The type I hematopoietic growth factor receptor family, of which c-Mpl is a member, consists of more than 20 molecules that bear 1 or 2 cytokine receptor motifs, an approximately 200–amino acid module containing 4 spatially conserved Cys residues, 14 β-sheets, and a juxtamembrane Trp-Ser-Xaa-Trp-Ser sequence (54
). In addition to the cytokine receptor motif(s), type I receptors contain a 20- to 25-residue transmembrane domain and a 70– to 500–amino acid intracellular domain containing short sequences that bind intracellular kinases and other signal-transducing molecules. The thrombopoietin receptor is expressed primarily in hematopoietic tissues, specifically in megakaryocytes, their precursors, and their progeny. For the most part, c-Mpl is constitutively expressed in these tissues, although receptor display is modulated by thrombopoietin binding and receptor internalization. A second potential level of c-Mpl regulation exists; multiple spliceoforms of the receptor have been described that vary in their biological activity, and 1 form can alter receptor catabolism. Although the proportion of the various isoforms of the receptor differs in different tissues, they have not yet been shown to exert a regulatory effect.
gene contains 12 exons and is organized like other members of the hematopoietic cytokine receptor family (55
). A site for initiation of c-Mpl
transcription resides 13 nucleotides upstream of the translation initiation codon, and although the promoter lacks conventional TATA and CAAT motifs, the 5′ flanking sequence contains consensus binding sequences for Ets and GATA transcription factors, proteins vital for the regulation of many megakaryocyte-specific genes. Analysis of c-Mpl
transcripts has identified several alternately spliced forms, including extracellular domain deletions (56
), an alternate intracellular domain (the K isoform; ref. 8
), and a prematurely truncated isoform containing a unique carboxyl terminus (the Mpl-tr isoform; ref. 57
). While potentially acting as a dominant-negative form of the receptor, and differentially expressed in certain cell types, the K isoform does not affect thrombopoietin signaling, as it does not interact with the wild-type receptor (58
). However, Mpl-tr may play a physiological role, as it is the only isoform expressed in both human and murine cells. Of note, expression levels of c-Mpl are low, with only 25–100 surface receptors present per platelet (59
). The origin of the poor expression of c-Mpl appears to be related to the c-Mpl-tr isoform, as its coexpression with full-length c-Mpl leads to rapid degradation of the latter (57
). However, whether this physiology is reflected in thrombopoietin signal regulation is, at present, only speculative.
Another aspect of c-Mpl regulation under intense study is its expression on hematopoietic cells of patients with myeloproliferative disorders (MPDs). While easily detectable on normal marrow megakaryocytes and platelets, the receptor is decreased on cells from patients with polycythemia vera and other myeloproliferative diseases (61
). While the molecular basis for this is not understood, it could be related to the hypersensitivity to cytokines and signaling abnormalities seen in these disorders. Another clue to this finding may lie in 2 recent observations, that coexpression of the signaling kinase JAK2 is vital for hematopoietic cytokine receptor expression (63
), and that the activity of this kinase is altered in a substantial number of patients with MPDs.
Upon binding, cognate ligand hematopoietic cytokine receptors such as c-Mpl are activated to transmit numerous biochemical signals. The molecular details of this process are now well understood based on studies of the erythropoietin receptor (EpoR). The EpoR exists in a homodimeric state in the absence of ligand, in a conformation that holds the cytoplasmic domains 73 Å apart (64
). Upon ligand binding, receptor conformation shifts, bringing the cytoplasmic domains within 39 Å of one another. Additional studies indicate that the membrane-proximal box1 and box2 cytoplasmic domains constitutively bind JAK family kinases, even in an inactive state. Upon ligand binding, the closer juxtaposition of the 2 tethered kinases allows their cross-activation, initiating signal transduction. The active JAK kinase then phosphorylates (a) tyrosine residues within the receptor itself; (b) molecules that promote cell survival and proliferation, including the STATs, PI3K, and the MAPKs; and (c) those that limit cell signaling, including the SHP1 and SHIP1 phosphatases and SOCSs (Figures and ).
Figure 2 Hematopoietic cytokine receptor architecture and mechanism of initial signaling. A stylized hematopoietic cytokine receptor is shown, depicting the 1 or 2 cytokine receptor motifs (C, Cys; WS, Trp-Ser-Xaa-Trp-Ser), the transmembrane domain, and the box1 (more ...)
Figure 3 Signaling pathways activated by thrombopoietin. A stylized drawing of c-Mpl is shown in the activated (phosphorylated) form. Once phosphorylated, Tyr112 serves as a docking site for STAT3 and STAT5, both activated by thrombopoietin in megakaryocytes, (more ...)
Additional insights into how the JAK kinases are regulated come from domain analysis of the proteins. All 4 members of the family (JAK1, JAK2, JAK3, and TYK2) display 3 major domains, JH1 (JAK homology 1), JH2, and FERM (four-point-one, ezrin, radixin, moesin), the latter responsible for binding to the cytoplasmic domain of the cytokine receptors (Figure ). The JH1 domain carries the kinase activity of JAKs, and while JH2 bears significant homology to JH1, its active site is altered and inactivated and is thus termed the pseudokinase
K domain. The function of JH2 was identified by differential expression studies; the JH1 domain is an active kinase when expressed alone, whereas the activity of a JH1/JH2 polypeptide is greatly blunted (65
). Thus, the JH2 domain regulates the kinase activity of JH1, a physiology put into structural terms by homology modeling of JH1/JH2; the JH2 domain interacts with the inactive, but not the active, conformation of the activation loop of JH1 (Figure ; ref. 66
), in a region of JH2 shown to be vital for kinase regulatory activity.
Figure 4 A molecular model of JAK2 JH1 and JH2 domains. Based on the tertiary structure of the dimer receptor tyrosine kinase FGF receptor-4, the model depicts the ATP-binding site (yellow), the kinase active site (orange), the activation loop of JH1 in both inactive (more ...)
Once c-Mpl is activated by thrombopoietin engagement, its multiple effects on HSCs, megakaryocytes, and platelets are mediated by a series of biochemical signaling events. Thrombopoietin activates both JAK2 and TYK2 in c-Mpl–bearing cell lines, although only JAK2 is essential for signaling and is the predominant isoform activated in primary megakaryocytes (67
). By generating a complex composed of the phosphatase SHP2, a scaffolding Gab/IRS protein, and the p85 regulatory subunit of PI3K, thrombopoietin stimulation of megakaryocytes and their precursors activates PI3K and its immediate downstream effector Akt (PKB) (Figure ) (68
). Blocking this pathway inhibits thrombopoietin-induced cell survival and proliferation (70
). In the mature platelet, the hormone enhances α-granule secretion and aggregation induced by thrombin in a PI3K-dependent fashion (71
). The pathways downstream of Akt in megakaryocytes and platelets are under study and include the transcription factor FOXO3a, the cell cycle inhibitor p27, and glycogen synthase kinase-3β (GSK3β). In addition to PI3K, thrombopoietin stimulates 2 of the MAPK pathways (Figure ), p42/p44 ERK1 and ERK2 (72
) and p38 MAPK (73
), events mediated by receptor phosphorylation, binding and phosphorylation of Grb2, SHC, and SOS, and exchange of GDP for GTP on Ras (74
). The functional consequences of these events include induction of the transcription factor HoxB4 and expansion of HSCs mediated by p38 MAPK (73
); translocation of the transcription factor HoxA9 from cytoplasm to nucleus, which also favorably affects HSC expansion (75
); the ERK1/2–induced proliferation and polyploidization of megakaryocytes (76
); and augmented thrombin-induced liberation of phospholipase A2
and platelet activation (77