Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes

Abstract

Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: Human Erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA-mediated knockdown of Jak2 led to Mpl trapping in the ER. Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially co-localize with ER-Tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically-encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER-Golgi and autolysosome secretory pathways, as well as recycling.

An incomplete trafficking defect to the cell-surface leads to paradoxical thrombocytosis for human and murine MPL P106L

“…For the second question, it has been shown in cell lines that MPL WT could exit the ER and enter the autophagy compartment before going to the cell surface in an immature N-glycosylated form. 41 Here, using surface immunoprecipitation, we detected an immature form of MPL P106L on the cell surface (supplemental Figure 6B) that partially colocalizes with LC3 (supplemental Figure 6A). Thus, for a small fraction of MPL P106L, there is an escape mechanism from the ER mediated via the nonconventional autophagy pathway, leading to low cell-surface MPL P106L and likely explaining the internalization defect observed.…”

Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms

“…1 However, CALR was previously thought to function as a chaperone in the endoplasmic reticulum (ER), and its relation to the c-MPL and JAK2 pathway remains poorly understood. 9 In this work, we used UT-7/TPO cells and induced pluripotent stem cell (iPS)-derived hematopoietic cells to study the mechanism(s) by which mutant calreticulin promotes development of MPNs.…”

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia

“…Note that WT Mpl migration during gel electrophoresis resolved the receptor into 2 different forms: the upper band represents mature glycosylated Mpl (indicating passage through the Golgi), and the lower band represents core-glycosylated Mpl (ER pattern). 13 Both forms were seen in cells expressing WT or K39N Mpl, but only the lower form was seen for Mpl mutants bearing the W272R mutation, indicating failure to progress from the ER to the Golgi.…”

“…13 This unconventional route bypasses the canonical ER-to-Golgi-to-plasma membrane delivery route for glycosylated proteins and can be promoted in cells by overexpressing Golgi reassembly stacking protein 55 (GRASP55). Because autophagy provides an alternative pathway for misfolded proteins to reach the cell surface, 22 we tested the hypothesis that overexpression of GRASP55 might partially restore Tpo-induced signaling in cells expressing the doubly substituted Mpl K39N/W272R.…”

“…11,12 An alternative pathway to the surface for Mpl is provided by an unconventional autophagy-linked secretory pathway. 13 It is important to determine the functional impact of CAMT mutations on Mpl signaling and trafficking, because clinical presentation, disease progression, and treatment options reflect the underlying cellular mechanisms. 6,14,15 In this study, the severity of CAMT type I disease in 3 siblings was associated with a homozygous double MPL K39N/W272R mutant that resulted in complete blocking of Mpl trafficking to the plasma membrane.…”