain in which the activation loop obstructs ATP and substrate binding. Steady-state kinetic analysis demonstrates that autophosphorylation results in a 200-fold increase in k(cat) and a 10-fold decrease in the K(m) for ATP. These studies provide a molecular basis for understanding the regulation of MuSK catalytic activity and suggest that an additional in vivo component may contribute to regulation via the juxtamembrane region.     

Phosphatidylinositol linkage of a truncated form of the platelet-derived growth factor receptor

The platelet-derived growth factor (PDGF) receptor is usually anchored to the plasma membrane through a membrane-spanning hydrophobic amino acid sequence that splits the molecule into two approximately equal pieces, an amino-terminal external domain that contains the binding site for PDGF and a carboxyl-terminal cytoplasmic domain that includes the tyrosine kinase coding sequences. Here we report the expression of a truncated PDGF receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains. Unexpectedly, this form of the receptor that lacks a hydrophobic membrane-anchoring sequence was bound to the membrane and was not secreted into the culture media. Conventional methods to dissociate noncovalent protein-protein interactions failed to release the protein from the membrane. When the transmembrane and cytoplasmic sequences were artificially deleted from the PDGF receptor, the truncated extracellular domain was anchored to the membrane through phospholipids and could be released by phospholipase C treatment. This truncated form of the receptor bound PDGF with an affinity 5-20-fold lower than the full-length receptor.