Extracellular Domain N-Glycosylation Controls Human Thrombopoietin Receptor Cell Surface Levels

Abstract

The thrombopoietin receptor (TpoR) is a type I transmembrane protein that mediates the signaling functions of thrombopoietin (Tpo) in regulating megakaryocyte differentiation, platelet formation, and hematopoietic stem cell renewal. We probed the role of each of the four extracellular domain putative N-glycosylation sites for cell surface localization and function of the receptor. Single N-glycosylation mutants at any of the four sites were able to acquire the mature N-glycosylated pattern, but exhibited a decreased Tpo-dependent JAK2–STAT response in stably transduced Ba/F3 or Ba/F3-JAK2 cell lines. The ability of JAK2 to promote cell surface localization and stability of TpoR required the first N-glycosylation site (Asn117). In contrast, the third N-glycosylation site (Asn298) decreased receptor maturation and stability. TpoR mutants lacking three N-glycosylation sites were defective in maturation, but N-glycosylation on the single remaining site could be detected by sensitivity to PNGaseF. The TpoR mutant defective in all four N-glycosylation sites was severely impaired in plasma membrane localization and was degraded by the proteasome. N-glycosylation receptor mutants are not misfolded as, once localized on the cell surface in overexpression conditions, they can bind and respond to Tpo. Our data indicate that extracellular domain N-glycosylation sites regulate in a combinatorial manner cell surface localization of TpoR. We discuss how mutations around TpoR N-glycosylation sites might contribute to inefficient receptor traffic and disease.

The Extracellular Domain of Notch2 Increases Its Cell-Surface Abundance and Ligand Responsiveness during Kidney Development

“…Importantly, N21 and N2 are equally abundant at the cell surface indicating that surface distribution is determined by sequences in the Notch ECD, not ICD. Although other transmembrane proteins may contain trafficking signals in their ECD (Albu and Constantinescu, 2011; Steiner et al, 2008; VandenBussche et al, 2009), the only indication that the Notch2 ECD may play a role in its trafficking comes from a study on the importance of S1 cleavage to the exocytosis of N1, but not N2 (Gordon et al, 2009). Second, in luciferase complementation imaging assays (Ilagan et al, 2011) that quantified the amount of NICD released from N21 and N1 in cultured human embryonic kidney cells in response to ligand or EGTA, N21 consistently released more NICD.…”

Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants

“…13 The TpoR mutant devoid of all 4 N-glycosylation sites (TpoR N1234tQ) failed to support STAT5 activation in the presence of CALR mutants ( Figure 4D). When individual Asn residues were restored in TpoR N1234tQ background, N117 (N1, the most N-terminal) residue was shown to be the most activating ( Figure 4H), N298 and N358 were not activating, and N178 supported weak activation only by CALR ins5 (Figure 4E-G).…”

“…The TpoR Asn mutants were as described. 13 Viral infections were performed as previously described. 11 shRNA against JAK2 and MPL/TpoR cloned into PRRLsin-PGK-eGFP-WPRE lentiviral vector (Généthon, Evry, France) were used as previously described.…”

Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes

“…At least for the closely related Epo receptor, residues in the N‐terminal pseudokinase domain linker also mediate interactions between Jak2 and its cognate receptor . Interactions between Jak2 and Mpl are influenced by the receptor's N‐glycosylation status, particularly at Asn117 . This may be related to conformational restrictions on the dimerization competency of the newly synthesized receptor in the ER, as suggested by recent structural studies .…”

“…This raises the potential for trapped Mpl to initiate stress response signals that contribute to MPN pathogenesis, as has recently been implicated in the context of breast cancer . Notably, Albu and Constantinescu recently showed that N‐glycosylation mutants of Mpl bind and respond to Tpo, suggesting that the immature Mpl glycoproteins are likely to be functional once delivered from autophagosomal compartments .…”