Effects of clinically relevant MPL mutations in the transmembrane domain revealed at the atomic level through computational modeling

Background

Mutations in the thrombopoietin receptor (MPL) may activate relevant pathways and lead to chronic myeloproliferative neoplasms (MPNs). The mechanisms of MPL activation remain elusive because of a lack of experimental structures. Modern computational biology techniques were utilized to explore the mechanisms of MPL protein activation due to various mutations.

Results

Transmembrane (TM) domain predictions, homology modeling, ab initio protein structure prediction, and molecular dynamics (MD) simulations were used to build structural dynamic models of wild-type and four clinically observed mutants of MPL. The simulation results suggest that S505 and W515 are important in keeping the TM domain in its correct position within the membrane. Mutations at either of these two positions cause movement of the TM domain, altering the conformation of the nearby intracellular domain in unexpected ways, and may cause the unwanted constitutive activation of MPL''s kinase partner, JAK2.

Conclusions

Our findings represent the first full-scale molecular dynamics simulations of the wild-type and clinically observed mutants of the MPL protein, a critical element of the MPL-JAK2-STAT signaling pathway. In contrast to usual explanations for the activation mechanism that are based on the relative translational movement between rigid domains of MPL, our results suggest that mutations within the TM region could result in conformational changes including tilt and rotation (azimuthal) angles along the membrane axis. Such changes may significantly alter the conformation of the adjacent and intrinsically flexible intracellular domain. Hence, caution should be exercised when interpreting experimental evidence based on rigid models of cytokine receptors or similar systems.     

Compound heterozygosity for two different amino-acid substitution mutations in the thrombopoietin receptor (c-mpl gene) in congenital amegakaryocytic thrombocytopenia (CAMT)

Congenital amegakaryocytic thrombocytopenia (CAMT) without physical anomalies is a rare disease, presenting isolated thrombocytopenia and megakaryocytopenia in infancy, which can evolve into aplastic anemia and leukemia. Recently, two heterozygous truncating mutations of the thrombopoietin (TPO) receptor MPL, coded by the c-mpl gene, were identified in a 10-year-old Japanese patient with CAMT transmitted in an autosomal recessive manner. Here, we report for the first time two different MPL amino-acid substitutions in a 2-year-old Italian boy with CAMT and compound heterozygosis for two (c-mpl point mutations. C-to-T transitions were detected on exons 5 and 12 at the 769 and 1904 cDNA nucleotide positions, respectively. The mutation in exon 5 substitutes an arginine with a cysteine (R257C) in the extracellular domain, 11 amino acids distant from the WSXWS motif conserved in the cytokine-receptor superfamily. The mutation in exon 12 substitutes a proline with a leucine (P635L) in the last amino acid of the C-terminal intracellular domain, responsible for signal transduction. As in the Japanese family, the mutations were both transmitted from the parents. TPO plasma levels were highly increased in the patient. The patient's 7-year-old brother, who was a candidate donor for allografting, turned out to be an asymptomatic heterozygous carrier of P635L and showed defective megakaryocyte colony formation from bone-marrow progenitor cells. The present study provides important confirmation that CAMT can be associated with (c-mpl) mutations.     

A microtubule associated protein (hNUDC) binds to the extracellular domain of thrombopoietin receptor (Mpl)

Human NUDC (hNUDC) was initially characterized as a nuclear migration protein based on the similarity of its C-terminus to that of fungal NUDC from Aspergillus nidulans. However, hNUDC is a 331 amino acid protein whereas fungal NUDC is 198 amino acids in length. The extra N-terminal portion of hNUDC has no known function or homology to other proteins. In this study, we report the binding of hNUDC to the extracellular domain of the thrombopoietin receptor (Mpl) as detected by the yeast two-hybrid system, GST pull-down, and co-immunoprecipitation. Our deletion analysis demonstrated that amino acids between positions 100 and 238 as the critical domain mediating the hNUDC and Mpl interactions as detected by the two-hybrid system and GST pull-down assay. Immunofluorescence staining of human megakaryocyte cells indicated that hNUDC and Mpl colocalized at all stages of megakaryocyte development. Substantial colocalization of hNUDC with microtubules was also detected around nuclei and elongated microtubular structures, especially in proplatelet extensions.     

Expression, purification and characterization of soluble human thrombopoietin receptor from Escherichia coli

The extracellular domain (edMpl) of human thrombopoietin (TPO) receptor, c-Mpl was expressed in Escherichia coli by changing some nucleotides before and after the translation initiation codon. The mutations increased the expression by approx. 15-fold. The inclusion bodies were solubilized in 8 M guanidine-HCl under reducing conditions and refolded using a glutathione-redox system. The monomeric form of edMpl was purified to near homogeneity by two successive steps of ion-exchange chromatography using DEAE-Sephacel and Mono Q columns. The purified monomeric edMpl inhibited the TPO-dependent cell proliferation, suggesting that it was binding to TPO. Also, antisera raised against the edMpl bound specifically to the soluble receptor secreted by mammalian cells.     

Point mutations within a dimer interface homology domain of c-Mpl induce constitutive receptor activity and tumorigenicity.

c-Mpl, a receptor for thrombopoietin (TPO), belongs to the haemopoietin/cytokine receptor superfamily, a group of cell surface molecules characterized by conserved sequence motifs within their ligand binding domains. A recurring mechanism for the activation of haemopoietin receptors is the formation of functional complexes by receptor subunit oligomerization. Within the growth hormone receptor, a cluster of extracellular amino acids forms a dimer interface domain that stabilizes ligand-induced homodimers. This domain appears to be functionally conserved in the erythropoietin (EPO) receptor because substitution of cysteines for residues in the analogous region causes EPO-independent receptor activation via disulfide-linked homodimerization. This report identifies an homologous domain within the c-Mpl receptor. The substitution of cysteine residues for specific amino acids in the dimer interface homology regions of c-Mpl induced constitutive receptor activity. Factor-dependent FDC-P1 and Ba/F3 cells expressing the active receptor mutants no longer required exogenous factors and proliferated autonomously. The results imply that the normal process of TPO-stimulated Mpl activation occurs through receptor homodimerization and is mediated by a conserved haemopoietin receptor dimer interface domain. Moreover, cells expressing activated mutant Mpl receptors were tumorigenic in transplanted mice. Thus, like v-mpl, its viral counterpart, mutated forms of the cellular mpl gene also have oncogenic potential.     

Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche

Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated beta1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche.     

Establishment of a novel mesenchymal stem cell-based regimen for chronic myeloid leukemia differentiation therapy

Chronic myeloid leukemia (CML) is characterized by the accumulation of malignant and immature white blood cells which spread to the peripheral blood and other tissues/organs. Despite the fact that current tyrosine kinase inhibitors (TKIs) are capable of achieving the complete remission by reducing the tumor burden, severe adverse effects often occur in CML patients treated with TKIs. The differentiation therapy exhibits therapeutic potential to improve cure rates in leukemia, as evidenced by the striking success of all-trans-retinoic acid in acute promyelocytic leukemia treatment. However, there is still a lack of efficient differentiation therapy strategy in CML. Here we showed that MPL, which encodes the thrombopoietin receptor driving the development of hematopoietic stem/progenitor cells, decreased along with the progression of CML. We first elucidated that MPL signaling blockade impeded the megakaryocytic differentiation and contributed to the progression of CML. While allogeneic human umbilical cord-derived mesenchymal stem cells (UC-MSCs) treatment efficiently promoted megakaryocytic lineage differentiation of CML cells through restoring the MPL expression and activating MPL signaling. UC-MSCs in combination with eltrombopag, a non-peptide MPL agonist, further activated JAK/STAT and MAPK signaling pathways through MPL and exerted a synergetic effect on enhancing CML cell differentiation. The established combinational treatment not only markedly reduced the CML burden but also significantly eliminated CML cells in a xenograft CML model. We provided a new molecular insight of thrombopoietin (TPO) and MPL signaling in MSCs-mediated megakaryocytic differentiation of CML cells. Furthermore, a novel anti-CML treatment regimen that uses the combination of UC-MSCs and eltrombopag shows therapeutic potential to overcome the differentiation blockade in CML.Subject terms: Chronic myeloid leukaemia, Mesenchymal stem cells     

Expression and characterization of the extracellular domain of guanylyl cyclase C from a baculovirus and Sf21 insect cells

Guanylyl cyclase (GC)-C, a single-transmembrane receptor protein for heat-stable enterotoxin, guanylin, and uroguanylin, and its N-terminal extracellular domain were prepared at a high level of expression from a system constructed of Sf21 insect cells and recombinant baculovirus. The recombinant GC-C, containing the complete sequence, retained its binding affinity to heat-stable enterotoxin with a KD value (6.2 x 10(-10) M) and cyclase catalytic activity at a level similar to those of GC-C expressed in mammalian cell lines, such as COS-7. The N-terminal extracellular domain was prepared in a form which contained the hexahistidine tail at its C-terminus and was purified as a homogenous protein by Con A and Ni-chelating affinity chromatography from the culture medium of the insect cells. The purified N-terminal extracellular domain of GC-C exhibited the high (KD = 4 x 10(-10) M) and low (KD = 7 x 10(-8) M) affinity sites in binding to heat-stable enterotoxin. These results clearly indicate that the N-terminal extracellular domain of GC-C possesses the same biochemical characteristics as the complete GC-C protein even in the membrane-free form. Moreover, the extracellular domain is able to form an oligomer in a ligand-dependent manner, suggesting that the N-terminal extracellular domains interact with one another in binding to ligands.     

Macrocyclization in the design of a conformationally constrained Grb2 SH2 domain inhibitor.

Grubbs' olefin metathesis reaction was utilized to prepare a macrocyclic variant of a linear Grb2 SH2 domain antagonist in an attempt to induce a beta-bend conformation known to be required for high affinity binding. In extracellular Grb2 SH2 domain binding assays, the macrocyclic analogue exhibited an approximate 100-fold enhancement in binding potency relative to its linear counterpart. The macrocycle was not as effective in whole cell binding assays as would be expected based on its extracellular binding potency.     

Constitutive activation of a variant of the env-mpl oncogene product by disulfide-linked homodimerization.

The myeloproliferative leukemia retrovirus (MPLV) has the v-mpl cellular sequences transduced in frame with the deleted and rearranged Friend murine leukemia virus env gene. The resulting env-mpl fusion oncogene is responsible for an acute myeloproliferative disorder induced in mice by MPLV. v-mpl is a truncated form of the c-mpl gene which encodes the receptor for thrombopoietin. We investigated the contribution of the Env-Mpl extracellular domain in the constitutive activation of this truncated cytokine receptor and found that the rearrangement of the env sequences in the env-mpl fusion gene was not required for oncogenicity. A pathogenic variant, DEL3MPLV, was generated, which differs from MPLV by the deletions of 22 amino acids of the Env signal peptide, all of the mature Env sequences, and 18 N-terminal amino acids of the v-Mpl extracellular domain. The resulting del3-mpl oncogene product conserves in its extracellular region the first 12 amino acids of the Env signal sequence including a cysteine residue, and 25 amino acids of the v-Mpl. We show here that a mutation converting this cysteine to a glycine completely abolishes del3-mpl oncogenicity and that the del3-mpl oncogene product is constitutively activated by disulfide-linked homodimerization.     

人血小板生成素受体c—MPL膜内部分的聚合作用

血小板生成素（ＴＰＯ）是调节血小板生成最主要的细胞因子,其生物学效应由其受体ｃ－ＭＰＬ介导。利用酵母双杂合系统研究ｃ－ＭＰＬ膜内部分在ＴＰＯ信号转导途径中的功能。首先用反转录ＰＣＲ（ＲＴ－ＰＣＲ）方法从人红白血病ＨＥＬ细胞系总ＲＮＡ中扩增并克隆Ｐ型ｃ－ＭＰＬ（ＭＰＬＰ）膜内部分ｃＤＮＡ,经测序验证后克隆至双杂合载体ｐＡＳ２和ｐＧＡＤ４２４中,重组质粒命名为ｐＡＳＭＭ和ｐＧＡＤＭＭ。将ｐＡＳＭＭ与ｐ     

Identification and characterization of an alternative splice variant of Mpl with a high affinity for TPO and its activation of ERK1/2 signaling

The thrombopoietin receptor is a crucial element in thrombopoietin-initiated signaling pathways, which stimulates the differentiation of normal hematopoietic progenitor cells, the maturation of megakaryocytes, and the generation of platelets. In this study, we identified a novel activating variant of thrombopoietin receptor, termed Mpl-D, in human megakaryoblastic leukemia Dami cells and demonstrated that the binding affinity of the Mpl-D receptor for thrombopoietin is enhanced. Cell cycle analysis revealed that in the presence of thrombopoietin, most Mpl-D expressing NIH3T3 (NIH3T3/Mpl-D) cells were prevalent in G1 phase while the S and G2/M populations were less frequently observed. Unexpectedly, thrombopoietin induced strong and prolonged ERK1/2 signaling in NIH3T3/Mpl-D cells compared with its receptor wild-type expressing NIH3T3 (NIH3T3/Mpl-F) cells. Further analysis of the mRNA levels of cyclin D1/D2 in NIH3T3/Mpl-D cells demonstrated markedly down-regulated expression compared to NIH3T3/Mpl-F cells in the presence of thrombopoietin. Thus, the prolonged activation of ERK1/2 by Mpl-D might lead to G1 cell cycle arrest through a profound reduction of cyclin D1/D2 in order to support cell survival without proliferation. We also provided tertiary structural basis for the Mpl-D and thrombopoietin interaction, which might provide insights into how Mpl-D effectively increases binding to thrombopoietin and significantly contributes to its specific signaling pathway. These results suggest a new paradigm for the regulation of cytokine receptor expression and function through the alternative splicing variant of Mpl in Dami cells, which may play a role in the pathogenesis of megakaryoblastic leukemia.     

Atrial natriuretic factor-receptor guanylate cyclase signal transduction mechanism

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC), like the other members of the membrane guanylate cyclase family, is a single transmembrane-spanning protein. The transmembrane domain separates the protein into two regions, extracellular and intracellular. The extracellular region contains the ANF-binding domain and the intracellular region the catalytic domain located at the C-terminus of the protein. Preceding the catalytic domain, the intracellular region is comprised of the following functional domains: juxtaposed 40 amino acids to the transmembrane domain is the ATP-regulated module (ARM) domain [also termed the kinase homology domain (KHD)], and the putative dimerization domain. The ANF-RGC signaling is initiated by hormone, ANF, binding to its extracellular binding site. The binding signal is transduced through the transmembrane domain to the intracellular portion where ATP binding to the ARM domain partially activates the cyclase and prepares it for subsequent steps involving phosphorylation and attaining the fully activated state. This chapter reviews the signaling modules of ANF-RGC.     

The thrombopoietin/MPL pathway in hematopoiesis and leukemogenesis

Hematopoietic stem cells (HSC) comprise a small percentage of total hematopoietic cells. Their ability to self-renewal is key to the continuous replenishment of the hematopoietic system with newly formed functional blood cell types while maintaining their multipotential capacity. Understanding the extrinsic signals that are essential to HSC maintenance will provide insights into the regulation of hematopoiesis at its most primitive stage, and with the knowledge applied, will potentially lead to improved clinical transplantation outcomes. In this review, we will summarize the current understanding of the role of the thrombopoietin/MPL signaling pathway in HSC maintenance during adult and fetal hematopoiesis. We will also speculate on the downstream key players in the pathway based on published data, and summarize the role of this pathway in leukemia.     

His499 Regulates Dimerization and Prevents Oncogenic Activation by Asparagine Mutations of the Human Thrombopoietin Receptor

Ligand binding to the extracellular domain of the thrombopoietin receptor (TpoR) imparts a specific orientation on the transmembrane (TM) and intracellular domains of the receptors that is required for physiologic activation via receptor dimerization. To map the inactive and active dimeric orientations of the TM helices, we performed asparagine (Asn)-scanning mutagenesis of the TM domains of the murine and human TpoR. Substitution of Asn at only one position (S505N) activated the human receptor, whereas Asn substitutions at several positions activated the murine receptor. Second site mutational studies indicate that His⁴⁹⁹ near the N terminus of the TM domain is responsible for protecting the human receptor from activation by Asn mutations. Structural studies reveal that the sequence preceding His⁴⁹⁹ is helical in the murine receptor but non-helical in peptides corresponding to the TM domain of the inactive human receptor. The activating S505N mutation and the small molecule agonist eltrombopag both induce helix in this region of the TM domain and are associated with dimerization and activation of the human receptor. Thus, His⁴⁹⁹ regulates the activation of human TpoR and provides additional protection against activating mutations, such as oncogenic Asn mutations in the TM domain.     

Double antenna structure of chicken prolactin receptor deduced from the cDNA sequence.

Chicken prolactin receptor (cPRLR) deciphered from the cDNA sequence showed a unique double antenna structure in its extracellular domain. The predicted cPRLR preprotein was composed of 831 amino acids and contained a signal peptide and a transmembrane region. The extracellular domain comprised 438 residues, and was divided into two tandemly repeated, highly homologous units, each of which corresponded to the extracellular domains of mammalian prolactin receptors. Both extracellular units of cPRLR possessed two structural features characteristic of the ligand binding units of cytokine/prolactin receptor family, namely two pairs of cysteine residues and a WSXWS motif. These findings strongly suggest that cPRLR contains two repeated ligand binding units, that is a double antenna structure. The cPRLR gene is expressed in a wide range of tissues of laying hen.     

The human growth hormone receptor. Secretion from Escherichia coli and disulfide bonding pattern of the extracellular binding domain

A gene fragment encoding the extracellular domain of the human growth hormone (hGH) receptor from liver was cloned into a plasmid under control of the Escherichia coli alkaline phosphatase promoter and the heat-stable enterotoxin (StII) signal peptide sequence. Strains of E. coli expressing properly folded hGH binding protein were identified by blotting colonies with 125I-hGH. The E. coli strain capable of highest expression (KS330) secreted 10 to 20 mg/liter of culture of properly processed and folded hGH receptor fragment into the periplasmic space. The protein was purified to near homogeneity in 70 to 80% yield (in tens of milligram amounts) using ammonium sulfate precipitation, hGH affinity chromatography, and gel filtration. The unglycosylated extracellular domain of the hGH receptor has virtually identical binding properties compared to its natural glycosylated counterpart isolated from human serum, suggesting glycosylation is not important for binding of hGH. The extracellular binding domain codes for 7 cysteines, and we show that six of them form three disulfide bonds. Peptide mapping studies show these disulfides are paired sequentially to produce short loops (10-15 residues long) as follows: Cys38-Cys48, Cys83-Cys94, and Cys108-Cys122. Cys241 is unpaired, and mutagenic analysis shows that the extreme carboxyl end of the receptor fragment (including Cys241) is not essential for folding or binding of the protein to hGH. High level expression of this receptor binding domain and its homologs in E. coli will greatly facilitate their detailed biophysical and structural analysis.     

Conserved high affinity ligand binding and membrane association in the native and refolded extracellular domain of the human glycine receptor alpha1-subunit

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel composed of ligand binding alpha- and gephyrin anchoring beta-subunits. To identify the secondary and quaternary structures of extramembraneous receptor domains, the N-terminal extracellular domain (alpha1-(1-219)) and the large intracellular TM3-4 loop (alpha1-(309-392)) of the human GlyR alpha1-subunit were individually expressed in HEK293 cells and in Escherichia coli. The extracellular domain obtained from E. coli expression was purified in its denatured form and refolding conditions were established. Circular dichroism and Fourier-transform-infrared spectroscopy suggested approximately 25% alpha-helix and approximately 48% beta-sheet for the extracellular domain, while no alpha-helices were detectable for the TM3-4 loop. Size exclusion chromatography and sucrose density centrifugation indicated that isolated glycine receptor domains assembled into multimers of distinct molecular weight. For the extracellular domain from E. coli, we found an apparent molecular weight compatible with a 15mer by gel filtration. The N-terminal domain from HEK293 cells, analyzed by sucrose gradient centrifugation, showed a bimodal distribution, suggesting oligomerization of approximately 5 and 15 subunits. Likewise, for the intracellular domain from E. coli, a single molecular mass peak of approximately 49 kDa indicated oligomerization in a defined native structure. As shown by [(3)H]strychnine binding, expression in HEK293 cells and refolding of the isolated extracellular domain reconstituted high affinity antagonist binding. Cell fractionation, alkaline extraction experiments, and immunocytochemistry showed a tight plasma membrane association of the isolated GlyR N-terminal protein. These findings indicate that distinct functional characteristics of the full-length GlyR are retained in the isolated N-terminal domain.