TYROSINE 462 OF THE MEMBRANE-PROXIMAL F′-G′ LOOP OF MURINE Mpl IS NOT ESSENTIAL FOR HIGH-AFFINITY BINDING OF THROMBOPOIETIN

Sabath, Diana F.; Lin, Nancy; Sabath, Daniel E.; Kaushansky, Kenneth; Broudy, Virginia C.

doi:10.1006/cyto.1999.0531

Cited by 9 publications

(10 citation statements)

References 31 publications

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“…1 and 2). This result is in keeping with the findings of several other independent approaches in the literature (33)(34)(35). It is also consistent with the fact that the Mpl-EC-D2 is not involved in ligand binding but is instead needed for receptor activation (36).…”

Section: Discussionsupporting

confidence: 92%

Identification of the Residues in the Extracellular Domain of Thrombopoietin Receptor Involved in the Binding of Thrombopoietin and a Nuclear Distribution Protein (Human NUDC)

Chen¹,

Yu²,

Zhang³

et al. 2010

Journal of Biological Chemistry

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Thrombopoietin (TPO) and its receptor (Mpl) have long been associated with megakaryocyte proliferation, differentiation, and platelet formation. However, studies have also shown that the extracellular domain of Mpl (Mpl-EC) interacts with human (h) NUDC, a protein previously characterized as a human homolog of a fungal nuclear migration protein. This study was undertaken to further delineate the putative binding domain on the Mpl receptor. Using the yeast two-hybrid system assay and co-immunoprecipitation, we identified that within the Mpl-EC domain 1 (Mpl-EC-D1), amino acids 102-251 were strongly involved in ligand binding. We subsequently expressed five subdomains within this region with T7 phage display. Enzymelinked immunosorbent binding assays identified a short stretch of peptide located between residues 206 and 251 as the minimum binding domain for both TPO and hNUDC. A series of sequential Ala replacement mutations in the region were subsequently used to identify the specific residues most involved in ligand binding. Our results point to two hydrophobic residues, Leu 228 and Leu 230 , as having substantial effects on hNUDC binding. For TPO binding, mutations in residues Asp 235 and Leu 239 had the largest effect on binding efficacy. In addition, deletion of the conservative motif WGSWS reduced binding capacity for hNUDC but not for TPO. These separate binding sites on the Mpl receptor for TPO and hNUDC raise interesting implications for the cytokine-receptor interactions. TPO2 has long been studied as the primary cytokine regulator of megakaryocyte development. Its activity is mediated by the thrombopoietin receptor (Mpl), a member of the cytokine receptor superfamily (1-4). Genetic disruption of either cytokine or receptor drastically interferes with platelet production, indicating the major role TPO and Mpl play in the activation of megakaryopoiesis and platelet production (5-7). However, despite the platelet volume decrease, the megakaryocytes and platelets that are produced remain morphologically normal and are adequate to prevent severe hemophilia in murine experiments (8). This observation leaves open the possibility for other cytokines and receptors to have an overlapping biological potency. One such candidate we have investigated as an additional ligand for Mpl is known as hNUDC. Despite having no detectable sequence similarity with TPO, hNUDC has been shown to have similar megakaryopoietic effects (9, 10). TPO and hNUDC also exhibited a communal signal transduction cascade in several megakaryocytic cells expressing Mpl (11,12).Human NUDC was initially identified and cloned as a nuclear migration protein based on the similarity of its C terminus to that of fungal NUDC from Aspergillus nidulans (13). Numerous studies have implicated hNUDC as an endogenous factor involved in cell mitotic spindle formation, cell proliferation, cell cytokinesis, and nuclear migration in a wide variety of experimental animal cells (14 -16). Although earlier studies have suggested the mechanisms by which hNUDC exerts i...

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Section: Discussionsupporting

confidence: 92%

Identification of the Residues in the Extracellular Domain of Thrombopoietin Receptor Involved in the Binding of Thrombopoietin and a Nuclear Distribution Protein (Human NUDC)

Chen¹,

Yu²,

Zhang³

et al. 2010

Journal of Biological Chemistry

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“…Recent studies demonstrate that the membranedistal, but not the membrane-proximal, CRH domain of c-Mpl is a receptor domain for TPO. Binding of radioactively labeled TPO to BaF3 cells expressing mutants of murine c-Mpl was abolished when the distal CRH domain was either deleted or replaced with a second copy of the proximal CRH domain (51). These studies also showed that BaF3 cells expressing these c-Mpl mutants grew constitutively even in the absence of TPO, indicating that the membrane distal CRH domain also functions to inhibit autophosphorylation in the absence of TPO.…”

Section: Implications For the Mechanism Of Signal Transduction By C-mplmentioning

confidence: 76%

Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment

Feese

Tamada

Kato

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO 163) to a 2.5-Å resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO 163 (1) predicted the existence of a potent, lineage-specific soluble factor, which they called thrombopoietin (TPO), that stimulates megakaryocytopoiesis and platelet production. It was not until 1994 that unequivocal evidence for the existence of this elusive molecule was provided by the nearly simultaneous isolation and cloning of TPO by five independent research groups (2-6). This cytokine has proven to be a primary factor in megakaryocytopoiesis from megakaryocyte colony formation to platelet production and the differentiation and proliferation of progenitor cells of multiple hematopoietic lineages (7). As such, TPO is being investigated for its potential to treat thrombocytopenia resulting from AIDS and chemotherapy and radiation treatments for cancer and leukemia and for the in vivo and ex vivo expansion of hematopoietic stem cells for bone marrow transplantation.Human TPO (hTPO) is a heavily glycosylated protein with two distinct regions. The 153-residue N-terminal region is homologous to human erythropoietin (EPO) with which it shares 23% sequence identity and is sufficient for receptor binding and signal transduction (2,3,8). The 179-residue C-terminal region has a large number of proline and glycine residues and six N-linked glycosylation sites. Its function is not known, although recent work indicates a role in secretion and protection from proteolysis (9, 10).The TPO receptor c-Mpl was first identified as an oncogene of the murine myeloproliferative leukemia virus (11, 12) that was able to immortalize hematopoietic progenitor cells and was later cloned from human and mouse (13,14). c-Mpl is expressed in some pluripotent hematopoietic stem cells (15) and in the megakaryocyte lineage from progenitor cells to platelets (16). It is a class I cytokine receptor of the hematopoietic superfamily of receptors and signals by the JAK͞STAT, Ras, and mitogenactivated protein kinase pathways (17-21). Class I hematopoietic receptors bind to their cytokine ligands by Ϸ200-aa Ig-like extracellular domains called cytokine receptor homology (CRH) or hematopoietic receptor domains that contain a distinctive WSXWS sequence motif (13).Cytokines possess two distinct interaction sites that bind with differing affinities [high affinity (nanomolar range) and low affinity (micromolar range)] to the same cytokinerecognition surface of the CRH domain. Crystal structures of human EPO and human growth hormone (hGH) in complex with the extracellular CRH domains of their receptors (22, 23) have shown the cytokine-CRH interaction in detail. However, unlike EPO receptor (EPOR) and hGH receptor (hGHR), which have only one CRH domain, c-Mpl belongs to a subset of hematopoietic ...

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“…IL-2R␣ cDNA (untagged) and human wild-type or JAK3 A572V mutant cDNAs were subcloned in pREX-IRES-CD4 (34). FLAG-tagged murine thrombopoietin receptor (TpoR) containing the IL-7R signal sequence (35) was cloned in pMX-IRES-GFP1.1 (30). Myc-tagged type II transforming growth factor-␤ (T␤RII) in pcDNA3 was described (36).…”

Section: Methodsmentioning

confidence: 99%

Ligand-independent Homomeric and Heteromeric Complexes between Interleukin-2 or -9 Receptor Subunits and the γ Chain

Malka

Hornakova

Royer

et al. 2008

Journal of Biological Chemistry

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TYROSINE 462 OF THE MEMBRANE-PROXIMAL F′-G′ LOOP OF MURINE Mpl IS NOT ESSENTIAL FOR HIGH-AFFINITY BINDING OF THROMBOPOIETIN

Cited by 9 publications

References 31 publications

Identification of the Residues in the Extracellular Domain of Thrombopoietin Receptor Involved in the Binding of Thrombopoietin and a Nuclear Distribution Protein (Human NUDC)

Identification of the Residues in the Extracellular Domain of Thrombopoietin Receptor Involved in the Binding of Thrombopoietin and a Nuclear Distribution Protein (Human NUDC)

Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment

Ligand-independent Homomeric and Heteromeric Complexes between Interleukin-2 or -9 Receptor Subunits and the γ Chain

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