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

Abstract

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...

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms

“…The 4 somatic mutations we identified were affecting the extracellular ligand binding domain of the MPL, 30,31 whereas the 2 germline mutations affected the membrane proximal extracellular domain which appears to play a role in negative regulation of receptor activation. We initially hypothesized that the mutations might affect the affinity to TPO, however, the luciferase assay we performed in g2A cells showed that all identified mutants activated STAT5-induced transcription in the absence of stimulation with TPO.…”

Structural Features and Chaperone Activity of the NudC Protein Family

“…This potentially includes both Hsp90-dependent pathway 40 and direct chaperone activity 40,44 . Finally, there is a set of papers exploring the hypothesis that NudC is a secreted protein which functions by binding to the extracellular domain of the thrombopoietin receptor 45–50 .…”

Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants

“…Importantly, a soluble extracellular domain where all the 4 Asn sites were mutated to Gln failed to exert inhibition ( Figure 5B). However, CALR del52 mutant could still induce activation of the Tpo-binding-deficient TpoR mutant (D261A/L265A) 36 ( Figure 5C). Altogether these results show that activation of TpoR by CALR mutants is dependent on TpoR N-glycosylation sites, but independent of the Tpo binding site.…”