Dimerization and activation of the kit receptor by monovalent and bivalent binding of the stem cell factor.

The protooncogene c-kit encodes a tyrosine kinase receptor for the stem cell factor (SCF). Mutants of c-kit were shown to confer a pleiotropic defective phenotype and often display negative dominance in heterozygous mice. To explore the involvement of receptor dimerization in this genetic phenomenon, we employed both a human ligand, which does not recognize the murine receptor, and a rodent SCF, which binds to the human receptor with 100-fold reduced affinity as compared with human SCF. SCF binding to living cells was found to induce rapid and complete receptor dimerization that involved activation of the catalytic tyrosine kinase function. Although receptor dimerization can be attributed to the dimeric nature of the ligand, no dissociation of Kit dimers occurred at high excess of SCF, suggesting that receptor-receptor interactions are also involved in dimer stabilization. This was supported by in vitro formation of heterodimers between the human and murine Kit proteins through monovalent binding of species-specific human SCF. By coexpression of human and mouse Kit in murine fibroblasts, we found that receptor heterodimerization in living cells involved an increase in the affinity of human Kit for rat SCF and also an accelerated rate of receptor down-regulation. When a human Kit mutant lacking the kinase insert domain was coexpressed with the murine wild-type receptor, we observed a significant decrease in both the activation of the intact tyrosine kinase and its coupling to an effector protein, namely phosphatidylinositol 3'-kinase. Our results favor a receptor activation model that assumes an initial step of monovalent ligand binding, followed by an intermediate receptor dimer bound by one arm of the ligand molecule. This model predicts the existence of an intrinsic receptor dimerization site and provides a structural basis for genetic dominance of mutant SCF receptors.     

Role of c-kit/SCF in cause and treatment of gastrointestinal stromal tumors (GIST)

c-Kit encodes for the receptor tyrosine kinase (RTK) and belongs to type III receptor family. This includes platelet derived growth factor (PDGF) alpha and beta and macrophage colony stimulating factor (mCSF) apart from others. Their characteristic features are the presence of five immunologlobulin like domains in the extracellular region and 70-100 residues long kinase insert domain in the cytoplasmic region. The RTKs activate several signaling pathways within the cells leading to cell proliferation, differentiation, migration or metabolic changes. The Kit ligand-stem cell factor (SCF) induces a rapid and complete receptor dimerization resulting in activation by autophosphorylation of the catalytic tyrosine kinase and generation of signal transduction leading to regulation of cell growth. Various mutations in c-kit such as insertions and deletions (without affecting reading frame) and point mutations in the inhibitory juxtamembrane (JM) domain encoded by exon 11 have been reported in gastrointestinal stromal tumors (GISTs). Thus, c-kit signaling is believed to play a role in tumorigenesis. Efforts are being made to control and treat these tumors by blocking kit signaling using Imatinib with varying degrees of success. This review deals with the features of c-kit, its ligand and roles in gastrointestinal stromal tumors.     

Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor beta receptor

Growth factor receptors are typically activated by the binding of soluble ligands to the extracellular domain of the receptor, but certain viral transmembrane proteins can induce growth factor receptor activation by binding to the receptor transmembrane domain. For example, homodimers of the transmembrane 44-amino acid bovine papillomavirus E5 protein bind the transmembrane region of the PDGF beta receptor tyrosine kinase, causing receptor dimerization, phosphorylation, and cell transformation. To determine whether it is possible to select novel biologically active transmembrane proteins that can activate growth factor receptors, we constructed and identified small proteins with random hydrophobic transmembrane domains that can bind and activate the PDGF beta receptor. Remarkably, cell transformation was induced by approximately 10% of the clones in a library in which 15 transmembrane amino acid residues of the E5 protein were replaced with random hydrophobic sequences. The transformation-competent transmembrane proteins formed dimers and stably bound and activated the PDGF beta receptor. Genetic studies demonstrated that the biological activity of the transformation-competent proteins depended on specific interactions with the transmembrane domain of the PDGF beta receptor. A consensus sequence distinct from the wild-type E5 sequence was identified that restored transforming activity to a non-transforming poly-leucine transmembrane sequence, indicating that divergent transmembrane sequence motifs can activate the PDGF beta receptor. Molecular modeling suggested that diverse transforming sequences shared similar protein structure, including the same homodimer interface as the wild-type E5 protein. These experiments have identified novel proteins with transmembrane sequences distinct from the E5 protein that can activate the PDGF beta receptor and transform cells. More generally, this approach may allow the creation and identification of small proteins that modulate the activity of a variety of cellular transmembrane proteins.     

Tyrosine protein kinases and spermatogenesis: truncation matters

Protein phosphorylation on serine/threonine or tyrosine residues represents a significant regulatory mechanism in signal transduction during spermatogenesis, oogenesis, and fertilization. There are several families of tyrosine protein kinases operating during spermatogenesis: the Src family of tyrosine protein kinases; the Fujinami poultry sarcoma/feline sarcoma (Fps/Fes) and Fes-related protein (Fer) subfamily of non-receptor proteins; and c-kit, the transmembrane tyrosine kinase receptor that belongs to the family of the PDGF receptor. A remarkable characteristic is the coexistence of full-length and truncated tyrosine kinases in testis. Most of the truncated forms are present during spermiogenesis. Examples include the truncated forms of Src tyrosine kinase hematopoietic cell kinase (Hck), FerT, and tr-kit. A feature of FerT and tr-kit is the kinase domain that ensures the functional properties of the truncated protein. FerT, a regulator of actin assembly/disassembly mediated by cortactin phosphorylation, is present in the acroplaxome, a cytoskeletal plate containing an F-actin network and linking the acrosome to the spermatid nuclear envelope. This finding suggests that Fer kinase represents one of the tyrosine protein kinases that may contribute to spermatid head shaping. The c-kit ligand, stem cell factor (SCF), which induces c-kit dimerization and autophosphorylation, exists as both membrane-associated and soluble. Although tyrosine protein kinases are prominent in spermatogenesis, a remarkable observation is the paucity of phenotypic alterations in spermatogenic cells in male mice targeted with Fer kinase-inactivating mutation. It is possible that the redundant functions of the tyrosine protein kinase pool present during spermatogenesis may explain the limited phenotypes of single mutant mice. The production of compound and viable mutant mice, lacking the expression of two or more tyrosine kinases, may shed light on this intriguing issue.     

Structure of the active core of human stem cell factor and analysis of binding to its receptor kit

Stem cell factor (SCF) is an early-acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane-bound forms. It transduces signals by ligand- mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet-derived growth factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand-binding portions composed of immunoglobulin-like repeats. We have determined the crystal structure of selenomethionyl soluble human SCF at 2.2 A resolution by multiwavelength anomalous diffraction phasing. SCF has the characteristic helical cytokine topology, but the structure is unique apart from core portions. The SCF dimer has a symmetric 'head-to-head' association. Using various prior observations, we have located potential Kit-binding sites on the SCF dimer. A superimposition of this dimer onto VEGF in its complex with the receptor Flt-1 places the binding sites on SCF in positions of topographical and electrostatic complementarity with the Kit counterparts of Flt-1, and a similar model can be made for the complex of PDGF with its receptor.     

Isolation and characterization of the alpha platelet-derived growth factor receptor from rat olfactory epithelium.

We have cloned and characterized a new member of the receptor tyrosine kinase family. The cDNA clone, isolated from a rat olfactory cDNA library, has considerable homology to the family of receptors that includes the colony-stimulating factor 1 receptor, the c-kit proto-oncogene, and the platelet-derived growth factor (PDGF) receptors. Analysis of DNA sequence homology, ligand-binding, and ligand-stimulated phosphorylation data suggests that this clone encodes the rat PDGF-A/B or alpha-receptor. Comparison of its sequence to those of other receptors allows us to postulate a mechanism for receptor dimerization and activation. The expression of the rat alpha-PDGF receptor in nonneuronal cells of the olfactory epithelium and in the olfactory bulb is consistent with a role for PDGF in glial cell generation.     

Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor

Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR-II) which has a serine/threonine kinase domain, and stem cell factor receptor (c-kit) which contains a tyrosine kinase domain form a complex in vitro and in vivo; the interaction is induced upon treatment of cells with BMP2 and SCF. Stem cell factor (SCF) modulated BMP2-dependent activation of Smad1/5/8 and phosphorylation of Erk kinase. SCF also enhanced BMP2-dependent differentiation of C2C12 cells. We found that BMPR-II was phosphorylated at Ser757 upon co-expression with and activation of c-kit. BMPR-II phosphorylation required intact kinase activity of BMPR-II. Abrogation of the c-kit/SCF-dependent phosphorylation of BMPR-II at the Ser757 interfered with the cooperative effect of BMP2 and SCF. Our data suggest that the complex formation between c-kit and BMPR-II leads to phosphorylation of BMPR-II at Ser757, which modulates BMPR-II-dependent signaling.     

Structural and Functional Properties of Platelet-Derived Growth Factor and Stem Cell Factor Receptors

The receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) are members of the type III class of PTK receptors, which are characterized by five Ig-like domains extracellularly and a split kinase domain intracellularly. The receptors are activated by ligand-induced dimerization, leading to autophosphorylation on specific tyrosine residues. Thereby the kinase activities of the receptors are activated and docking sites for downstream SH2 domain signal transduction molecules are created; activation of these pathways promotes cell growth, survival, and migration. These receptors mediate important signals during the embryonal development, and control tissue homeostasis in the adult. Their overactivity is seen in malignancies and other diseases involving excessive cell proliferation, such as atherosclerosis and fibrotic diseases. In cancer, mutations of PDGF and SCF receptors—including gene fusions, point mutations, and amplifications—drive subpopulations of certain malignancies, such as gastrointestinal stromal tumors, chronic myelomonocytic leukemia, hypereosinophilic syndrome, glioblastoma, acute myeloid leukemia, mastocytosis, and melanoma.The type III tyrosine kinase receptor family consists of platelet-derived growth factor (PDGF) receptor α and β, stem cell factor (SCF) receptor (Kit), colony-stimulating factor-1 (CSF-1) receptor, and Flt-3 (Blume-Jensen and Hunter 2001). Members of this receptor family are characterized by five Ig-like domains in their extracellular part, a single transmembrane domain, and an intracellular part consisting of a rather well-conserved juxtamembrane domain, a tyrosine kinase domain with a characteristic inserted sequence without homology with kinases, and a less well-conserved carboxy-terminal tail. The ligands for these receptors are all dimeric molecules, and on binding they induce receptor dimerization. Although the overall mechanisms for the activation of the type III tyrosine kinase receptors and the signaling pathways they induce are similar, the receptors are expressed on different cell types and thus have different functions in vivo.Here we will describe the structural and functional properties of the PDGF receptors and Kit.     

A recombinant ectodomain of the receptor for the stem cell factor (SCF) retains ligand-induced receptor dimerization and antagonizes SCF-stimulated cellular responses.

The stem cell factor (SCF) is a polypeptide ligand that is essential for the development of germ cells, hematopoietic progenitor cells, and melanocyte precursors. It binds to a tyrosine kinase membrane receptor that is encoded by the c-kit proto-oncogene. We have constructed an expression vector that directs the synthesis of the entire extracellular ligand-binding domain of the Kit/SCF receptor. When expressed and amplified in Chinese hamster ovary cells, a secreted 90-kDa glycoprotein could be harvested from the growth medium of the cells in a soluble form. This extracellular portion of the Kit/SCF receptor, denoted Kit-X, was recognized by antibodies specific to the SCF receptor; and when injected into animals, it raised antibodies that were reactive with the complete membrane form of the receptor. Direct binding and covalent cross-linking of radiolabeled SCF showed that Kit-X fully retained high affinity ligand binding and also underwent efficient dimerization in the presence of the ligand. The capacity of Kit-X to act as an antagonist of SCF was assayed on cultured cells that overexpress the receptor. Simultaneous addition of SCF and Kit-X to these cells resulted in a stoichiometric inhibition of SCF binding and a consequent decrease in autophosphorylation of the SCF receptor on tyrosine residues. The inhibition extended to later SCF-mediated responses, including the association of the receptor with phosphatidylinositol 3'-kinase and coupling to the Raf1 protein kinase. These results indicate that the recombinant ectodomain of the Kit-SCF receptor can be used as a specific antagonist of SCF actions and may enable detailed molecular analysis of ligand-receptor interactions.     

Tec kinase associates with c-kit and is tyrosine phosphorylated and activated following stem cell factor binding.

Stem cell factor (SCF) plays a crucial role in hematopoiesis through its interaction with the receptor tyrosine kinase c-kit. However, the signaling events that are activated by this interaction and involved in the control of growth or differentiation are not completely understood. We demonstrate here that Tec, a cytoplasmic, src-related kinase, physically associates with c-kit through a region that contains a proline-rich motif, amino terminal of the SH3 domain. Following SCF binding, Tec is tyrosine phosphorylated and its in vitro kinase activity is increased. Tyrosine phosphorylation of Tec is not detected in the response to other cytokines controlling hematopoiesis, including colony-stimulating factor-1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3). Conversely, the cytoplasmic kinase JAK2 is activated by IL-3 but not by SCF stimulation. The activation of distinct cytoplasmic kinases may account for the synergy seen in the actions of SCF and IL-3 on hematopoietic stem cells.     

The role of stem cell factor and of alternative c-kit gene products in the establishment, maintenance and function of germ cells

The c-kit gene plays a fundamental role during the establishment, the maintenance and the function of germ cells. In the embryonal gonad the c-kit tyrosine kinase receptor and its ligand Stem Cell Factor (SCF) are required for the survival and proliferation of primordial germ cells. In the postnatal animal, c-kit/SCF are required for the production of the mature gametes in response to gonadotropic hormones, i.e. for the survival and/or proliferation of the only proliferating germ cells of the testis, the spermatogonia, and for the growth and maturation of the oocytes. Finally, a truncated c-kit product, tr-kit, specifically expressed in post-meiotic stages of spermatogenesis and present in mature spermatozoa, causes parthenogenetic activation when microinjected into mouse eggs, suggesting that it might play a role in the final function of the gametes, fertilization.     

Specific locations of hydrophilic amino acids in constructed transmembrane ligands of the platelet-derived growth factor beta receptor

The 44 amino acid E5 transmembrane protein is the primary oncogene product of bovine papillomavirus. Homodimers of the E5 protein activate the cellular PDGF beta receptor tyrosine kinase by binding to its transmembrane domain and inducing receptor dimerization, resulting in cellular transformation. To investigate the role of transmembrane hydrophilic amino acids in receptor activation, we constructed a library of dimeric small transmembrane proteins in which 16 transmembrane amino acids of the E5 protein were replaced with random, predominantly hydrophobic amino acids. A low level of hydrophilic amino acids was encoded at each of the randomized positions, including position 17, which is an essential glutamine in the wild-type E5 protein. Library proteins that induced transformation in mouse C127 cells stably bound and activated the PDGF beta receptor. Strikingly, 35% of the transforming clones had a hydrophilic amino acid at position 17, highlighting the importance of this position in activation of the PDGF beta receptor. Hydrophilic amino acids in other transforming proteins were found adjacent to position 17 or at position 14 or 21, which are in the E5 homodimer interface. Approximately 22% of the transforming proteins lacked hydrophilic amino acids. The hydrophilic amino acids in the transforming clones appear to be important for driving homodimerization, binding to the PDGF beta receptor, or both. Interestingly, several of the library proteins bound and activated PDGF beta receptor transmembrane mutants that were not activated by the wild-type E5 protein. These experiments identified transmembrane proteins that activate the PDGF beta receptor and revealed the importance of hydrophilic amino acids at specific positions in the transmembrane sequence. Our identification of transformation-competent transmembrane proteins with altered specificity suggests that this approach may allow the creation and identification of transmembrane proteins that modulate the activity of a variety of receptor tyrosine kinases.     

Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family--oncogenic activation of v-kit involves deletion of extracellular domain and C terminus.

The protein kinase domains of v-kit, the oncogene of the acute transforming feline retrovirus HZ4-FeSV (HZ4-feline sarcoma virus), CSF-1R (macrophage colony stimulating factor receptor) and PDGFR (platelet derived growth factor receptor) display extensive homology. Because of the close structural relationship of v-kit, CSF-1R and PDGFR we predicted that c-kit would encode a protein kinase transmembrane receptor (Besmer et al., 1986a; Yarden et al., 1986). We have now determined the primary structure of murine c-kit from a DNA clone isolated from a brain cDNA library. The nucleotide sequence of the c-kit cDNA predicts a 975 amino acid protein product with a calculated mol. wt of 109.001 kd. It contains an N-terminal signal peptide, a transmembrane domain (residues 519-543) and in the C-terminal half the v-kit homologous sequences (residues 558-925). c-kit therefore contains the features which are characteristic of a transmembrane receptor kinase. Comparison of c-kit, CSF-1R and PDGFR revealed a unique structural relationship of these receptor kinases suggesting a common evolutionary origin. The outer cellular domain of c-kit was shown to be related to the immunoglobulin superfamily. The sites of expression of c-kit in normal tissue predict a function in the brain and in hematopoietic cells. N-terminal sequences which include the extracellular domain and the transmembrane domain as well as 50 amino acids from the C-terminus of c-kit are deleted in v-kit. These structural alterations are likely determinants of the oncogenic activation of v-kit.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Stem cell factor (SCF) can regulate the activation and expansion of murine intraepithelial lymphocytes

Murine intraepithelial lymphocytes (IEL) express c-kit, the receptor for stem cell factor (SCF). SCF induced a low but significant proliferative response in IEL, but not in splenic T cells. SCF stimulation of IEL resulted in an expansion of the c-kit(+), TCRgammadelta(+)cell population. SCF-induced proliferation was dependent upon SCF-c-kit interactions, since antibody to c-kit blocked this response, and IEL obtained from c-kit mutant (W/W(v)) mice failed to respond to SCF. SCF acted synergistically with anti-TCRgammadelta and with concavalin A (Con A) to induce proliferation and interferon gamma (IFN-gamma) production in IEL. Finally, mice injected with SCF had a significant increase in the number of IEL in the small intestine. SCF-treated mice had increased numbers of TCRalphabeta(+)and TCRgammadelta(+)cell populations, as well as increased numbers of c-kit(+)and c-kit(-)IEL. These data suggest that SCF-c-kit interactions play an important role in regulating IEL expansion and activation.     

Chimeric cytokine receptor can transduce expansion signals in interleukin 6 receptor alpha (IL-6Ralpha)-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors

We generated transgenic mice expressing chimeric receptors, which comprise extracellular domains of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor and transmembrane and cytoplasmic domains of the mouse leukemia inhibitory factor receptor. In suspension cultures of lineage-negative (Lin(-)), 5-fluorouracil-resistant bone marrow cells of the transgenic mice, a combination of hGM-CSF and stem cell factor (SCF) induced exponential expansions of mixed colony-forming unit. The combination of hGM-CSF and SCF was effective on enriched, Lin(-)Sca-1(+)c-kit(+) progenitors and increased either mixed colony-forming unit or cobblestone area-forming cells. In case of stimulation with hGM-CSF and SCF, interleukin-6 (IL-6) and SCF, or IL-11 and SCF, the most efficient expansion was achieved with hGM-CSF and SCF. When Lin(-)Sca-1(+)c-kit(+)CD34(-) further enriched progenitors were clone sorted and individually incubated in the presence of SCF, hGM-CSF stimulated a larger number of cells than did IL-6, IL-6 and soluble IL-6 receptor (IL-6R), or IL-11. These data suggest the presence of IL-6Ralpha-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors. Such primitive progenitors are equipped with signal transduction molecules and can expand when these chimeric receptors are genetically introduced into the cells and stimulated with hGM-CSF in the presence of SCF.     

A loss-of-function mutation of c-kit results in depletion of mast cells and interstitial cells of Cajal, while its gain-of-function mutation results in their oncogenesis

Loss-of-function mutations of the c-kit receptor tyrosine kinase (KIT) result in depletion of mast cells and interstitial cells of Cajal (ICCs). In contrast, gain-of-function mutations of KIT induce neoplasms of mast cells and ICCs. In humans, the sites of mutations are different between mast cell neoplasms and those of ICCs. The former were found in the juxtamembrane domain between the transmembrane and tyrosine kinase domains, and the latter in the tyrosine kinase domain. Moreover, the mechanism of constitutive activation is different. Point mutations and/or deletions in the juxtamembrane domain induced the KIT dimerization, and the dimerized KIT was activated. A point mutation at the particular aspartic acid in the tyrosine kinase domain induced spontaneous activation without forming dimers. Mutations of the c-kit gene are a good model for understanding the relationship between mutations and diseases in both humans and mice.     

Ligand-induced interaction between alpha- and beta-type platelet-derived growth factor (PDGF) receptors: role of receptor heterodimers in kinase activation.

Two types of PDGF receptors have been cloned and sequenced. Both receptors are transmembrane glycoproteins with a ligand-stimulatable tyrosine kinase site. We have shown earlier that ligand-induced activation of the beta-type PDGF receptor is due to the conversion of the monomeric form of the receptor to the dimeric form [Bishayee et al. (1989) J. Biol. Chem. 264, 11699-11705]. In the present studies, we have established the ligand-binding specificity of two receptor types and extended it further to investigate the ligand-induced association state of the alpha-receptor and the role of alpha-receptor in the activation of beta-receptor. These studies were conducted with cells that express one or the other type of PDGF receptor as well as with cells that express both types of receptors. Moreover, ligand-binding characteristics of the receptor were confirmed by immunoprecipitation of the receptor-125I-PDGF covalent complex with type-specific anti-PDGF receptor antibodies. These studies revealed that all three isoforms of PDGF bind to alpha-receptor, and such binding leads to dimerization as well as activation of the receptor. In contrast, beta-receptor can be activated only by PDGF BB and not by PDGF AB or PDGF AA. However, by using antipeptide antibodies that are specific for alpha- or beta-type PDGF receptor, we demonstrated that in the presence of alpha-receptor, beta-receptor kinase can be activated by PDGF AB. We present here direct evidence that strongly suggests that such PDGF AB induced activation of beta-receptor is due to the formation of a noncovalently linked alpha-beta receptor heterodimer.     

Role of Glutamine 17 of the Bovine Papillomavirus E5 Protein in Platelet-Derived Growth Factor β Receptor Activation and Cell Transformation

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) β receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF β receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF β receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF β receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF β receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF β receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF β receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF β receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF β receptor.     

Platelet-derived growth factor (PDGF)-induced disulfide-linked dimerization of PDGF receptor in living cells.

It is well established that epidermal growth factor and platelet-derived growth factor (PDGF) are able to induce noncovalent dimerization of their surface receptors. It is thought that receptor dimerization plays an important role in activation of the tyrosine kinase function and in the process of receptor autophosphorylation. Here we show that the addition of either PDGF-BB or PDGF-AA to intact 3T3 cells induces formation of 400- and 430-kDa species, respectively, recognized by either anti-PDGF receptor antibodies or anti-phosphotyrosine antibodies. Interestingly, the 400- and the 430-kDa species are detected in nonreducing gels but not in reducing gels. Moreover, an alkylating agent, N-ethylmaleimide, inhibits PDGF-induced formation of high-molecular-mass species. Comparisons of V8 protease peptide maps of [35S]methionine-labeled PDGF receptors and high-molecular-mass proteins indicate that they represent dimers of PDGF receptors. It appears therefore that in addition to noncovalent dimerization, PDGF receptors undergo ligand-dependent disulfide-linked dimerization.