Regulation of cell differentiation by hNUDC via a Mpl-dependent mechanism in NIH 3T3 cells

Thrombopoietin receptor (Mpl) belongs to the cytokine receptor surperfamily with a large extracellular N-terminal portion responsible for cytokine recognition and binding. Thrombopoietin (TPO) has so far been the only widely studied cytokine for Mpl. However we have recently identified human NUDC (hNUDC), previously described as a human homolog of a fungal nuclear migration protein, as another putative binding partner of Mpl. The purpose of this study is to test the extent of the functioning of hNUDC by identifying protein-protein interactions with Mpl in mammalian cells. The full-length cDNAs encoding Mpl and hNUDC were cloned into pEGFP-N1 and pDsRed2-N1 respectively which were subsequently expressed as Mpl-EGFP (green) and hNUDC-DsRed (red) fusion proteins. Using ELISA and immunofluorescence studies, we have demonstrated the direct binding of hNUDC to cell surface-captured Mpl. We also observed that hNUDC induced significant changes in cellular morphology in NIH 3T3 cells stably transfected with pMpl-EGFP. Interestingly, these morphological changes were characteristic of cells undergoing megakaryocyte differentiation. Extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2) have been shown to mediate such megakaryocyte-like differentiation. In addition, co-expression of Mpl-EGFP and hNUDC-DsRed led to the release of hNUDC-DsRed into the culture medium.     

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.     

Thrombopoietin and hematopoietic stem cells

Thrombopoietin (TPO) is the cytokine that is chiefly responsible for megakaryocyte production but increasingly attention has turned to its role in maintaining hematopoietic stem cells (HSCs). HSCs are required to initiate the production of all mature hematopoietic cells, but this differentiation needs to be balanced against self-renewal and quiescence to maintain the stem cell pool throughout life. TPO has been shown to support HSC quiescence during adult hematopoiesis, with the loss of TPO signaling associated with bone marrow failure and thrombocytopenia. Recent studies have shown that constitutive activation mutations in Mpl contribute to myeloproliferative disease. In this review, we will discuss TPO signaling pathways, regulation of TPO levels and the role of TPO in normal hematopoiesis and during myeloproliferative disease.Key words: thrombopoietin, TPO, Mpl, hematopoietic stem cell, hematopoiesis, Jak2, MPLW515K, MPLW515L     

Mutational and Cysteine Scanning Analysis of the Glucagon Receptor N-terminal Domain

The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp⁶³, Arg¹¹⁶, and Lys⁹⁸ are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp⁶³ and Arg¹¹⁶ form a salt bridge, whereas Lys⁹⁸ is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.     

Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation

Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.     

Ligand Binding Pocket Formed by Evolutionarily Conserved Residues in the Glucagon-like Peptide-1 (GLP-1) Receptor Core Domain

Glucagon-like peptide-1 (GLP-1) plays a pivotal role in glucose homeostasis through its receptor GLP1R. Due to its multiple beneficial effects, GLP-1 has gained great attention for treatment of type 2 diabetes and obesity. However, little is known about the molecular mechanism underlying the interaction of GLP-1 with the heptahelical core domain of GLP1R conferring high affinity ligand binding and ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R, we determined that the evolutionarily conserved amino acid residue Arg³⁸⁰ flanked by hydrophobic Leu³⁷⁹ and Phe³⁸¹ in extracellular loop 3 (ECL3) may have an interaction with Asp⁹ and Gly⁴ of the GLP-1 peptide. The molecular modeling study showed that Ile¹⁹⁶ at transmembrane helix 2, Met²³³ at ECL1, and Asn³⁰² at ECL2 of GLP1R have contacts with His¹ and Thr⁷ of GLP-1. This study may shed light on the mechanism underlying high affinity interaction between the ligand and the binding pocket that is formed by these conserved residues in the GLP1R core domain.     

Thrombopoietin signal transduction: studies from cell lines and primary cells

Thrombopoietin (TPO) and its receptor Mpl support all of the developmental step necessary for megakaryocytopoiesis. In the past few years, the signaling pathways utilized by this member of the cytokine receptor family have been extensively studied, especially JAK/STAT, Ras/MAP kinase, Shc, and other adapter molecules. Many if not most of the secondary signaling pathways activated by thrombopoietin have also been identified upon binding of other hematopoietic growth factors to their cognate receptors, making the study of Mpl signaling representative of the field in general. However, identifying unique molecules or combinations of signals that direct megakaryocyte development has been an elusive goal and has led some investigators to conclude that there is little specificity during Mpl signal transduction. In this article we review the data regarding Mpl signaling with particular attention to the methods employed and critical interpretation of the data generated. Future studies will have to focus on primary bone marrow cells and intact animal models rather than transformed cell lines. Furthermore, it is likely that a comprehensive, integrative analysis of the many pathways activated by ligand binding will be necessary to understand the physiology of cytokine signaling.     

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.     

By Interacting with the C-terminal Phe of Apelin,Phe255 and Trp259 in Helix VI of the Apelin Receptor Are Critical for Internalization

Apelin is the endogenous ligand of the orphan seven-transmembrane domain (TM) G protein-coupled receptor APJ. Apelin is involved in the regulation of body fluid homeostasis and cardiovascular functions. We previously showed the importance of the C-terminal Phe of apelin 17 (K17F) in the hypotensive activity of this peptide. Here, we show either by deleting the Phe residue (K16P) or by substituting it by an Ala (K17A), that it plays a crucial role in apelin receptor internalization but not in apelin binding or in Gα_i-protein coupling. Then we built a homology three-dimensional model of the human apelin receptor using the cholecystokinin receptor-1 model as a template, and we subsequently docked K17F into the binding site. We visualized a hydrophobic cavity at the bottom of the binding pocket in which the C-terminal Phe of K17F was embedded by Trp¹⁵² in TMIV and Trp²⁵⁹ and Phe²⁵⁵ in TMVI. Using molecular modeling and site-directed mutagenesis studies, we further showed that Phe²⁵⁵ and Trp²⁵⁹ are key residues in triggering receptor internalization without playing a role in apelin binding or in Gα_i-protein coupling. These findings bring new insights into apelin receptor activation and show that Phe²⁵⁵ and Trp²⁵⁹, by interacting with the C-terminal Phe of the pyroglutamyl form of apelin 13 (pE13F) or K17F, are crucial for apelin receptor internalization.     

Subunit Symmetry at the Extracellular Domain-Transmembrane Domain Interface in Acetylcholine Receptor Channel Gating

Transmitter molecules bind to synaptic acetylcholine receptor channels (AChRs) to promote a global channel-opening conformational change. Although the detailed mechanism that links ligand binding and channel gating is uncertain, the energy changes caused by mutations appear to be more symmetrical between subunits in the transmembrane domain compared with the extracellular domain. The only covalent connection between these domains is the pre-M1 linker, a stretch of five amino acids that joins strand β10 with the M1 helix. In each subunit, this linker has a central Arg (Arg^3′), which only in the non-α-subunits is flanked by positively charged residues. Previous studies showed that mutations of Arg^3′ in the α-subunit alter the gating equilibrium constant and reduce channel expression. We recorded single-channel currents and estimated the gating rate and equilibrium constants of adult mouse AChRs with mutations at the pre-M1 linker and the nearby residue Glu⁴⁵ in non-α-subunits. In all subunits, mutations of Arg^3′ had similar effects as in the α-subunit. In the ϵ-subunit, mutations of the flanking residues and Glu⁴⁵ had only small effects, and there was no energy coupling between ϵGlu⁴⁵ and ϵArg^3′. The non-α-subunit Arg^3′ residues had Φ-values that were similar to those for the α-subunit. The results suggest that there is a general symmetry between the AChR subunits during gating isomerization in this linker and that the central Arg is involved in expression more so than gating. The energy transfer through the AChR during gating appears to mainly involve Glu⁴⁵, but only in the α-subunits.     

Thrombopoietin signal transduction requires functional JAK2, not TYK2

The Janus family of tyrosine kinases (JAKs) plays a critical role in signal transduction by members of the cytokine receptor superfamily. In response to ligand-receptor interaction, these nonreceptor tyrosine kinases are rapidly phosphorylated and activated, triggering tyrosine phosphorylation and activation of downstream signaling intermediates. Upon binding to its receptor, the product of the proto-oncogene c-mpl, thrombopoietin (TPO) activates both JAK2 and TYK2 in multiple cell lines as well as megakaryocytes and platelets. To study whether one or both of these kinases are essential for TPO signal transduction, we engineered a parental human sarcoma cell line (2C4) as well as sarcoma cell lines that are deficient in JAK2 expression (gamma2A) or TYK2 expression (U1A) to express the wild-type Mpl receptor. The ability of TPO to induce tyrosine phosphorylation of Mpl and multiple intracellular substrates in each cell line was then examined. Our results demonstrate that JAK2-deficient cells (gamma2A-Mpl) are unable to initiate TPO-mediated signaling. In contrast, cells that are TYK2-deficient (U1A-Mpl) are able to induce tyrosine phosphorylation of Mpl, JAK2, STAT3, and Shc as efficiently as parental cells (2C4-Mpl). These data indicate that JAK2 is an essential component of Mpl signaling and that, in the absence of JAK2, TYK2 is incapable of initiating TPO-induced tyrosine phosphorylation.     

Characterization of the Ligand Binding Functionality of the Extracellular Domain of Activin Receptor Type IIB

The single transmembrane domain serine/threonine kinase activin receptor type IIB (ActRIIB) has been proposed to bind key regulators of skeletal muscle mass development, including the ligands GDF-8 (myostatin) and GDF-11 (BMP-11). Here we provide a detailed kinetic characterization of ActRIIB binding to several low and high affinity ligands using a soluble activin receptor type IIB-Fc chimera (ActRIIB.Fc). We show that both GDF-8 and GDF-11 bind the extracellular domain of ActRIIB with affinities comparable with those of activin A, a known high affinity ActRIIB ligand, whereas BMP-2 and BMP-7 affinities for ActRIIB are at least 100-fold lower. Using site-directed mutagenesis, we demonstrate that ActRIIB binds GDF-11 and activin A in different ways such as, for example, substitutions in ActRIIB Leu⁷⁹ effectively abolish ActRIIB binding to activin A yet not to GDF-11. Native ActRIIB has four isoforms that differ in the length of the C-terminal portion of their extracellular domains. We demonstrate that the C terminus of the ActRIIB extracellular domain is crucial for maintaining biological activity of the ActRIIB.Fc receptor chimera. In addition, we show that glycosylation of ActRIIB is not required for binding to activin A or GDF-11. Together, our findings reveal binding specificity and activity determinants of the ActRIIB receptor that combine to effect specificity in the activation of distinct signaling pathways.     

Molecular Basis for Benzodiazepine Agonist Action at the Type 1 Cholecystokinin Receptor

Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu^7.39 that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a “trigger” for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu^7.39, whereas the role of this residue was less clear for chemically distinct agonists.     

Mutational Analysis Identifies Residues Crucial for Homodimerization of Myeloid Differentiation Factor 88 (MyD88) and for Its Function in Immune Cells

Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu¹⁸³, Ser²⁴⁴, and Arg²⁸⁸ impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88_151–189 and GFP-MyD88_168–189), comprising the Glu¹⁸³ residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.     

Thrombopoietin and hematopoietic stem cells

Thrombopoietin (TPO) is the cytokine that is chiefly responsible for megakaryocyte production but increasingly attention has turned to its role in maintaining hematopoietic stem cells (HSCs). HSCs are required to initiate the production of all mature hematopoietic cells, but this differentiation needs to be balanced against self-renewal and quiescence to maintain the stem cell pool throughout life. TPO has been shown to support HSC quiescence during adult hematopoiesis, with the loss of TPO signaling associated with bone marrow failure and thrombocytopenia. Recent studies have shown that constitutive activation mutations in Mpl contribute to myeloproliferative disease. In this review, we will discuss TPO signaling pathways, regulation of TPO levels and the role of TPO in normal hematopoiesis and during myeloproliferative disease.     

Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1)

Discoidin domain receptor 1 (DDR1) belongs to a unique family of receptor tyrosine kinases that signal in response to collagens. DDR1 undergoes autophosphorylation in response to collagen binding with a slow and sustained kinetics that is unique among members of the receptor tyrosine kinase family. DDR1 dimerization precedes receptor activation suggesting a structural inhibitory mechanism to prevent unwarranted phosphorylation. However, the mechanism(s) that maintains the autoinhibitory state of the DDR1 dimers is unknown. Here, we report that N-glycosylation at the Asn²¹¹ residue plays a unique role in the control of DDR1 dimerization and autophosphorylation. Using site-directed mutagenesis, we found that mutations that disrupt the conserved ²¹¹NDS N-glycosylation motif, but not other N-glycosylation sites (Asn²⁶⁰, Asn³⁷¹, and Asn³⁹⁴), result in collagen I-independent constitutive phosphorylation. Mass spectrometry revealed that the N211Q mutant undergoes phosphorylation at Tyr⁴⁸⁴, Tyr⁵²⁰, Tyr⁷⁹², and Tyr⁷⁹⁷. The N211Q traffics to the cell surface, and its ectodomain displays collagen I binding with an affinity similar to that of the wild-type DDR1 ectodomain. However, unlike the wild-type receptor, the N211Q mutant exhibits enhanced receptor dimerization and sustained activation upon ligand withdrawal. Taken together, these data suggest that N-glycosylation at the highly conserved ²¹¹NDS motif evolved to act as a negative repressor of DDR1 phosphorylation in the absence of ligand. The presence of glycan moieties at that site may help to lock the collagen-binding domain in the inactive state and prevent unwarranted signaling by receptor dimers. These studies provide a novel insight into the structural mechanisms that regulate DDR activation.     

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.     

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.     

Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway.

Thrombopoietin (TPO) is the major regulator of both growth and differentiation of megakaryocytes. We previously showed that both functions can be generated by TPO in the megakaryoblastic cell line UT7, in which murine Mpl was introduced, and are independently controlled by distinct regions of the cytoplasmic domain of Mpl. Particularly, residues 71 to 94 of this domain (deleted in the mutant mpl delta3) were found to be required for megakaryocytic maturation but dispensable for proliferation. We show here that TPO-induced differentiation in UT7 cells is tightly dependent on a strong, long-lasting activation of the mitogen-activated protein kinase (MAPK) pathway. Indeed, (i) in UT7-mpl cells, TPO induced a strong activation of extracellular signal-regulated kinases (ERK) which was persistent until at least 4 days in TPO-containing medium; (ii) a specific MAPK kinase (MEK) inhibitor inhibited TPO-induced megakaryocytic gene expression; (iii) the Mpl mutant mpl delta3, which displayed no maturation activity, transduced only a weak and transient ERK activation in UT7 cells; and (iv) TPO-induced megakaryocytic differentiation in UT7-mpl delta3 cells was partially restored by expression of a constitutively activated mutant of MEK. The capacity of TPO to trigger a strong and prolonged MAPK signal depended on the cell in which Mpl was introduced. In BAF3-mpl cells, TPO triggered a weak and transient ERK activation, similar to that induced in UT7-mpl delta3 cells. In these cells, no difference in MAPK activation was found between normal Mpl and mpl delta3. Thus, depending on the cellular context, several distinct regions of the cytoplasmic domain of Mpl and signaling pathways may contribute to generate quantitative variations in MAPK activation.     

A structure-function analysis of serine/threonine phosphorylation of the thrombopoietin receptor, c-Mpl

Thrombopoietin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Numerous studies have shown that TPO binding leads to JAK2 kinase activation and Tyr phosphorylation of c-Mpl and several intracellular signaling intermediates, events vital for the biological activity of the hormone. In contrast, virtually nothing is known of the role of Ser or Thr phosphorylation of c-Mpl. By using phosphoamino acid analysis we found that Ser residues of c-Mpl were constitutively phosphorylated in receptor-bearing cells, levels that were increased following exposure of cells to TPO. To identify which residues were modified, and to determine the functional consequences of their phosphorylation, we generated a series of Ser to Ala mutations of a truncated c-Mpl receptor (T69) capable of supporting TPO-induced cell growth. Of the eight Ser within T69 we found that at least four are phosphorylated in TPO-stimulated cells. The mutation of each of these residues alone had minimal effects on TPO-induced proliferation, but substitution of all of the phosphoserine residues with Ala reduced the capacity of the receptor to support cell growth by over 50%. Additionally, the Ser at cytoplasmic position 18 is not detectably phosphorylated. However, the mutation of Ser-18 to Ala nearly abrogates TPO-induced proliferation and co-precipitation of JAK2 with Mpl. This study provides the first systematic analysis of the role of Ser residues in c-Mpl signaling.