TPO Receptor David J. Kuter* and Junzhi Li Hematology Unit, COX 640, Massachusetts General Hospital, 100 Blossom Street, Boston, MA 02114, USA * corresponding author tel: 617-726-8743, fax: 617-724-3166, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.20008.
SUMMARY The thrombopoietin receptor, c-mpl, is a member of the hematopoietin/cytokine receptor superfamily and consists of two duplicated hematopoietin receptor domains (HRD). The proximal HRD is responsible for receptor dimerization and consequent signal transduction and the distal HRD may regulate its action. The thrombopoietin receptor is found on many hematopoietic progenitors but is primarily found on megakaryocytes and platelets. The platelet receptor is of high binding affinity (120±200 pM) but low surface density (30±60 sites per cell). Receptor binding by thrombopoietin results in the promotion of cell growth/cellular differentiation, the prevention of apoptosis, and the internalization and degradation of the receptor±ligand complex. Loss of the distal HRD by fusion with the murine leukemia virus env protein results in unregulated expression of c-mpl and a myeloproliferative syndrome in mice. Mice deficient in the thrombopoietin receptor produce 10±15% as many megakaryocytes and platelets as wild-type mice but also have 25±35% the normal number of erythroid and myeloid progenitors despite normal peripheral red and white blood cell counts. No human abnormalities of c-mpl have yet been described and therapeutic use of the soluble receptor has not yet been demonstrated.
BACKGROUND
Discovery In 1986 Wendling et al. reported that a murine retrovirus, the myeloproliferative leukemia virus
(MPLV), caused an acute hematological disorder in most mice strains that was characterized by multilineage proliferation and differentiation. Cells in this disease became growth factor-independent and showed autonomous growth in vitro. By sequencing the DNA encoding the envelope gene of the MPLV a novel oncogene called v-mpl was found that was in phase with two parts of the Friend murine leukemia virus envelope gene. v-mpl showed close structural analogies with the hematopoietin receptor superfamily but was not related to any known receptor. In infected cells the env-mpl fusion gene produced a truncated transmembrane receptor for a then unidentified hematopoietic growth factor (Souryi et al., 1990). When the full-length cellular homolog, c-mpl, was cloned in 1992 (Vigon et al., 1992) it was found to be a novel hematopoietin receptor that was present in a large number of hematopoietic cells and most megakaryocytic cell lines (Vigon et al., 1993b,c). Subsequently it was shown that c-mpl was present in purified populations of platelets and megakaryocytes and that antisense oligonucleotides against c-mpl inhibited megakaryocyte growth (Methia et al., 1993). This close association between c-mpl and megakaryocyte growth led to the use of recombinant c-Mpl protein on an affinity column to purify its ligand (Bartley et al., 1994; de Sauvage et al., 1994; Kato et al., 1995). It is now known that the c-Mpl ligand is indeed the hematopoietic growth factor long called thrombopoietin (Kelemen et al., 1958). Final proof of this association was provided by experiments in mice in which the c-mpl genes were eliminated; such animals had less than 10% of the normal number of platelets and megakaryocytes (Gurney et al., 1994).
1954 David J. Kuter and Junzhi Li
Alternative names The terms thrombopoietin (TPO) receptor (TPOR), c-Mpl, c-Mpl receptor, Mpl, and Mpl receptor are used interchangeably.
Structure The TPO receptor is a member of the hematopoietin/ cytokine receptor superfamily, which is characterized by a highly conserved HRD in the extracellular region (Vigon et al., 1992; Drachman and Kaushansky, 1995). Although the HRD of different members of this family shows only about 20±30% amino acid sequence homology, each HRD contains two fibronectin type III (FNIII)-like domains, each of which consists of seven strands. Two to four amino acid residues link the two FNIII-like domains. The Nterminus of the first FNIII-like domain contains four highly conserved cysteine residues which form two disulfide bonds between strands A/B and C 0 /E, respectively. Substitution or deletion of any of these four cysteine residues abolishes receptor binding of the ligand, presumably due to disruption of proper conformation at the ligand binding site. At the C-terminus of the second FNIII-like domain, a 5 amino acid sequence, termed the WSXWS motif, is highly conserved in the hematopoietin/cytokine receptor superfamily. Although the WSXWS motif is located away from the ligand binding site in the three-dimensional structural model, mutation of this motif also results in inactivation of ligand binding. Along with a few other members of the hematopoietin/cytokine receptor superfamily, like the leukemia-inhibitory factor receptor and the chain of the IL-3 receptor, the TPO receptor has two duplicated HRDs (referred to hereafter as the membraneproximal HRD and the membrane-distal HRD). In addition, the TPO receptor is unique in having a 50 amino acid insertion between strands C 0 and E in the second FNIII-like domain of the membranedistal HRD. Structure±function analysis has demonstrated that the membrane proximal HRD is responsible for the dimerization and consequent signal transduction that occurs upon ligand binding (Alexander et al., 1995). Whether both proximal and distal HRD can bind ligand is unclear. Recent data (Sabath et al., 1999) using mutants of c-mpl suggest that the distal HRD binds thrombopoietin and is responsible for thrombopoietin-dependent growth. Furthermore, in the absence of ligand the distal HRD seems to inhibit receptor activation; cells transfected with a c-mpl construct lacking the distal HRD displayed
thrombopoietin-independent growth, just like v-mpl, and did not bind thrombopoietin. Four isoforms of the TPO receptor mRNA have been identified by cDNA sequencing; all of them arise from alternative splicing of c-mpl RNA (Vigon et al., 1992; Kiladjian et al., 1997; Li et al., 2000). Of these, the P isoform is the only one demonstrated to be a functional receptor. After cleavage of the signal peptide, the mature wild-type TPO receptor (P form) is composed of an extracellular domain of 463 amino acids, a transmembrane domain of 22 amino acids, and intracellular domain of 122 amino acids. The biological and/or physiological role of the other three alternative RNA splicing isoforms is completely unclear. The K isoform lacks much of the cytoplasmic domain and presumably would lack the ability to initiate signal transduction pathways. A third RNA isoform lacks the transmembrane region and would be predicted to be a soluble receptor (Skoda et al., 1993). The existence of the protein products from these two RNA isoforms has not been demonstrated in vivo. The fourth, called c-mpl-del, lacks 72 bp in the extracellular region of c-mpl and arises as a consequence of alternative RNA splicing between exons 8 and 9 (Kiladjian et al., 1997; Li et al., 2000). c-mpl-del mRNA and protein are expressed in platelets, megakaryocytes, and CD34 progenitor cells but the protein does not seem to be transported to the cell surface and hence it lacks biological activity.
Main activities and pathophysiological roles Like all other members of the hematopoietin/cytokine receptor superfamily, the extracellular domain of c-Mpl must provide two essential functions in order to permit TPO signal transduction: a high-affinity binding site for TPO and a dimerization site upon ligand binding. As a model system for analyzing the TPO receptor, platelets have a single class of binding sites (approximately 50±60 per platelet) for TPO with binding affinity (Kd) of 100±200 pM (Li et al., 1996, 1999b; Broudy et al., 1997; Fielder et al., 1997). Receptor binding initiates at least three major events in cells. The first is initiation of signal transduction pathways that promote cell growth and/or cellular differentiation. The second is the activation of signal transduction pathways that prevent apoptosis. The third is internalization and degradation of the receptor±ligand complex for regulating the circulating TPO level (Kuter and Rosenberg, 1995; Fielder et al., 1996; Kuter, 1996a; Broudy et al., 1997; Fielder et al., 1997; Li et al., 1999).
TPO Receptor 1955 The consequences of receptor binding vary with the cell type. For megakaryocyte colony-forming cells (Meg-CFC), receptor activation inhibits apoptosis and increases the rate of mitosis. For early megakaryocytes, receptor binding inhibits apoptosis, stimulates megakaryocyte endomitosis, and promotes cytoplasmic maturation (Kuter, 1996a, 1997). This results in an increase in the number, size, and ploidy of bone marrow megakaryocytes. For late, mature megakaryocytes receptor binding may actually inhibit platelet shedding (Choi et al., 1996; Nagahisi et al., 1996; Sheridan et al., 1997). For platelets, receptor binding produces a decreased threshold for platelet activation (Chen et al., 1995; Harker et al., 1996; Kubota et al., 1996; Montrucchio et al., 1996). For platelets and possibly megakaryocytes, receptor binding results in internalization and degradation of thrombopoietin. This is the major mechanism by which circulating thrombopoietin levels are regulated (Kuter, 1996a,b, 1997; Li et al., 1999b). For other early, nonmegakaryocyte bone marrow progenitors and the pluripotential stem cell, receptor activation prevents apoptosis and modestly increases the progenitor number. This pluripotential effect is most strikingly illustrated in mice in which c-mpl has been eliminated. Although the circulating red and white blood cell numbers were not altered, the number of erythroid and myeloid progenitor cells was reduced to 20±30% of normal (Carver-Moore et al., 1996). Finally, there is no TPO effect (and probably no TPO receptor) on the more mature nonmegakaryocyte precursors in the bone marrow. At the present time there are no human disorders directly attributable to changes in c-Mpl structure or expression. The initial description of the murine v-mpl-env fusion protein in the MPLV-infected mice was associated with a myeloproliferative syndrome in which all lineages were stimulated. Deletion of all but the 43 most membrane proximal amino acids of the extracellular domain was responsible for this oncogenic effect. The structural basis for this oncogene activation has recently been explored (Sabath et al., 1999). The intact c-mpl cDNA was compared with a c-mpl construct lacking the distal HRD. Upon transfection of the wild-type c-mpl cDNA into Baf3 cells, the cells exhibited thrombopoietin-dependent growth and bound thrombopoietin. However, the cells transfected with the c-mpl construct lacking the distal HRD displayed thrombopoietin-independent growth, just like v-mpl, and did not bind thrombopoietin. These results suggest that the distal HRD imposes some inhibitory effect on the proximal HRD, binds thrombopoietin, and is responsible for thrombopoietin dependence; absence of the distal HRD confers thrombopoietin-independent growth.
In polycythemia vera (Moliterno et al., 1998) and in essential thrombocythemia (Li et al., 1996) the amount of thrombopoietin receptor on platelets seems to be reduced. Whether this plays any pathophysiological role is unclear. Platelets from patients with polycythemia vera failed to undergo tyrosine phosphorylation upon exposure to thrombopoietin and had reduced levels of c-Mpl on their surface. Platelets from patients with essential thrombocythemia had a normal binding affinity for thrombopoietin but had only 5.6 receptors/platelet, compared with the 56 receptors/platelet found on normal platelets (Li et al., 1996). The thrombopoietin receptor is found on cells and cell lines from many nonlymphoid hematological malignancies but not on cells from purely lymphoid malignancies or nonhematopoietic solid tumors (Columbyova et al., 1995; Drexler and Quentmeier, 1996; Graf et al., 1996; Matsumura et al., 1996; Quentmeier et al., 1996; Bredoux et al., 1997; Hirai et al., 1997). There is a suggestion that the presence of c-mpl in acute myeloid leukemia cells confers a poor prognosis (Vigon et al., 1993a; Wetzler et al., 1997).
GENE
Accession numbers The accession numbers of the GenBank database for both human genomic DNA and cDNA of the TPO receptor are available from National Center for Biotechnology Information (NCBI: www.ncbi.org). These are U68159 (exons 1±6), U68160 (exons 7 and 8), U68161 (exons 9 and 10), and U68162 (exons 11 and 12) for the genomic DNA, M90102 and M90103 for the P and K forms of the cDNA, respectively. The accession numbers for mouse genomic and cDNA are Z22657, X73677, and Z22649, respectively.
Sequence See Figure 1 for the human genomic DNA sequence.
PROTEIN
Accession numbers The accession numbers for cDNA-derived P and K forms of the human TPO receptor protein are AAA69971 and AAA69972, respectively. The accession number for the genomic DNA-derived TPO
Figure 1 Human genomic DNA sequence. 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901
CAAGGGGGCA TGAAGACCAT ATGCAGGTTC GCCCACCTAA GGCAGCCGTG TATTCATGCG TGGGGCAGAG TTATGCAGGT GGGCAAATCT ACTATTGTGC AAGTGCTCAG GATACAGAGG CACCAAAAGG CCCTGGCCCC GACGTGGGGC GCTTCCTGAA GGCCCTCTTC CAGCCAAGGT GGGAGGTGAT TCCCCTTCCA ATGTCTCCTT ACCTCACTTG ATGCCTACCC TAGCTGAGTC CTAAGTACCT TACATAACCC CAGAAGTTGG AGGGACCTCT ATGCCCCACT TTCTTTCCGC GTCCTCTTTG GCTGCCCCCA TACCCCTATT TGGCATCTAG CAAATCATTT CTAGGTGATG GCAAGCCATC CTCCTCAGCC CAATTCAGCC CAGAGTTCTG GGCCATGGGT AATCAGTGAT TGGTCCCACG TCACTCAGCC TGGACCAAAG TATCTCCTAC CTAGGTTCGT TCCTACTTTG GATTTCGCAA TGCATATCTA GGTTGGAGGC AAGGCTTCAG AACTCCTACT GGATCCTGGT GAATAGGGAG AGCTCTGGGA TTCAGGGAAA GAAATCTGAA CTTTAGTGGC GGCAGCAACA GCCCCAGAGA CAATCTTGCA TCCAAACCAT TATCTTCTCA ATGTCACTGG CTGCTAACAG
GGGTAAGGAG TGCTTCTCCA AACTTTAATA TGCTGTTGCT GAGGCCACCC CAAAGTCACA AGTTATTTCT GGGTTCTGAA TTAACTTCTC GGATTGTCTA TAAAAGTTGC CTGAGTTGCC CCTGTGTCCT TGGCCCCAGT TGTATCTGAC GGGAGGATGG ATGGTCACCT GAGGTGCACA GCAGGGCCCC CATAAACATG GCTGGCATCA CTTCTGGGAT GCGGTAGGTG CCACTCCAGC ACTTTTTGAA CTAATCCCAC GCATGGGCCC TCTATGCCAA TTGGAACCCG TGCACCTCTG TGGACAGTGT GTCCAGCTCC ACCAGACCCC AGCTAGAAAT ATTCATCCAT GGGATATGGA ATACTGCAAA TGCCCTCCAG CCCAGCACCC ATGTGCCCTG GGGAGCCAGC TTCCTGAGGT GTCATACAGC TCTGCTCTGG CAGACCTCCC CTTCAATCTT CCTTGGAGAG GGTCATTCCC CAAAACCCTG TCCAGCAAGG TCTCTCAGCT CTCTGACAGC GGCTGCAGCT CCCTCCCTGT ATGGGGAGGA ACCATGGTCC GAAGAGAGAA CACCCTATAC ACTTGGACTG GGACCATGCT CAGGTGAGAG GAAAAAAAGA ACAGCTTTCA ACCCCTCTCT GACCATTGCA AGTGATCTTT
TGTGAGCCAT ACTTTAAGTT GGCCTGGGGT CCTGGTCTAC ATAATGGAGA GAACTATTAA CAGCAGATTA GGTTGAACCT TGTATCTCCG AGTGAATGGA TATTCTGATG TTTTGGTACA CTCTTTCAAA GTGGTCTGGA AGGAACCTGA GCTAAGGCAG CCTGCCTCCT GAGGGTGGAG CGGAGGGGAG CCTGGGAGGA GACTCAGAGC GAGGAAGAGG CTGGACTGTG AGCTTTCCTG CAGATGTGCT CTATCCCAGG AGGTCTGGGT CAGGGAGAAG ATACGTGTGC GGTGAAGAAT AGGTAAGAGC CGGAATCAGA CTGAGGCACC GCCCAACAGA TCAAGAGTTA GAGAAAAAGG GAAGTCACTG GAGAATGACA CTCTCTGCAG TCTTGCCCTC CAGGGGAACT ACGAACTCCG TGATTGCCAC ACCAGTCTCC CAAGTAGAGA GCCCCAGGAA TTAGTATAGG ACTGACAAAA AACACCACCT AGCTGAGCTC GACAGGCAGA AGAGGGTGGA GCGCAGCGAA GACTGTGGAC GATAAAAGAA TTCCTGATGA TAGGAGTCAA AGTAGGGGCA CAATGCTTTA AGCTCCCAAG CTGAACTGCT AGAGAGTGTT ATGCTCTCTT GTTCCCATTG ACATCCTACA GTTAAGCTCA
CTCCAATCTG ACATTCAAAC GGGGCCTAAG AGACCACATT GGGCATTTCC AGTGGAAATA GCCTCCCAAG CCTGAGCTTT TTTCCTCATC TATAAAGTGC CTATTATACC CGGTCAAATA TTCCTCCTTC TGGGCCCCAG GGGGCTGGCC GCACACAGTG CCTGGCCCCT ATCACCTATG GTAGAGTAAG CCCAGGGCCA CCCTGAAGTG CAGCGCCCAG CCCCACTCCC CCTGTCCGAG TTGGATGTAT CAGTGAGAAG CCTCAGGCGT CCCCGTGCTT CAGTTTCCAG GTGTTCCTAA CATCCTCCTG CTTGCCTGTG CCAAGACTCC TCATTCACAC CAGAATACCT GATCCCTGCT TTCTCATGAG GCCTACAATT TCCAGAGGCT AGGCCTGCCG TCAGATCAGC CTATGGCCCC AGAAACCTGC ATGTGCTCAG AGTATGCTGA AGGACAGACC CTCAGATATG GCAAGGCTTT GAAACCCACC CTCTCCACAG CCTAGATTGT AGCTGCCTCA CCTGATGGGA CTGCCTGGAG TATCTCTAGG TCTCGAACTT TGTTCTAAGT CACGGGCCCT CCTTGGACCT GCTTCTTCTA GATTGAGGTT CTTGGTCCTC ATCTATTCTG GGAATGCTTT GTCTAATTCA AACTGTGCAT
AGCAAACAGG CATCTGGGGA ATTCTGCATT TTGAGTAGCA AGTTCCAGGG CCCACCATTT AGCATAGCAC ATGTCTTAGT TCTATAATGG TTAACACACG ATTCTATAAT TACAACCCCC CCTCCTGCCC AGGGGCAGGG TGGGAGGGGA GCGGAGAAGA CAAAACCTGG CCCCAGGAAG AGGCTCTCCT ACTCACCAGC TTTCTCCCGA TGGGACATAC CATGTATCTG GACCACTCTG GTGGGCCCCA AAAAATGGCA CCGCATGGTG GCCCCCTGAG ACCAGGAGGA ACCAGACTCG TCACCCTGCC CCCTTCCAGC CTTGTCATTC CCTGTNNCTC ACTGTGTGCC TACAAGAAGC AGAAGAACAG TTTGAATCCA GAGCCATAGA GCTCCCCCCA TGGGAGGAGC AGAGATCCCA TGCCCTGCTC CCCACAATGC CCTTCTTCTG ATACTTTGGG AGCACGCCTA CAGGCCTCCA AACTTAGCCC AGATGCTGTG GAAGCTGGGA TCTCAGGACT TCTCCCTCGG ATGCAGGTGA GAAGCCTGGG GCCACTGGAC TATATGTGTA GATGGGACTT GAAGAATGTT CCACAGCAGG GGTGTCATGG TTCACTCTCC TCATCCTCCA GGTTTAGTTT CCTCCAATGT GACCTT
ATAAAAGTAC TTTTGGCAAA TCTAATAAGT AGGGATTAGA AGAATGTGTT GGCATGGTTG AGCATGGCTG ATGTAATCTT AAATAACAAG GCAGCATAGT CGTTAGCACA ATCTCCCTCA ACTCCCCCTC ACAGGGACAG TTGGGGCCCA TGCCCTCCTG CCCAAGTCAG AGGGAGCCCT GCTGGTCCCC TGTTCCTTAG ACATTTGAGG CAGCTGCTGT TCCCTGCACT AATACAAGCC GCTCCAGCCC GTACTAGAGA GCTGTGTAGG TTCCCAGAGC AGTGCGTCTC GACTCAGCGA CCCTCCACTT TCAGCACGGA CTCCCAGCCT CCATCGTCAA AGACACTGGG ACTGAGTCTA AATCAGGAGC GAAGCTGCCC CTGTGGTACT GTATCATCAA CAGCTCCAGA AGAACTCCAC TGCAGAGGCC CCTGGCAAGA CCCCACCTCT ATTCCAGAC CTTAGGGGCT AATTAATGGA CTGGTCTGTG CAAATATAAG TTTTCCTCCC CCAGCCTGGC TGGCTCCTGG GTCAACAAAG CTAGATCTGA AGGAACTATG GAAATTATCT ACTTCTTTGA ACCTGTCAAT GCACGGTGCT GAGTGAGCCA TTCCTTTGTC ATCGATATTT AGCCTTAATC ATTCCCCATG
TPO Receptor 1957 Figure 1 (Continued ) Exons 7 and 8: 1 AAAATGCAAA 61 ATAAAGACAG 121 GCAGGCCATC 181 TCAATGACTC 241 AGAAAACAAA 301 ATGACAGCAT 361 ACCTGGGCTC 421 CATAGTTCCC 481 TGACCGATGG 541 TGACATCCCT 601 GCATCTGGAA 661 ACTCCGATAC 721 ACAGACCTCA 781 TGTCTATGTA 841 GTCTCTCTTG 901 CTTTGGGATA 961 TCCTCTCCTG 1021 CCTAGGCTCT 1081 CCATGGTTTC 1141 AAAGGAATTT 1201 TGAAATGAGG 1261 AAATACCTAG 1321 GTTTGTCTAC 1381 GTT Exons 9 and 10: 1 CCTTGCACTA 61 GTAAGAGACG 121 ACAGTTCATA 181 TAACGCAAAT 241 CTGAATATAT 301 TAGGGTGTGC 361 CGTAGCTCTC 421 CAGAGCGTGA 481 CGTGTTTTCT 541 GCAGGATTTG 601 CTTCTTTGCT 661 CCCGCGATCT 721 TCCCTGGAGC 781 AAGCCCCGGC 841 TGGGGCGGGG 901 GGGCCGACGC 961 GGGCTGGCTG 1021 CTCCTAGCCT 1081 CTGGGCCTGC 1141 GCAGGAGACT 1201 GCTCTGTGAC 1261 CACGTCACCT 1321 ACCTCGAACA 1381 AGCCAAACTG 1441 TCCAAACAAT 1501 CCTCTTGCCC 1561 TGAACCTATC 1621 CCTATGATGA 1681 TCTACCCTGT 1741 AGTGCCTTTG
AACAAACGAA AGGGAAAGCT GTTCTTGTAG TGTGGGGCTG TCCAGGACTA TATTCACATC CCCTTTCTGG ACCCCCACTG CTCTGGTGGC GTAGTGCGCC TTGGAGTGGC ACAGGAGAAG CTACGCAGGG TCCAGTCTCT GGCATCTGAT TCAGTCTTCC GCTCCTCCTT TTTCTCCTCT AATGTCCATA ACAAACCACA ATGAAGCTAT CATACTGCCT TTTTATTCTC
CAAATGAACA CTGGAGGCCC GATGGGAAGC GGTCTTAGGT CAGACCCCAC CTTGTGGAGG ATCCACCAGG AATCTGACCC ACAATGCCTT TCCCCACCCC AGCACCCATC GCCATCAGGA GATCCCTGGG GCTAGTATAT ATTTCTGGCT CCGATTTTCC TCCAGCCTTC GATAAAATAA CATTAACAAT GGCAAATTAC CTACCTTATG GGCATAAGGA ATTTGCCACA
AAGGAAAAAA CTGGAAGGAA CTTGGGATTA ACCCCATCTG AGTTCTCTCG TGACCACAGC CTGGTAAGAA TGTGCCCAGG GTGCACAGAA AAACTTGCAC GTCCTGGGCA CTGGAAGGTA GTTGGCCATG CTATGTTTAT ACTCCTACTT TCCTACCTCT CACAAAATGC ATATGTTCTC CGCAATTTTA CAAAATGTTT GAGTTATAGT AGGTGCTCAA CATATATACA
CAAAAAGAAA GGAGTTAAAT GTCTCTGAGG GGAGAACTGC CTGCCACTTC CCCGGGTACT CTTTCTTCCT ATCCCCAACT GGACTTAAGC TGGAGGGAGA GCCCAAGAGA TGGTCAAGCA CCTGTTAGCA CAGATTCAAC AACATTTCTC CTGACTGAAC TGTGCTTCCT CCCAGGTGAG CCATTCTAAC GAAGTCTGTT AAAGATTAAA TATACATTAC ACTATGTACA
ACATCATGGC GATAATCCCT CAGGCCTGAT GAAGAGGAAG AAGTCACGAA GTTCACAGCT CATTCTTCCA CTGACCCTTC TGCTCCCTGC TCTCCAGTGG CCTGTTATCA ACAAATGCCC GGAGTGAAAG TGGTATCTTA TTCCCTTTGT TTTCTCACTC AGAGTCTCAT TGCTTTAACT TCCCATATGG TCCTCATTGG TACTATATCT TTAAATATTA ACATCTTATG
ATTAATTGTT GTGTTTTATT AATGTTTGCT CTGGCATCCT CTGTTTCTGG GTGTACCGGG TGAGGTGAGG TCCCGTTAAG GCCGGTGGGG GGTCAAACAG CAGGTGCTGG CGCTACCGTT TCGTGGTCGG CGCACCAAAG CTCGGAGAGG TGCGCAGGTG GATGAGGGCG GGATCTCCTT TGCTGCTGAG GGCGGTGGAC CCCAGATCTC CTGGGACTCG GCCCCGCCTC CCCACTCCAC GGCCCCCTTT CTCCGATCTA AATTCCCTCC CAAGCGAGGC GCATCAGCCT CACATAACGT
GTGATTATTT GCTTTCTCCC GCCTTCCTGC CTGCAGCATG GGGTGTCCAT ATCCCTGCCG TCTGTGTCTC GAGGCTCTCG CTCTTTGTGG ACGCTGGGCT AGCCGCCTCT TACAGCTGCG ACCCAACTAG CCGCACAGCG GGCGAGNNGG CCCGCAGTCC GGGCTCCGGC GGTGACCGCT GTGGCAGTTT CAGGTGGAGC CGTCCACCGC CCGCGGCTCC CTGCTGCTCA CCCCATCCAA CTCTAGCCCT GCTACACAGC TTAAATCCTT TCTGCAAAAT TTCCAAACTG CCGCTCTGCC
ACCTGTTTGG CAGTGCCTAA TTGTACACAT AGTATTATTT CGCCCGACCT AGGGTGTACC AGGAGGTGGG GTTAGGGCGC GAATCTCCGA ATCGAAGCCC CGGGGCCCGA CGCCAGGCTC GGTGGAGACC CCTGCGCAGG GGCGNGGAGA CAGGGGCGGC CCGGGTGGGC CTGCATCTAG CCTGCACACT CGAACCGGTG CCGTGCGCAC TTACACTCTA CCTCGGCGAC TCTGCCGTCA ACAGACACGA CACCAGAATA TCAATAGCTC CGGCCCCTGC GTTTTCTCCA TGAAATAGCC
ATGTCTTTCT CCCAATGTTT GATGCTTGTA GTGGCATGGT GGAGTTGTGA TGGGTGTTGG CCTGGCACGT TCTATCCTGT CCGCCTGGGG CGACGCCGGG GGAGGGACCC AACGGCCCCA GCCACCGAGA GACTGGGCGC GGGCGGGGCC GAGGGGCGGG CGAAGTCTGA TGCTGGGCCT ACAGGTACCG TAAACAGGCA CTACGGCTTC ACACGCCCAC TAGGCCACCG GTCCCACCTC CCTGACTCAC ATCTTTCTAA CCAGGATATA CAGCAAGCAC CAAGTCAGGT TCCCT
CATTAGATAT GGAGTATCAC CCCCAGCGCC CTGTGTGGCT GAACACCCGG TGTTAGGATA TTCTCTCGGG TGCTGGGAAG ATTCGGAGCT CCACCGCACG TGGAGCTGCG CCTACCAAGG CCGGTGAGGC CGGGTGCGAG CTGACCTTGC GCCAGAGTAG CCCTTTTTGT CAGCGCCGTC CCCCCGCCAG TTCTTGGTTC GCACTTCCTG TATACCGCCC TCCACCCTTC CTAAACCTAG TGAAACAGAA AATGCGCACA GTCCAGGCTC TTCAGCCTGT TTTACTTCTC
1958 David J. Kuter and Junzhi Li Figure 1 (Continued ) Exons 11 and 12: 1 CTGCAGATAA AGAGGGAGTA 61 GAGGCCTACA ATGAGGTGAG 121 GTTTGAGGAA ACTGCAGCAC 181 CTGAGGTTTT CTTCTTACTC 241 TATCTCCAAG CCTTACTCCC 301 CTGCCAATCC ACTGCCATGG 361 CATGCCCTGT GGCCCTCACT 421 ACTGCAGCCC TGAGCCCGGT 481 TGGTACTGGA TCCTTGCCCC 541 CCATCCCCAG GCACTACCCC 601 CCTCCTCCCA CAGGATCTGC 661 CACAGTCTCA GATACCTGTG 721 CTCAGAGAGG ACTCCTTTGC 781 GCAGCCTTCT TGCCTGGGGA 841 GTCCTGCTGT ACCACCCACA 901 GCCTTGAGGA CAGGCTCCTC 961 TCTGTGAACT TCCCTACCCT 1021 CCTCTGTCTG CCCTCACAAT 1081 TAAAACCAGG ACCCTTTCTC 1141 TCTCTCCTCT TTGATGTCAA 1201 TTACTCTTGA GACTACTTCA 1261 CAAAGTGCAC CTCAAATCTT 1321 CCTTTGATCT CGCTGTAAGA 1381 CCTACCTTGG TCCTGCCTCT 1441 GGTCTCACTC TGTCACCCAG 1501 CACCTCCGGG TTCAAGCGAT 1561 CACACCACCA CACACAGCTA 1621 GGAGCCTTGC CTGTTGCCAG 1681 TGCCTCCCGG GTTCAAGCCA 1741 GCCACCATGC CTAATTTTTT 1801 GATGGTCTCG ATATCTGATC 1861 ACAGGTGTGA CCCACTGCGC 1921 TTTTGCCATG TTGCCCAGGC 1981 GCCTCCCAAA GTGTTAGGAT 2041 GTTAAGAGAA TAAACTAGAT 2101 ACTAGCTGTG CAACCTTGGG 2161 TAAAACAGGG ATAATAACAG 2221 AAAGCACAGT CCCTAGGACA 2281 TCCTTCTTAC TCTCCTCTTC 2341 TTCTTAACAG TCTCCAATCC 2401 GTTCTTATCT CTAGCTCAGA 2461 TTACATATCT GTCCCCTGCT 2521 TTTGTCCTTT TCCCCCAGCA 2581 AATTTTTTCT AAATGAATGA 2641 TCCTCATCCT CTAAACCGTT 2701 ACTCTCCTAA CACATTCTAA 2761 TCCAAATATA CCCACTGTAT 2821 GGGTAATATG ATGACAGTGT
CTGTGTTTCA GGAATGAATC CCTTTGTTGA TACTCCCCTG CGGCCTCACT CTCAGTCTGC TCCAGACCTG GAGTGTGCTT AACAATACAA AGCACTACCC TTTAATCCAG AAGAAGTGGA CCCTGTGTTC CCATGCCCCT TTGCCAACCA ACTCCCAGTT ACCCCCACAA TAGGCTTCAT CACAGGCAGG ACGCCTTGAA ATTAGTTCCC CTAATTCCAA TTTGTCAAGG TTGAGTATAT GCTGCAGTGC TCTTGTGCCT ATTTTTTTTT ACTGGAGTGC TTCTGCCTCA TCTATTTTTA TCGTGATCCG ACAGCCCCAG TGGTCTTGAA TACAGGCATG CTAGAATCAG CACATAACTT CACCCCATAG TAGTAAATGA TAGCATTTCT CATCTATTCT CATCTACTAC ACTAGATTGT CCTAGAGTAG AATCTTTCTG CATAGGCAGC TTCTCAAACA CAGTCAGGGT AATAAACT
receptor protein is AAB08424. Those for the mouse TPO receptor are CAA80365 and CAA52031.
Description of protein Human full-length cDNAs encode 635 and 579 amino acid residues for the P and K forms of the TPO receptor, respectively (Vigon et al., 1992). The P and
GGCAAGGTAG CAGTGTTCCA ACCTGCCCAC CCCAACTTTA GCTCCCCCAC TTCTCTTCCT CACCGGGTCC CCCTCCCCTG CTTGTTCAAG CAGCCCTTCT CGCCTCTCCT ACCCAGCCTC CTCCCAGGCC GTCTGTGTGC TTCCTACCTA CCCTGGACAG CACAAGCACC TGCACTGATC CTCATTTCAC AACAAGCCTC CTACTACACT GATCCAATAG CTGACTACTC TAGTAGGTTT AATGGCGCGA CGGCCTCCCT TTTTTTTTTT AGTGGCACGA GCCTCCCAAG GGAGAGACCG CCTGCCTCTG CTAATTTTCA CTCCTAACCT AGCCACTGCG AGCTGGATTC AATGTCTTTG AGTTGTGACG TTCATATATC TCCAATTATT CTGCCTTTAC AGCACTGTGA GAGCTCCTTG TGCTTGGTGC GACAAGGTTA AGTAAATGTG AATGGTTTTG CTTGGAAGAA
TCAGGGATGA GACAGAACAA CACTGACTTC TTGCCTTGAC CCCAACTGCT TCTCCCCCAG TAGGCCAGTA TGCCCACCAC GTCCTTGCCC CCTTCCTGTA CATCTCTCCC CTTGAAATCC CAGATGGACT CCACCCATGG CCACTAAGCT AGCTAAACTC CCAGACCTCA TTACTCTACT TAAGCTCCTC CACTTCCCCA TTGCTAGTGA GATCTCGTTA ACTTCTCCTT TTTTTATTTG TCTCAGCTCA AGTAGCTGGG TTTTTTTTTT TCTCGGCTCA TAGCTGGGAG GTTTTCACCA CCTCCCAAAG TATTTTTAGT CGGGTGATCC CCCGGCTGAG AATTCCTGTC AGCCTTAGTT AGGATTGAGA CGAACTACTG ACAGTCCTTC TATATGTTGA TGCTTTATGC AGGGAAAGGA ATGATAGTAG CCTTAATCCC CCAGACAGTT TTGTCATTGT ATAGATGACA
CATTTGAACA ACATTGGAGT CCTCCCCTAC CATGCTCTTA GTCCTCCTCC GAGACTGAGG CCTTAGGGAC CAACCCTGCC TACCAGTGTG CAGTCCAGCC AGCCCAAGGC TCCCCAAGTC ACCGAAGATT CTGAGTCAGG ATTGGCAGCA TCGAGACTTC CCTCCATCCC GCTGCTGACA CTTTACTTCC CACTTCCCAT ACTGCCCAGG ATCATCAGTT TAAATTCTTT TTTGAGACAG CTGCAACCTC ATTACAGGCG TTTTTTAGAC CTGCAACCTC TACAGCGTCT CGTTGGCCAG TGCTGGGATT AGAGACAGGG ACCCACCTTG TGTACTAGTA CTTCACATTT TTTTCATCTG TAATCTAAGT TTATAATTAT AAGATTCCAT CCATTCCAAA AACTAACTGT ACATGATTTA GCCTTCAATA ATGCCAGCTG CTGATTATGG TTTTNCTGGC CACTCAAATT
K forms share identical amino acid sequence from residues 1 to 522 at the N-terminus, but are completely different after residue 523. The P and K forms result from alternative TPO receptor mRNA splicing at exon 10±11 ( Vigon et al., 1992). The first 25 amino acid residues at the N-terminus encode signal peptides. A hydrophobic region from residues 492 to 513 in the transmembrane region separates the extracellular domain residues 26±491 from the intracellular
TPO Receptor 1959 Figure 1 (Continued ) Human cDNA sequence for TPO receptor P form: 1 ATGCCCTCCT GGGCCCTCTT CATGGTCACC TCCTGCCTCC 61 GCCCAAGTCA GCAGCCAAGA TGTCTCCTTG CTGGCATCAG 121 TTCTCCCGAA CATTTGAGGA CCTCACTTGC TTCTGGGATG 181 GGGACATACC AGCTGCTGTA TGCCTACCCG CGGGAGAAGC 241 TCCCAGAGCA TGCCCCACTT TGGAACCCGA TACGTGTGCC 301 GTGCGTCTCT TCTTTCCGCT GCACCTCTGG GTGAAGAATG 361 ACTCAGCGAG TCCTCTTTGT GGACAGTGTA GGCCTGCCGG 421 GCCATGGGTG GGAGCCAGCC AGGGGAACTT CAGATCAGCT 481 ATCAGTGATT TCCTGAGGTA CGAACTCCGC TATGGCCCCA 541 GGTCCCACGG TCATACAGCT GATTGCCACA GAAACCTGCT 601 CACTCAGCCT CTGCTCTGGA CCAGTCTCCA TGTGCTCAGC 661 GGACCAAAGC AGACCTCCCC AAGTAGAGAA GCTTCAGCTC 721 TGCCTCATCT CAGGACTCCA GCCTGGCAAC TCCTACTGGC 781 GATGGGATCT CCCTCGGTGG CTCCTGGGGA TCCTGGTCCC 841 CCTGGAGATG CAGTGGCACT TGGACTGCAA TGCTTTACCT 901 TGTCAATGGC AGCAACAGGA CCATGCTAGC TCCCAAGGCT 961 CGGTGCTGCC CCAGAGACAG GTACCCCATC TGGGAGAACT 1021 AATCCAGGAC TACAGACCCC ACAGTTCTCT CGCTGCCACT 1081 ATTATTCACA TCCTTGTGGA GGTGACCACA GCCCCGGGTA 1141 TCCCCTTTCT GGATCCACCA GGCTGTGCGC CTCCCCACCC 1201 ATCTCCAGTG GGCATCTGGA ATTGGAGTGG CAGCACCCAT 1261 ACCTGTTATC AACTCCGATA CACAGGAGAA GGCCATCAGG 1321 CCTCTCGGGG CCCGAGGAGG GACCCTGGAG CTGCGCCCGC 1381 CTGCGCGCCA GGCTCAACGG CCCCACCTAC CAAGGTCCCT 1441 ACTAGGGTGG AGACCGCCAC CGAGACCGCC TGGATCTCCT 1501 GTGCTGGGCC TCAGCGCCGT CCTGGGCCTG CTGCTGCTGA 1561 TACAGGAGAC TGAGGCATGC CCTGTGGCCC TCACTTCCAG 1621 CAGTACCTTA GGGACACTGC AGCCCTGAGC CCGCCCAAGG 1681 GAAGAAGTGG AACCCAGCCT CCTTGAAATC CTCCCCAAGT 1741 CCCCTGTGTT CCTCCCAGGC CCAGATGGAC TACCGAAGAT 1801 ACCATGCCCC TGTCTGTGTG CCCACCCATG GCTGAGTCAG 1861 ATTGCCAACC ATTCCTACCT ACCACTAAGC TATTGGCAGC
TCCTGGCCCC ACTCAGAGCC AGGAAGAGGC CCCGTGCTTG AGTTTCCAGA TGTTCCTAAA CTCCCCCCAG GGGAGGAGCC GAGATCCCAA GCCCTGCTCT CCACAATGCC TGACAGCAGA TGCAGCTGCG TCCCTGTGAC TGGACCTGAA TCTTCTACCA GCGAAGAGGA TCAAGTCACG CTGTTCACAG CAAACTTGCA CGTCCTGGGC ACTGGAAGGT GATCTCGCTA GGAGCTCGTG TGGTGACCGC GGTGGCAGTT ACCTGCACCG CCACAGTCTC CCTCAGAGAG TGCAGCCTTC GGTCCTGCTG AGCCTTGA
TCAAAACCTG CCTGAAGTGT AGCGCCCAGT CCCCCTGAGT CCAGGAGGAA CCAGACTCGG TATCATCAAG AGCTCCAGAA GAACTCCACT GCAGAGGCCT CTGGCAAGAT GGGTGGAAGC CAGCGAACCT TGTGGACCTG GAATGTTACC CAGCAGGGCA AGAGAAAACA AAATGACAGC CTACCTGGGC CTGGAGGGAG AGCCCAAGAG GCTGGAGCCG CCGTTTACAG GTCGGACCCA TCTGCATCTA TCCTGCACAC GGTCCTAGGC AGATACCTGT GACTCCTTTG TTGCCTGGGG TACCACCCAC
Human cDNA sequence for TPO receptor K form: 1 ATGCCCTCCT GGGCCCTCTT CATGGTCACC TCCTGCCTCC 61 GCCCAAGTCA GCAGCCAAGA TGTCTCCTTG CTGGCATCAG 121 TTCTCCCGAA CATTTGAGGA CCTCACTTGC TTCTGGGATG 181 GGGACATACC AGCTGCTGTA TGCCTACCCG CGGGAGAAGC 241 TCCCAGAGCA TGCCCCACTT TGGAACCCGA TACGTGTGCC 301 GTGCGTCTCT TCTTTCCGCT GCACCTCTGG GTGAAGAATG 361 ACTCAGCGAG TCCTCTTTGT GGACAGTGTA GGCCTGCCGG 421 GCCATGGGTG GGAGCCAGCC AGGGGAACTT CAGATCAGCT 481 ATCAGTGATT TCCTGAGGTA CGAACTCCGC TATGGCCCCA 541 GGTCCCACGG TCATACAGCT GATTGCCACA GAAACCTGCT 601 CACTCAGCCT CTGCTCTGGA CCAGTCTCCA TGTGCTCAGC 661 GGACCAAAGC AGACCTCCCC AAGTAGAGAA GCTTCAGCTC 721 TGCCTCATCT CAGGACTCCA GCCTGGCAAC TCCTACTGGC 781 GATGGGATCT CCCTCGGTGG CTCCTGGGGA TCCTGGTCCC 841 CCTGGAGATG CAGTGGCACT TGGACTGCAA TGCTTTACCT 901 TGTCAATGGC AGCAACAGGA CCATGCTAGC TCCCAAGGCT 961 CGGTGCTGCC CCAGAGACAG GTACCCCATC TGGGAGAACT 1021 AATCCAGGAC TACAGACCCC ACAGTTCTCT CGCTGCCACT 1081 ATTATTCACA TCCTTGTGGA GGTGACCACA GCCCCGGGTA 1141 TCCCCTTTCT GGATCCACCA GGCTGTGCGC CTCCCCACCC 1201 ATCTCCAGTG GGCATCTGGA ATTGGAGTGG CAGCACCCAT 1261 ACCTGTTATC AACTCCGATA CACAGGAGAA GGCCATCAGG 1321 CCTCTCGGGG CCCGAGGAGG GACCCTGGAG CTGCGCCCGC 1381 CTGCGCGCCA GGCTCAACGG CCCCACCTAC CAAGGTCCCT 1441 ACTAGGGTGG AGACCGCCAC CGAGACCGCC TGGATCTCCT 1501 GTGCTGGGCC TCAGCGCCGT CCTGGGCCTG CTGCTGCTGA 1561 TACAGGTACC GCCCCCGCCA GGCAGGAGAC TGGCGGTGGA 1621 AAACAGGCAT TCTTGGTTCG CTCTGTGACC CCAGATCTCC 1681 TACGGCTTCG CACTTCCTGC ACGTCACCTC TGGGACTCGC
TCCTGGCCCC ACTCAGAGCC AGGAAGAGGC CCCGTGCTTG AGTTTCCAGA TGTTCCTAAA CTCCCCCCAG GGGAGGAGCC GAGATCCCAA GCCCTGCTCT CCACAATGCC TGACAGCAGA TGCAGCTGCG TCCCTGTGAC TGGACCTGAA TCTTCTACCA GCGAAGAGGA TCAAGTCACG CTGTTCACAG CAAACTTGCA CGTCCTGGGC ACTGGAAGGT GATCTCGCTA GGAGCTCGTG TGGTGACCGC GGTGGCAGTT CCAGGTGGAG GTCCACCGCC CGCGGCTCCT
TCAAAACCTG CCTGAAGTGT AGCGCCCAGT CCCCCTGAGT CCAGGAGGAA CCAGACTCGG TATCATCAAG AGCTCCAGAA GAACTCCACT GCAGAGGCCT CTGGCAAGAT GGGTGGAAGC CAGCGAACCT TGTGGACCTG GAATGTTACC CAGCAGGGCA AGAGAAAACA AAATGACAGC CTACCTGGGC CTGGAGGGAG AGCCCAAGAG GCTGGAGCCG CCGTTTACAG GTCGGACCCA TCTGCATCTA TCCTGCACAC CCGAACGTGT CGTGCGCACC TACACTCTAA
1960 David J. Kuter and Junzhi Li Figure 1 (Continued ) Mouse cDNA for TPO receptor: 1 CCTCTTCATG GTCACCTCCT GCCTCCTCTT 61 CCAAGATGTC TTCTTGCTGG CCTTGGGCAC 121 TGAGGACCTC ACCTGCTTCT GGGATGAGGA 181 GCTGTATGCC TACCGAGGAG AGAAGCCCCG 241 CACCTTTGGA ACCCGGTATG TGTGCCAGTT 301 TCCGCTGCAC CTCTGGGTGA AGAATGTGTC 361 GTTTGTGGAT AGTGTGGGCC TGCCAGCTCC 421 CCAACCAGGG GAACTTCAGA TCCACTGGGA 481 GAGGCATGAA CTCCGCTATG GCCCCACGGA 541 TCAGCTGCTC TCCACAGAAA CCTGCTGCCC 601 TCTTGACCAG CCTCCGTGTG TTCATCCGAC 661 CTCCCCAGCT GGAGAAGCTC CATTTCTGAC 721 CCTCCAGGCT AGCAAATCCT ACTGGCTCCA 781 TCGTGGCTCC TGGGGACCCT GGTCCTTCCC 841 GACAATTGGA CTTCAGTGCT TTACCTTGGA 901 ACAAGACCGC ACTAGCTCCC AAGGCTTCTT 961 AGACAGGGAC CCCACCTGGG AGAAATGTGA 1021 CGCTCTCGTC TCCCGCTGCC ACTTCAAGTC 1081 AGAGGTGACC ACAGCGCAAG GTGCCGTTCA 1141 CCAGGCTGTG CTCCTTCCCA CCCCGAGCCT 1201 GGAGTTGGAG TGGCAGCACC AGTCATCTTG 1261 GTACACGGGA GAAGGCCGTG AGGACTGGAA 1321 AGGGACCCTA GAGCTGCGCC CCCGAGCTCG 1381 CGGCCCCACC TACCAAGGTC CCTGGAGCGC 1441 CTCCGAGACT GCTTGGATCA CCTTGGTGAC 1501 CCTTCTGGGC CTACTGCTGC TAAAGTGGCA 1561 TGCTTTGTGG CCCTCGCTTC CAGACCTACA 1621 TGCAGCCCTA AGTCCTTCTA AGGCCACGGT 1681 CCTCCTGGAA ATCCTCCCTA AATCCTCAGA 1741 ACCTCAGATG GACTACAGAG GACTGCAACC 1801 TCCACCCATG GCTGAGACGG GGTCCTGCTG 1861 ACCACTAAGC TATTGGCAGC AGCCCTGAAG 1921 TTCCTACACA CTACCTTATC CATCCTCAAC 1981 CTCTGGCTTT ATAACACTGA TCACTCCAAG 2041 CTGCAG
domain residues 514±635. There are four potential Nlinked glycosylation sites in the extracellular domain located at residues 117, 178, 298, and 358, respectively. The extracellular domain contains a duplicated HRD which is highly conserved in the hematopoietin/ cytokine receptor superfamily. Each HRD contains two highly conserved disulfide bonds and two FNIIIlike domains, each of which consists of seven strands. A 5 amino acid motif called WSXWS is found at the C-terminus of the second FNIII-like domain in both HRDs of the TPO receptor ± a finding which is characteristic of the hematopoietin/ cytokine receptor family. In addition, the TPO receptor is unique in having a 50 amino acid insertion in the second FNIII-like domain of the membranedistal HRD. A novel variant TPO receptor called c-Mpl-del in which 24 amino acid residues are deleted in the membrane-proximal HRD has been described (Li et al., 2000). Functional analysis of c-Mpl-del protein demonstrated that it was expressed but did not transport to the cell surface and was therefore not functional.
GGCCCTTCCA AGAGCCCCTG AGAGGCAGCA TGCATGCCCC TCCAGCCCAG CCTCAACCAG CCCCAGGGTC GGCCCCTGCT TTCCAGCAAC CACTTTGTGG AGCATCCCAA AGTGAAGGGT GCTACGCAGC TGTGACTGTG TCTGAAGATG CCGTCACAGC AGAGGAGGAA ACGAAATGAC CAGCTACCTG GCACTGGAGG GGCAGCTCAA GGTGCTGGAG CTACAGCTTG CTGGTCTCCC TGCTCTGCTC ATTTCCTGCG CCGGGTCCTA TACCGATAGC GAGCACTCCT TTGCCTGCGG CACCACACAC GCAGTCCCCA ACCATCCATT ATGGCTGCTC
AACCAGGCAC AACTGCTTCT CCCAGTGGGA CTGTATTCCC GTAGAAGTGC ACTTTGATCC ATCAAGGCCA CCTGAAATCA GCCACTGCCC ATGCCGAACC CCGCATGGAC GGAAGCTGTC CAACCCGACG GATCTTCCAG GTCACCTGCC AGGACGAGGT CCGCGTCCAG AGTGTTATTC GGCTCCCCTT GAGGTCTCAA GAGACCTGCT CCATCTCTCG CAGCTGCGTG CCAGCTAGGG CTGGTGCTGA CACTACAGGA GGCCAGTACC TGTGAAGAAG TTACCTCTGT ACCATGCCCC ATTGCCAACC TGCTACTGCA CTGTTGCCAC ACAAATCCAG
AAGTCACCAG CCCAAACATT CATACCAGCT AGAGTGTGCC GCCTCTTCTT AGCGGGTGCT GGGGTGGGAG GTGACTTCCT CCTCCGTCAT CAGTCCCTGT CAGTGAGGAC TCGTCTCAGG GGGTCTCCCT GAGATGCAGT AGTGGCAGCA GCTGCCCCAC GATCACAGCC ACATCCTTGT TTTGGATCCA GTGGAAGGCT ACCAGCTCCG GTGCCCGGGG CCAGGCTCAA TGTCCACGGG GCCTCAGTGC GACTGAGGCA TCAGAGACAC TGGAACCCAG GTCCCTCCCA TGTCTGTGTG ACTCCTACCT GACCTATACA CCCACTCCCC AGCTCTGTCT
The intracellular domain contains two short membrane-proximal motifs, box 1 and box 2, which are conserved in the hematopoietin/cytokine superfamily. Deletion of either box 1 or box 2 results in a defective TPO receptor (Gurney et al., 1995). The membrane-distal region of the intracellular domain shares no homology with other cytokine receptors. Although there is no enzymatic motif found in the intracellular domain, it does contain five tyrosine residues which are conserved in both human and mouse and which play a critical role in the TPO receptor-mediated signal transduction pathways.
Relevant homologies and species differences The genes for the human and mouse TPO receptors have been cloned and extensively studied. Comparison of the amino acid sequences of these TPO receptors reveals 81% amino acid identity (Foster et al., 1994). Further comparison of the intracellular domains
TPO Receptor 1961 shows that there is 91% identity (Vigon et al., 1993c), suggesting that the TPO-induced signaling pathways should be very similar in these two species.
Affinity for ligand(s) Most information about TPO receptor binding affinity for their ligands has been obtained from experiments on human platelets (Broudy et al., 1997; Fielder et al., 1997; Li et al., 1999). Scatchard analysis has shown that platelets contain a single class of binding sites for TPO with affinity constant (Kd) ranging from 120 to 200 pM. Each platelet contains 30±60 binding sites on the surface. After binding to platelets, c-Mpl ligands are internalized within 60 minutes and then degraded. Although megakaryocytes and their progenitor cells have been experimentally demonstrated to have TPO receptors on the cell surface (Broudy et al., 1997; Solar et al., 1998), the precise quantification of their number and binding affinity has not been done.
Cell types and tissues expressing the receptor The thrombopoietin receptor can be directly demonstrated or its presence inferred on early bone marrow hematopoietic progenitor cells of all lineages as well as on the pluripotential stem cell (Carver-Moore et al., 1996; Rasko et al., 1997; Ratajczak et al., 1997; Tanimukai et al., 1997; Yoshida et al., 1997; Yamada et al., 1998; Solar et al., 1998). It is present on common early erythroid/megakaryocyte progenitors (Kaushansky et al., 1995, 1996; Kobayashi et al., 1995; Carver-Moore et al., 1996; Drexler and Quentmeier, 1996; Graf et al., 1996; Kieran et al., 1996; Bonsi et al., 1997; Drexler et al., 1997; Goncalves et al., 1997; Higuchi et al., 1997; Komatsu et al., 1997; Miura et al., 1998). It is present on late megakaryocyte progenitors but not on late erythroid progenitors. It is not present on mature erythrocytes, neutrophils, eosinophils, basophils, or lymphocytes but it is present on platelets (Sasaki et al., 1995a). c-Mpl has been reported to be present on monocytes (but contamination by associated platelets could not be discounted; Sasaki et al., 1995a). The receptor is found on cells and cell lines from many nonlymphoid hematological malignancies but not on cells from purely lymphoid malignancies or nonhematopoietic solid tumors (Columbyova et al., 1995; Drexler et al., 1996; Graf et al., 1996; Matsumura et al., 1996; Quentmeier et al., 1996; Bredoux et al., 1997; Hirai et al., 1997).
Regulation of receptor expression There is no clinical or experimental situation in which regulation of c-Mpl has been convincingly demonstrated. A 200 bp portion of the c-mpl promoter has been demonstrated to be adequate for high-level expression of the gene in cell lines (Deveaux et al., 1996). GATA-1 and two members of the Ets family (Ets-1 and Fli-1) have specific binding sites and all three seem necessary for high-level expression in tissue culture cells. As mentioned previously, platelets from patients with essential thrombocythemia or polycythemia vera have reduced amounts of c-Mpl on the cell surface (Li et al., 1996; Moliterno et al., 1998). Whether this is due to altered expression or ligand-induced downregulation of the receptor is unclear.
Release of soluble receptors mRNA that lacks the c-mpl transmembrane region is found in cells that express c-mpl but the existence of its protein product has not been convincingly demonstrated. Like other alternatively spliced transcripts, the protein product may not be efficiently translated or exported from the endoplasmic reticulum (Li et al., 2000). In whole plasma and serum a c-Mpl protein that is not associated with cells can be demonstrated but it is unclear whether this is the predicted soluble receptor or simply the extracellular portion of the full-length, wild-type platelet receptor that has been cleaved in vivo or during sample preparation. In unpublished studies, the circulating level of c-Mpl protein not associated with cells is directly proportional to the platelet count.
SIGNAL TRANSDUCTION
Associated or intrinsic kinases As predicted by its cDNA structure, the TPO receptor does not contain any known intrinsic kinase activity ± a finding that is a characteristic feature of the hematopoietin/cytokine receptor family. Therefore its signal transduction following ligand binding and dimerization must utilize cytoplasmic enzymes such as nonreceptor protein tyrosine kinases and other signaling molecules. These molecules are able to associate by protein±protein interaction with the TPO receptor to form a signaling complex. Indeed, upon exposure to TPO, a number of tyrosine kinases and
1962 David J. Kuter and Junzhi Li signaling adapter molecules undergo tyrosine phosphorylation.
Cytoplasmic signaling cascades Signal transduction starts with c-Mpl ligand binding to c-Mpl on the cell surface. Upon dimerization, the receptors transduce the ligand-specific signal by transient and covalent chemical modifications of intracellular signaling molecules. These molecules are either enzymatic proteins (e.g. kinases or phosphatases) or nonenzymatic adapter proteins. In most cases they contain multiple domains for protein±protein interaction, such as the Src-homology 2 domain (SH2) which binds to phosphotyrosine. One commonly seen modification is tyrosine phosphorylation. Tyrosine phosphorylation of these molecules could change their molecular conformation, leading to regulation of either enzymatic activity or affinity for interaction with other bio-macromolecules, or both. Frequently, tyrosine phosphorylation also induces translocation of the signaling molecule in the cells. Finally, the ligand-initiated signaling cascades usually affect cellular proliferation, differentiation/maturation, and/or apoptosis. Treatment of cells with TPO induces tyrosine phosphorylation not only on the intracellular domain of the TPO receptor but also on a number of protein tyrosine kinases and signaling adaptor molecules. Although the biological specificity of the signaling pathways for TPO is still unsolved, there are at least three signaling cascades through which TPO may mediate its effects. Like most hematopoietic cytokines, TPO stimulates the tyrosine phosphorylation of two members of JAK kinase family, JAK2 (125 kDa) (Drachman et al., 1995; Miyakawa et al., 1995) and TYK2 (130 kDa) (Ezumi et al., 1995; Sattler et al., 1995), as well as two members of the STAT (signal transducer and activator of transcription) family, STAT5 (92 kDa) and STAT3 (90 kDa) (Miyakawa et al., 1996). STAT family proteins are well-known cytoplasmic substrates for the JAK family tyrosine kinases. Upon JAK2 phosphorylation and activation, STAT molecules are phosphorylated on their tyrosine residues and multimerized by protein±protein interactions via binding of the phosphotyrosine of one molecule to the SH2 domain of another. Then the STAT signaling complexes translocate into the nucleus to modulate the transcription of their downstream genes. Although evidence suggests that JAK2 constitutively associates with the TPO receptor via box 1 and box 2 motifs (Mu et al., 1995; Drachman and Kaushansky, 1997), the role of the JAK2/STAT5
signaling pathway has been challenged by the observation that a c-mpl deletion mutant was able to convey a proliferative signal without activation of JAK2/ TYK2 kinases or phosphorylation of STAT5 (Dorsch et al., 1997). TPO-induced phosphorylation of Shc (60 kDa) is another hallmark signaling pathway (Drachman et al., 1995; Miyakawa et al., 1995; Mu et al., 1995). Shc is an adapter protein containing multiple protein interaction domains, including SH2 and SH3 domains, and plays important roles in activation of the Ras signaling pathway in various systems. Shc phosphorylation and association with the TPO receptor are independent of JAK2 activation, but dependent on TPO receptor phosphorylation on the tyrosine-599 of the intracellular domain, which provides a docking site for association with the SH2 domain of Shc (Alexander et al., 1996a; Drachman and Kaushansky, 1997). It is still unclear which tyrosine kinase contributes to Shc phosphorylation. However, it appears that the signaling complexes between the TPO receptor and Shc probably further activate the Ras signaling pathway. Indeed, a growing body of evidence strongly supports this model. Firstly, a dominant negative Ras mutant inhibited TPO-induced megakaryocytic differentiation and reduced cell proliferation (Matsumura et al., 1998). Secondly, TPO treatment induced phosphorylation of MAPK (Mu et al., 1995; Rouyez et al., 1997), Raf1 (Dorsch et al., 1997), and Sos (Sasaki et al., 1995b), key components of the Ras signaling pathway. Thirdly, the expression of c-fos and c-myc was upregulated by TPO treatment (Dorsch et al., 1997; Kunitama et al., 1997). Furthermore, the TPO-initiated Shc/Ras/Raf/ MAPK pathway is primarily responsible for induction of megakaryocytic differentiation (Alexander et al., 1996a; Dorsch et al., 1997; Rouyez et al., 1997). TPO-induced activation of phosphatidylinositol-3 0 (PI-3) kinase (85 kDa, regulatory subunit) is a third important signaling pathway (Chen et al., 1995; Dorsch et al., 1997; Sattler et al., 1997; Zauli et al., 1997). The activation of PI-3 kinase is independent of either JAK2 activation or Shc phosphorylation (Dorsch et al., 1997), but is associated with TPOinduced tyrosine phosphorylation of Vav (95 kDa) and Cbl (120 kDa; Oda et al., 1996; Sattler et al., 1997). These two proto-oncogene products are expressed primarily in multiple lineages of hematopoietic cells, including megakaryocytes and platelets. As demonstrated in human B cells, it is more likely that PI-3 kinase activation is a downstream event of Vav phosphorylation. Protein kinase C is also associated with PI-3 kinase activation (Kunitama et al., 1997; Hong et al., 1998). Two biological roles of TPO-induced Vav/PI-3 signaling pathway have been
TPO Receptor 1963 implicated. One is to provide a mitogenic signal to cells (Dorsch et al., 1997). The other is to increase the adherence of cells to the extracellular matrix by activation of integrin on the cell surface (Chen et al., 1995; Cui et al., 1997; Gotoh et al., 1997; Zauli et al., 1997). Addition of the PI-3 kinase inhibitor, wortmanin, abolished both biological effects of PI-3 kinase.
DOWNSTREAM GENE ACTIVATION
Transcription factors activated TPO exerts its wide range of biological activities by initiation of several critical signaling pathways that lead to activation of a group of transcription factors. These activated transcription factors come from two basic sources. One is the conversion of preexisting, inactive transcription factors into active ones by covalent chemical modification. The best example of this is TPO-induced activation of STAT3/STAT5. A second is the synthesis of new transcription molecules by induction of gene expression. The best example of this is TPO-induced upregulation of c-fos and c-myc. However, no matter what their source, these TPOinduced and activated transcription factors are also used by many other hematopoietic cytokines. For example, the JAK/STAT signaling pathway is used by almost all hematopoietic cytokines. As soon as the TPO receptor was discovered, TPO receptor-mediated induction of megakaryocytespecific genes expressed in both CD34 cell cultures and c-mpl-transfected hematopoietic progenitor cell lines was described (Morita et al., 1996). For example, treatment of c-mpl-transfected UT7 cells with TPO could increase the expression of GPIIb/IIIa (Porteu et al., 1996). This TPO effect was associated with increased expression and binding activity of PU.1/ Spi-1, a member of the Ets transcription factor family (Doubeikovski et al., 1997). Overexpression of PU.1/ Spi-1 enhanced GPIIb promoter activity, suggesting that PU.1/Spi-1 was a TPO-inducible transcription factor responsible for regulation of megakaryocytespecific gene expression. Further analysis of the GPIIb gene promoter by transfection of a series of the promoter fragments indicated that the TPOresponsive cis element was within an enhancer region of the promoter, which included GATA- and Etsbinding sites. PU.1/Spi-1 was strongly and preferentially bound to this region. Another protein, named nucleosome assembly protein (NAP), is reportedly upregulated in
hematopoietic cells by TPO (Cataldo et al., 1999). Although this protein is thought to affect megakaryocyte polyploidation, the mechanism for regulation of its gene expression by TPO is unclear.
BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY
Unique biological effects of activating the receptors TPO receptor binding initiates at least three major events in cells. The first is initiation of signal transduction pathways that promote cell growth and/or cellular differentiation. The second is the activation of signal transduction pathways that prevent apoptosis. The third is internalization and degradation of the receptor±ligand complex (Fielder et al., 1996, 1997; Broudy et al., 1997; Li et al., 1999). All of these have been discussed above.
Phenotypes of receptor knockouts and receptor overexpression mice Homozygous c-mpl knockout mice had platelet and megakaryocyte numbers that were 10±15% of normal but had normal numbers of white and red blood cells (Gurney et al., 1994; Alexander et al., 1996b). They had a compensatory rise in the circulating level of thrombopoietin. Analysis of the bone marrow of these mice (Alexander et al., 1996b; Carver-Moore et al., 1996) showed that while the megakaryocyte progenitor cell (Meg-CFC) levels were about 5% of normal, the erythroid (E-CFC) and myeloid (G-CFC) progenitor cell levels were also reduced to 20±30% of normal. Heterozygous knockout animals were entirely normal. c-mpl is the only member of the hematopoietin receptor family to be identified as a proto-oncogene. TPO and c-Mpl provide a very potent hematopoietic stimulus and excess in vivo activation of either produces serious consequences. Ligand-dependent activation of c-Mpl via overexpression of TPO causes increased platelet reactivity (Montrucchio et al., 1996; Fontenay-Roupie et al., 1998), myelofibrosis (Yan et al., 1995, 1996; Villeval et al., 1997; Abina et al., 1998), a myeloproliferative syndrome (Villeval et al., 1997), and osteosclerosis (Yan et al., 1996; Villeval et al., 1997). As described in the introduction to this
1964 David J. Kuter and Junzhi Li
THERAPEUTIC UTILITY
concentration, reduce the stimulatory effect on megakaryocyte growth, and decrease platelet production. A similar experiment had been previously performed in rats in which the infusion of excess platelets (containing c-Mpl) was followed by a reduction in TPO levels, megakaryocyte growth, and platelet production (Jackson et al., 1984; Kuter and Rosenberg, 1990). Also, after transfusing platelets into thrombocytopenic patients with aplastic anemia, the elevated TPO levels were reduced. It should be noted that all of these sMpl experiments have been done with the extracellular portion of recombinant murine c-Mpl and not with the `native sMpl'. The latter is a naturally occurring c-mpl mRNA splice variant that is predicted to encode a protein containing both intracellular and extracellular regions of c-Mpl but lacking the transmembrane domain. When thrombocytopenic irradiated mice were treated with multiple injections of the extracellular portion of recombinant murine c-Mpl, platelet recovery was delayed, suggesting that the injected sMpl blocked the interaction of endogenous TPO with the c-Mpl on megakaryocytes. The platelets produced in these sMpl-treated animals did not show a reduction in JAK2 protein in platelets ± something which was observed in platelets from thrombocytopenic control animals and attributed to the action of the high concentration of endogenous TPO on the c-Mpl receptor (Nishiyama et al., 1998). Paradoxically, infusion of the sMpl into normal mice for 7 days caused a 50% increase in the platelet count but no change in the red or white blood cell number (Sheridan et al., 1997); infusion of control 1% normal mouse serum or heat-inactivated sMpl had no effect. These results were interpreted as being due to removal of the normal inhibitory effect TPO has on the shedding of platelets from megakaryocytes (Sheridan et al., 1997). It had previously been demonstrated that excess TPO suppressed in vitro proplatelet formation from megakaryocytes (alleviated by addition of sMpl) and that excess TPO suppressed in vivo platelet production (Choi et al., 1996; Nagahisa et al., 1996). Whether longer infusion of sMpl would eventually have reduced total megakaryocyte growth and platelet production was not studied.
Effect of treatment with soluble receptor domain
Effects of inhibitors (antibodies) to receptors
The effect of infusing soluble murine c-Mpl (sMpl) into mice has produced strikingly variable results. Presumably infusion of the soluble receptor would bind the available TPO in the circulation, lower its
Antibodies that activate the c-Mpl receptor have been made. A murine monoclonal antibody, BAH-1, made against human megakaryocyte cells binds the c-Mpl receptor. It stimulates Meg-CFC growth in vitro just
review, expression in mice of the fusion protein envmpl made by the myeloproliferative leukemia virus (MPLV) produced a hematological disorder characterized by multilineage proliferation and differentiation (Wendling et al., 1986; Wendling and Tambourin, 1991). Cells in this disease became growth factor-independent and showed autonomous growth in vitro. Furthermore MPLV could directly transform committed and multipotential hematopoietic progenitor cells in vitro leading to the creation of factor-independent immortalized lines that could differentiate spontaneously (Wendling and Tambourin, 1991). However, overexpression of c-mpl in mice using a murine retroviral vector produced hepatosplenomegaly, massive expansion of erythroblasts, no early leukocytosis or thrombocytosis, thrombocytopenia at late stage, and death within 9± 12 weeks (Cocault et al., 1996). The enormous stimulation of erythroblasts and the lack of a multilineage myeloproliferative syndrome in these mice differ from the effects seen in either the TPO or v-mpl overexpression mice.
Human abnormalities No human abnormalities of c-Mpl have yet been described. c-mpl gene structure is normal in patients with essential thrombocythemia (Li et al., 1996) and other myeloproliferative disorders. The amount of c-Mpl on platelets in polycythemia vera and essential thrombocythemia are markedly reduced (Li et al., 1996; Moliterno et al., 1998). The pathophysiological significance of this is unknown. The receptor is found on cells from many nonlymphoid hematological malignancies but not on cells from purely lymphoid malignancies or nonhematopoietic solid tumors (Columbyova et al., 1995; Drexler et al., 1996; Graf et al., 1996; Matsumura et al., 1996; Quentmeier et al., 1996; Bredoux et al., 1997; Hirai et al., 1997). There is a suggestion that the presence of c-mpl in acute myeloid leukemia cells confers a poor prognosis (Vigon et al., 1993a; Wetzler et al., 1997).
TPO Receptor 1965 like TPO and in vivo expands the number of megakaryocyte progenitor cells in myelosuppressed mice (Deng et al., 1997). Such agonistic antibodies will probably have no advantage over recombinant TPO or TPO peptide mimetics in stimulating platelet production in vivo but could therapeutically serve to target cells expressing c-Mpl for the purpose of drug delivery or bone marrow purging. Antibodies that may inhibit c-Mpl have also been described. One patient with persistent hypomegakaryocytic thrombocytopenia and elevated TPO levels was found to have antibody that was felt to bind to either c-Mpl or c-Mpl ligand (Nichol et al., 1996). Although TPO peptide mimetics have been developed that activate c-Mpl (Cwirla et al., 1997), no competitive antagonist peptides have yet been published.
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