Oncogene (2002) 21, 3359 ± 3367 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc The molecular and cellular biology of thrombopoietin: the primary regulator of platelet production Kenneth Kaushansky*,1 and Jonathan G Drachman1 1Division of Hematology, University of Washington School of Medicine, 1959 NE Paci®c Street, Seattle, Washington, WA 98195, USA The term thrombopoietin (TPO) was ®rst coined in 1958 represents the receptor binding domain (RBD) of the and used to describe the humoral substance responsible hormone; the carboxyl-terminal domain bears no for causing the platelet count to rise in response to resemblance to any known proteins, but contains thrombocytopenic stimuli. Despite much progress during multiple sites of both N- and O-linked carbohydrate the 1980s in the puri®cation and characterization of the (Homan et al., 1996). This latter feature accounts for humoral regulators of lymphocyte, erythrocyte, mono- the large discrepancy between the predicted and actual cyte and granulocyte production, the successful search to molecular weight of the protein; nearly 50% of the 70- purify and molecularly clone thrombopoietin did not kDa TPO molecule is carbohydrate. Like EPO, growth begin until the oncogene v-mpl was discovered in 1990. hormone and other members of the hematopoietic Since that time the proto-oncogene c-mpl was identi®ed growth factor family, the amino-terminal domain of and, based on homology arguments, believed to encode a TPO is predicted to fold into a left-handed four-helix hematopoietic cytokine receptor, a hypothesis later bundle protein, oriented up-up-down-down, with two proven when the cytoplasmic domain was linked to the overhand loops connecting the ®rst two and last two ligand binding domain of the IL-4 receptor and shown to helices (Wells, 1996). Our laboratory and others have support the IL-4 induced growth of hematopoietic cells demonstrated that the amino-terminal RBD of TPO is (Skoda et al., 1993). Finally, two dierent strategies adequate for receptor binding, signaling, and support- using c-mpl lead to the identi®cation of a novel ligand for ing cellular proliferation (Bartley et al., 1994; Linden the receptor in 1994 (de Sauvage et al., 1994; Lok et al., and Kaushansky, 2000). Several of the residues critical 1994; Bartley et al., 1994), a protein that displays all the for function of the hormone have been mapped in this biologic properties of TPO. This review attempts to region (Pearce et al., 1997; Park et al., 1998). Not distill what has been learned of the molecular and surprisingly, the human ± mouse inter-species homology cellular biology of TPO and its receptor during the past of TPO is greatest in the RBD (93%), although the several years, and links this information to several new carboxyl-terminal carbohydrate rich region retains insights into human disease and its treatment. 74% homology, suggesting that it also serves an Oncogene (2002) 21, 3359 ± 3367. DOI: 10.1038/j/onc/ important physiologic function. One such function is 1205323 to promote survival of the hormone in the circulation (Harker et al., 1996). Recently, our laboratory and Keywords: thrombopoietin; hematopoiesis; megakaryo- others have shown that this region also functions to cyte; signal transduction enhance secretion (Linden and Kaushansky, 2000; Muto et al., 2000). Molecular biology of thrombopoietin Molecular biology of Mpl Using three separate strategies ®ve groups puri®ed or Alternative splicing generates multiple forms of Mpl cloned TPO and reported their results in 1994 (reviewed in Kaushansky et al., 1995). The cloned The gene encoding the TPO receptor, c-mpl, is located human TPO cDNA predicts a polypeptide of 353 on human chromosome 1p34 and is composed of 12 amino acids including a 21 amino acid secretory leader exons (Mignotte et al., 1994). The corresponding sequence (Kaushansky, 1998), that can conveniently be murine gene is on chromosome 4 and has similar divided into two domains: the amino-terminal 154 organization and spice sites (Alexander and Dunn, residues of the mature polypeptide bear striking 1995). Variation in splicing at the 3' end of the sequence homology with erythropoietin (EPO) and molecule results in the formation of three distinct mRNA species in humans. The predominant form encodes the full-length protein (P-form) with a *Correspondence: K Kaushansky; transmembrane domain and 122 cytoplasmic residues. E-mail: [email protected] A second species of human Mpl mRNA is due to a The molecular biology of Mpl and TPO K Kaushansky and JG Drachman 3360 read through beyond the exon 10 splice donor site. The divided into two domains of seven b-strands, forming resulting K-form of the receptor, or Mpl-K, diverges antiparallel b-sheets (de Vos et al., 1992). Four from the native sequence after the ninth cytoplasmic conserved cysteine residues and a pentapeptide Trp- amino acid and terminates within intron 10 with 66 Ser-Xaa-Trp-Ser motif near the transmembrane do- predicted cytoplasmic residues (Vigon et al., 1992). main further characterize the CRM. Recent evidence This protein has no known biological activity, has not demonstrates that the membrane distal CRM exerts a been shown to transduce a proliferative signal, and negative regulatory role on signaling, as its deletion or could potentially act as a dominant negative receptor substitution results in constitutive proliferative signal- by heterodimerizing with Mpl-P after ligand binding. ing (Sabath et al., 1999). Despite extensive investiga- The third mRNA species, found in both human and tion, Mpl appears to function as a homodimer after murine cells, encodes a potentially secreted form of ligand binding, and does not form heterologous Mpl due to alternative splicing of exon 8 directly to signaling complexes with any of the known signaling exon 11, eliminating the juxtamembrane WSXWS chains (i.e. bc, gc and gp130). This stoicheometry is motif, the transmembrane domain, and the initial similar to that of the EPO, G-CSF, prolactin, and cytoplasmic residues (Vigon et al., 1992; Skoda et al., growth hormone receptors. 1993; Mignotte et al., 1994). It is not known whether Immediately downstream of the hydrophobic trans- this protein is actually secreted by hematopoietic cells membrane domain (25 residues) is a relatively short nor whether it is able to bind TPO. However, an signaling domain (121 amino acids in mice; 122 aa in engineered, puri®ed soluble form of Mpl has been humans). Although this portion of the receptor is well shown in vitro to bind exogenous TPO and block conserved across species (91% identity between murine cytokine activity (Kaushansky et al., 1995). Addition- and human proteins), it does not have signi®cant ally, alternative splice donor sites in exon 4 of murine homology to other known proteins. Like other mpl (but not human) result in the deletion of eight members of the hematopoietic cytokine receptor amino acids (24 nucleotides) in the extracytoplasmic family, the signaling region does not encode a tyrosine domain (Alexander et al., 1995). The signi®cance of kinase domain, tyrosine phosphatase, or other enzy- this altered form of the receptor is not known. matic functions. Therefore, all of the signaling events initiated by TPO binding to Mpl must be due to homodimerization and the recruitment of signaling The mpl promoter molecules to docking sites on the signaling complex The mpl promoter regulates lineage-speci®c expression (see below). on the cell surface of megakaryocytic precursors, mature megakaryocytes, platelets, and hematopoietic stem cells. Analysis of the promoter sequence reveals The regulation of thrombopoietin levels the presence of Ets, Sp1, and GATA-1 binding sites (Alexander et al., 1995; Deveaux et al., 1996; Mignotte The transient induction of severe thrombocytopenia et al., 1994). Transcription is not induced by any results in a relatively rapid restoration of platelet levels known external signals, such as the binding of TPO to followed by a brief period of rebound thrombocytosis. its receptor. Both a 700 and 200 nucleotide fragment of In both acute, antibody-mediated and myelosuppres- the human mpl promoter are sucient to direct sive therapy-induced thrombocytopenia, plasma con- lineage-speci®c expression in cell lines (Deveaux et al., centrations of TPO vary inversely with platelet counts, 1996; Mignotte et al., 1994) while a 2.0 kb fragment of rising to maximal levels within 24 hours of the onset of the murine mpl promoter is sucient to direct lineage- profound thrombocytopenia (Chang et al., 1996; speci®c expression in transgenic mice (Ziegler et al., Emmons et al., 1996). Two models have been advanced 1998). However, when cDNA encoding mpl was to explain the regulation of TPO levels. In the ®rst, transgenically expressed in mpl7/7 mice using the production of the hormone is constitutive, but its 2.0 kb promoter, the progeny had an unexpectedly consumption is determined by the mass of Mpl high platelet count (Ziegler et al., 1998 and our receptors accessible to the plasma (i.e. those present unpublished results). This result suggests that there on platelets and megakaryocytes (Kuter and Rosen- may be additional regulatory promoter elements up- berg, 1995; Stoel et al., 1996). In this way, stream of the 2.0 kb fragment or that the untranslated thrombocytopenia reduces peripheral blood TPO regions of mpl may modulate gene expression. destruction, resulting in elevated blood levels of the hormone, which drive megakaryopoiesis and platelet recovery. The weight of current evidence supports an The Mpl receptor is a homodimer important role for this mechanism in the regulation of The Mpl receptor is a type I transmembrane protein TPO production; platelets can deplete plasma of the and belongs to the hematopoietic cytokine receptor cytokine, TPO mRNA levels in the liver and kidney do superfamily. The extracytoplasmic domain, like the not vary in states of profound thrombocytopenia or common beta chain (bc) shared by the IL-3, IL-5, and thrombocytosis, and TPO knockout mice display a GM ± CSF receptors, is characterized by duplication of gene dosage eect indicating that active regulation of the cytokine receptor module (CRM).