Functional dissection of structural domains in the receptor for colony-stimulating factor-1.

Receptor tyrosine kinases (RTKs) transduce external signals to the interior of the cell via a cytoplasmic kinase domain. We demonstrated previously that ligand-induced kinase activation of the colony-stimulating factor-1 receptor (CSF-1R) occurs via receptor oligomerization without propagation of conformational changes through the transmembrane (TM) domain (Lee, A. W., and Nienhuis, A. W. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7270-7274). We have now examined the role of the different subdomains in the metabolic and signaling properties of CSF-1R. Two types of chimeric receptors have been utilized, Glyfms A, with the extracellular and TM domains of glycophorin A (GpA) and the cytoplasmic domain of CSF-1R, and Glyfms B, where only the extracellular domain originates from GpA. Glyfms A was found to exhibit a higher basal level of in vitro kinase activity, an increased associated phosphatidylinositol (PtdIns) 3-kinase activity and to support enhanced cellular mitogenesis, compared with wild-type CSF-1R or to Glyfms B. The constitutive activation of Glyfms A is consistent with the hypothesis that the TM domain may play a role in receptor oligomerization. Cross-linking with anti-GpA antibodies activated the kinase function of Glyfms B leading to an increase in PtdIns 3-kinase association and to the transmission of a mitogenic signal. Our results indicate that an activated kinase domain contains the major determinant for coupling with PtdIns 3-kinase, independent of extracellular and TM sequences and of ligand binding. Both chimeric receptors underwent internalization in the presence of anti-GpA antibodies but were not degraded, including the tyrosine-phosphorylated and kinase-active population. These results suggest that structural determinants in the extracellular domain must be important for targeting internalized receptors for lysosomal degradation.     

Crystal structure of the tyrosine kinase domain of colony-stimulating factor-1 receptor (cFMS) in complex with two inhibitors

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.     

The kinase insert domain of colony stimulating factor-1 receptor is dispensable for CSF-1 induced phosphatidylcholine hydrolysis.

Mouse NIH 3T3 fibroblasts transfected with human colony stimulating factor-1 receptor produced diacylglycerol in response to CSF1 and this correlated with elevated phosphatidylcholine hydrolyzing activity measured in an in vitro assay. Treatment of cells with the isoflavone derivative genistein attenuated PC hydrolysis in vitro suggesting a role for CSF1R tyrosine kinase activity. A CSF1R mutant lacking 67 amino acids of the kinase insert domain, which may affect the association of receptor with certain substrates, stimulated PC hydrolysis in response to CSF1. Coupling to PC hydrolysis is likely a general property of CSF1R and the kinase insert domain is dispensable for this activity.     

Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors

Inhibitors of receptor tyrosine kinases are implicated as therapeutic agents for the treatment of many human diseases including cancer, inflammation and diabetes. Cell-based assays to examine inhibition of receptor tyrosine kinase mediated intracellular signaling are often laborious and not amenable to high-throughput cell-based screening of compound libraries. Here we describe the development of a nonradioactive, sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the activation and inhibition of ligand-induced phosphorylation of the colony-stimulating factor-1 receptor (CSF-1R) in 96-well microtiter plate format. The assay involves the capture of the Triton X-100 solubilized human CSF-1R, from HEK293E cells overexpressing histidine epitope-tagged CSF-1R (CSF-1R/HEK293E), with immobilized CSF-1R antibody and detection of phosphosphorylation of the activated receptor with a phosphotyrosine specific antibody. The assay exhibited a 5-fold increase in phosphorylated CSF-1R signal from CSF-1R/HEK293E cells treated with colony-stimulating factor (CSF-1) relative to treated vector control cells. Additionally, using a histidine epitope-specific capture antibody, this method can also be adapted to quantify the phosphorylation state of any recombinantly expressed, histidine-tagged receptor tyrosine kinase. This method is a substantial improvement in throughput and quantitation of CSF-1R phosphorylation over conventional immunoblotting techniques.     

IFN-gamma/lipopolysaccharide activation of macrophages is associated with protein kinase C-dependent down-modulation of the colony-stimulating factor-1 receptor.

IFN gamma/LPS treatment increases macrophage tumoricidal and microbicidal activity and inhibits CSF-1-induced macrophage proliferation. The mechanism underlying the latter effect was investigated in the CSF-1-dependent mouse macrophage cell line, BAC-1.2F5. IFN-gamma and LPS together dramatically reduced the total number of CSF-1 receptors (CSF-1R) via selective degradation of the cell surface form. Processing and transport of intracellular CSF-1R to the cell surface were unaffected. IFN-gamma alone had no effect but significantly enhanced LPS-induced CSF-1R down-regulation. The reduction in CSF-1R number was protein kinase C-dependent and involved changes in serine phosphorylation of the receptor at different sites. CSF-1R down-modulation by this mechanism may be important in switching off the energy-consuming processes of CSF-1R-mediated proliferation and chemotaxis in activated macrophages.     

Protein kinase domain of CTR1 from Arabidopsis thaliana promotes ethylene receptor cross talk

Ethylene controls many aspects of plant growth and development. Signaling by the gaseous phytohormone is initiated by disulfide-linked membrane-bound receptors, and the formation of heteromeric receptor clusters contributes to the broad range of ethylene responsiveness. In Arabidopsis thaliana, the TCS-like ethylene receptors interact with the cytosolic serine/threonine kinase constitutive triple response 1 (CTR1), a proposed mitogen-activated protein kinase kinase kinase. In the absence of the hormone, the receptor and therefore CTR1 are active. Hence, ethylene acts as an inverse agonist of its signaling pathway. The three-dimensional structures of the active, triphosphorylated and the unphosphorylated, inactive kinase domain of CTR1 in complex with staurosporine illustrate the conformational rearrangements that form the basis of activity regulation. Additionally, in analytical ultracentrifugation experiments, active kinase domains form back-to-back dimers, while inactive and activation loop variants are monomers. Together with a front-to-front activation interface, the active protein kinase dimers thereby engage in interactions that promote CTR1-mediated cross talk between ethylene receptor clusters. This model provides a structural foundation for the observed high sensitivity of plants to ethylene.     

Expression of c-Myc in response to colony-stimulating factor-1 requires mitogen-activated protein kinase kinase-1

The mitogen-inducible gene c-myc is a key regulator of cell proliferation and transformation. Yet, the signaling pathway(s) that regulate its expression have remained largely unresolved. Using the mitogen-activated protein kinase kinase (MEK1/2) inhibitor PD98059 and dominant negative forms of Ras (N17) and ERK1 (K71R), we found that activation of Ras and extracellular signal-regulated kinase (ERK) is necessary for colony-stimulating factor-1 (CSF-1)-mediated c-Myc expression and DNA synthetic (S) phase entry. Quiescent NIH-3T3 cells expressing a partially defective CSF-1 receptor, CSF-1R (Y809F), exhibited impaired ERK1 activation and c-Myc expression and failed to enter the S phase of the cell division cycle in response to CSF-1 stimulation. Ectopic expression of a constitutively active form of MEK1 in cells expressing CSF-1R (Y809F) rescued c-Myc expression and S phase entry, but only in the presence of CSF-1-induced cooperating signals. Therefore, MEK1 participates in an obligate signaling pathway linking CSF-1R to c-Myc expression, but other signals from CSF-1R must cooperate with the MEK/ERK pathway to induce c-Myc expression and S phase entry in response to CSF-1 stimulation.     

Assessment of expression of the receptor for colony-stimulating factor-1 (fms) in bovine trophoblast.

Bovine trophoblast was employed to assess the questions of whether the receptor for CSF-1 is expressed by noninvasive trophoblast and whether expression changes with differentiation within placentomes. Bovine placental poly(A) mRNA contained sequences cross-reactive with cDNA probes to c-fms and v-fms. Using a monoclonal antibody to v-fms, immunohistochemistry of postattachment bovine trophoblast showed expression of an fms-like protein between Day 29 and term. Expression occurred in both the intercotyledonary and cotyledonary trophoblast. Reactivity that was fms-like was also demonstrated on preattachment conceptuses flushed at Days 14 and 7 of gestation and on Day 7 embryos derived from in vitro oocyte maturation and fertilization. Unexpectedly, in the second half of pregnancy some cells, including binucleate cells, showed nuclear rather than cytoplasmic reactivity to the antibody. These data indicate expression of an fms-like protein in bovine placenta that does not correlate with properties of trophoblast cell invasiveness or major morphological differentiation. The data do support a universal role for this protein during mammalian placental development.     

Mouse mesangial cells produce colony-stimulating factor-1 (CSF-1) and express the CSF-1 receptor

CSF-1 stimulates the survival, proliferation, and differentiation of mononuclear phagocytes and may also play a role in placental development. The expression of CSF-1 and the CSF-1 receptor (CSF-1R) and their regulation were examined in cultures of mouse mesangial cells (MC). The concentration of CSF-1 in the medium of cultured MC increased linearly with time over 24 h. IFN-gamma stimulated and dibutyryl cyclic AMP inhibited CSF-1 production in a dose-dependent manner. MC expression of CSF-1 mRNA was shown by Northern blot analysis, and CSF-1 mRNA levels were increased within 4 h of IFN-gamma addition and inhibited within 4 h of dibutyryl cyclic AMP addition. Indirect immunofluorescence indicated that 90% of the untreated cultured MC expressed CSF-1. In addition, CSF-1R expression by MC was demonstrated by immunofluorescence with anti-receptor antibody, specific binding of [125I] CSF-1, and expression of the CSF-1R mRNA by Northern blot analysis. Thus, mouse MC, specialized pericytes of non-bone marrow origin, not only produce CSF-1 but also express receptors for CSF-1. The effects of CSF-1 on MC may be important in the control of immune function in the glomerulus.     

Protein structural domain identification

A simple method for the definition of protein structural domains is described that requires only alpha-carbon coordinate data. The basic method, which encodes no specific aspects of protein structure, captures the essence of most domains but does not give high enough priority to the integrity of beta-sheet structure. This aspect was encouraged both by a bias toward attaining intact beta-sheets and also as an acceptance condition on the final result. The method has only one variable parameter, reflecting the granularity level of the domains, and an attempt was made to set this level automatically for each protein based on the best agreement attained between the domains predicted on the native structure and a set of smoothed coordinates. While not perfect, this feature allowed some tightly packed domains to be separated that would have remained undivided had the best fixed granularity level been used. The quality of the results was high and, when compared with a large collection of accepted domain definitions, only a few could be said to be clearly incorrect. The simplicity of the method allowed its easy extension to the simultaneous definition of domains across related structures in a way that does not involve loss of detail through averaging the structures. This was found to be a useful approach to reconciling differences among structural family members. The method is fast, taking less than 1 s per 100 residues for medium-sized proteins.     

Biology and action of colony-stimulating factor-1

Colony-stimulating factor-1 (CSF-1), also known as macrophage colony-stimulating factor, controls the survival, proliferation, and differentiation of mono-nuclear phagocytes and regulates cells of the female reproductive tract. It appears to play an autocrine and/or paracrine role in cancers of the ovary, endometrium, breast, and myeloid and lymphoid tissues. Through alternative mRNA splicing and differential post-translational proteolytic processing, CSF-1 can either be secreted into the circulation as a glycoprotein or chondroitin sulfate-containing proteoglycan or be expressed as a membrane-spanning glycoprotein on the surface of CSF-1-producing cells. Studies with the op/op mouse, which possesses an inactivating mutation in the CSF-1 gene, have established the central role of CSF-1 in directly regulating osteoclastogenesis and macrophage production. CSF-1 appears to preferentially regulate the development of macrophages found in tissues undergoing active morphogenesis and/or tissue remodeling. These CSF-1 dependent macrophages may, via putative trophic and/or scavenger functions, regulate characteristics such as dermal thickness, male fertility, and neural processing. Apart from its expression on mononuclear phagocytes and their precursors, CSF-1 receptor (CSF-1R) expression on certain nonmononuclear phagocytic cells in the female reproductive tract and studies in the op/op mouse indicate that CSF-1 plays important roles in female reproduction. Restoration of circulating CSF-1 to op/op mice has preliminarily defined target cell populations that are regulated either humorally or locally by the synthesis of cell-surface CSF-1 or by sequestration of the CSF-1 proteoglycan. The CSF-1R is a tyrosine kinase encoded by the c-fms proto-oncogene product. Studies by several groups have used cells expressing either the murine or human CSF-1R in fibroblasts to pinpoint the requirement of kinase activity and the importance of various receptor tyrosine phosphorylation sites for signaling pathways stimulated by CSF-1. To investigate post-CSF-1R signaling in the macrophage, proteins that are rapidly phosphorylated on tyrosine in response to CSF-1 have been identified, together with proteins associated with them. Studies on several of these proteins, including protein tyrosine phosphatase 1C, the c-cbl proto-oncogene product, and protein tyrosine phosphatase-phi are discussed. Mol Reprod Dev 46:4–10, 1997. © 1997 Wiley-Liss, Inc.     

The Src-like adaptor protein 2 regulates colony-stimulating factor-1 receptor signaling and down-regulation

Src-like adaptor protein 2 (SLAP-2) is a hematopoietic adaptor protein previously implicated as a negative regulator of T-cell antigen receptor (TCR)-mediated signaling. SLAP-2 contains an SH3 and an SH2 domain, followed by a unique carboxyl-terminal tail, which is important for c-Cbl binding. Here we describe a novel role for SLAP-2 in regulation of the colony-stimulating factor 1 receptor (CSF-1R), a receptor tyrosine kinase important for growth and differentiation of myeloid cells. SLAP-2 co-immunoprecipitates with c-Cbl and CSF-1R in primary bone marrow-derived macrophages. Using murine myeloid cells expressing CSF-1R (FD-Fms cells), we show that SLAP-2 is tyrosine-phosphorylated upon stimulation with CSF-1 and associates constitutively with both c-Cbl and CSF-1R. In addition, we show that expression of a dominant negative form of SLAP-2 impairs c-Cbl association with the CSF-1R and receptor ubiquitination. Impaired c-Cbl recruitment also correlated with changes in the kinetics of CSF-1R down-regulation and trafficking. CSF-1-mediated differentiation of FD-Fms cells and activation of downstream signaling events was also enhanced in cells stably expressing dominant negative SLAP-2. Together, these results demonstrate that SLAP-2 plays a role in c-Cbl-dependent down-regulation of CSF-1R signaling.     

Protein kinase C-mediated desensitization of the neurokinin 1 receptor

An understanding of the mechanisms that regulate signaling bythe substance P (SP) or neurokinin 1 receptor (NK1-R) is of interestbecause of their role in inflammation and pain. By using activators andinhibitors of protein kinase C (PKC) and NK1-R mutations of potentialPKC phosphorylation sites, we determined the role of PKC indesensitization of responses to SP. Activation of PKC abolishedSP-induced Ca²⁺ mobilization in cells that expresswild-type NK1-R. This did not occur in cells expressing aCOOH-terminally truncated NK1-R (NK1-R324), which may correspond toa naturally occurring variant, or a point mutant lacking eightpotential PKC phosphorylation sites within the COOH tail (NK1-RSer-338, Thr-339, Ser-352, Ser-387, Ser-388, Ser-390, Ser-392,Ser-394/Ala, NK1-RKC4). Compared with wild-type NK1-R, thet_1/2 of SP-induced Ca²⁺mobilization was seven- and twofold greater in cells expressing NK1-R324 and NK1-RKC4, respectively. In cells expressing wild-type NK1-R, inhibition of PKC caused a 35% increase in thet_1/2 of SP-induced Ca²⁺mobilization. Neither inhibition of PKC nor receptor mutation affecteddesensitization of Ca²⁺ mobilization to repeated challengewith SP or SP-induced endocytosis of the NK1-R. Thus PKC regulatesSP-induced Ca²⁺ mobilization by full-length NK1-R and doesnot regulate a naturally occurring truncated variant. PKC doesnot mediate desensitization to repeated stimulation or endocytosis ofthe NK1-R.

     

Role of colony-stimulating factor-1 in bone metabolism

Colony-stimulating factor-1 (CSF-1) is a cytokine required for proliferation, differentiation, activity, and survival of cells of the mononuclear phagocytic system. The growth factor is synthesized as a soluble, matrix, or membrane associated molecule. The specific functions of these forms are not clear. However, some data suggest a dependence of the development of various populations of tissue macrophages on the locally expressed and presented cytokine. Deficiency in CSF-1, as is the case in the murine mutant strain op/op, results in low numbers of macrophages and monocytes and, most striking, leads to osteopetrosis due to a virtual absence of osteoclasts. Using the op/op mutation as a model, CSF-1 was established as one of the growth factors for osteoclasts. The expression of CSF-1 receptors, encoded by the proto-oncogene c-fms, by osteoclast precursors and osteoclasts, suggested an effect of this cytokine not only during osteoclast formation but also on the mature cells. In fact, CSF-1 was shown to inhibit the resorbing activity, to stimulate migration, and to support survival of isolated osteoclasts in vitro. By these actions on cells of the osteoclast lineage, CSF-1 induces recruitment of new osteoclasts, leading to a net increase of bone resorption, and might govern the spatial distribution of resorption sites within the bone. During these processes, locally expressed and presented forms of the growth factor may play a crucial role, as will be discussed in this article. © 1994 Wiley-Liss, Inc.     

A mutational analysis of phosphatidylinositol-3-kinase activation by human colony-stimulating factor-1 receptor.

Colony-stimulating factor-1 (CSF1) is a cell lineage-specific hemopoietin required for the growth, differentiation, and survival of macrophages and their precursors. The human CSF1 receptor (CSF1R) is a 150-kDa transmembrane glycoprotein whose cytoplasmic tyrosine kinase domain is split by a kinase insert (KI) region of approximately 70 amino acids. We tested the ability of CSF1R KI domain deletion mutants to stimulate phosphatidylinositol-3-kinase (PtdIns-3-kinase), an enzyme whose activity is augmented by tyrosine kinase oncogenes and receptor tyrosine kinases, and to support mitogenesis in transfected cells. Receptor immunoprecipitates from CSF1-stimulated cells contained greater than 5-fold more PtdIns-3-kinase activity compared to nonstimulated cells. High performance liquid chromatography analysis of the PtdIns-3-kinase product scraped from thin layer chromatography plates indicated that PtdIns-3-P was produced. CSF1R KI domain deletion mutants retained tyrosine kinase activity in vitro. Receptor immunoprecipitates of two partially overlapping 28 and 30 amino acid KI deletion mutants of CSF1R retained some PtdIns-3-kinase activity, in contrast to immunoprecipitates of CSF1R lacking 67 amino acids of the KI domain. Each deletion mutant stimulated CSF1-dependent DNA synthesis in transfected cells at much reduced levels compared to wild-type receptor expressing cells. These data suggest a role for the CSF1R KI domain in PtdIns-3-kinase association and for CSF1-induced thymidine incorporation into DNA.     

Fluorescence polarization assay for inhibitors of the kinase domain of receptor interacting protein 1

Necrotic cell death is prevalent in many different pathological disease states and in traumatic injury. Necroptosis is a form of necrosis that stems from specific signaling pathways, with the key regulator being receptor interacting protein 1 (RIP1), a serine/threonine kinase. Specific inhibitors of RIP1, termed necrostatins, are potent inhibitors of necroptosis. Necrostatins are structurally distinct from one another yet still possess the ability to inhibit RIP1 kinase activity. To further understand the differences in the binding of the various necrostatins to RIP1 and to develop a robust high-throughput screening (HTS) assay, which can be used to identify new classes of RIP1 inhibitors, we synthesized fluorescein derivatives of Necrostatin-1 (Nec-1) and Nec-3. These compounds were used to establish a fluorescence polarization (FP) assay to directly measure the binding of necrostatins to RIP1 kinase. The fluorescein-labeled compounds are well suited for HTS because the assays have a dimethyl sulfoxide (DMSO) tolerance up to 5% and Z' scores of 0.62 (fluorescein-Nec-1) and 0.57 (fluorescein-Nec-3). In addition, results obtained from the FP assays and ligand docking studies provide insights into the putative binding sites of Nec-1, Nec-3, and Nec-4.     

Expression and characterization of the integral membrane domain of bacterial histidine kinase SCO3062 for structural studies

Bacterial histidine kinases play an important role in the response to external stimuli. Structural studies of the histidine kinase transmembrane domain are challenging due to difficulties in protein expression and sample preparation. After carrying out expression screening of a series of histidine kinases, we investigated sample preparation methods for obtaining high quality samples of the periplasmic and transmembrane domain (PTD) of the bacterial histidine kinase SCO3062. Various sample conditions were tested for their ability to give homogeneous NMR spectra of the SCO3062 PTD with well-resolved resonances. Circular dichroism and 3D ¹⁵N-edited NOESY spectrum results demonstrate that the SCO3062 PTD is predominantly α-helical. This method should be applicable to the NMR analysis of other transmembrane proteins.     

Grb2-independent recruitment of Gab1 requires the C-terminal lobe and structural integrity of the Met receptor kinase domain

The Gab1 docking protein forms a platform for the assembly of a multiprotein signaling complex downstream from receptor tyrosine kinases. In general, recruitment of Gab1 occurs indirectly, via the adapter protein Grb2. In addition, Gab1 interacts with the Met/hepatocyte growth factor receptor in a Grb2-independent manner. This interaction requires a Met binding domain (MBD) in Gab1 and is essential for Met-mediated epithelial morphogenesis. The Gab1 MBD has been proposed to act as a phosphotyrosine binding domain that binds Tyr-1349 in the Met receptor. We show that a 16-amino acid motif within the Gab1 MBD is sufficient for interaction with the Met receptor, suggesting that it is unlikely that the Gab1 MBD forms a structured domain. Alternatively, the structural integrity of the Met receptor, and residues upstream of Tyr-1349 located in the C-terminal lobe of the kinase domain, are required for Grb2-independent interaction with the Gab1 MBD. Moreover, the substitution of Tyr-1349 with an acidic residue allows for the recruitment of the Gab1 MBD and for phosphorylation of Gab1. We propose that Gab1 and the Met receptor interact in a novel manner, such that the activated kinase domain of Met and the negative charge of phosphotyrosine 1349 engage the Gab1 MBD as an extended peptide ligand.