N‐Glycosylation‐defective receptor for erythropoietin can transduce the ligand‐induced cell proliferation signal

Abstract

. apacity by the epidermal growth factor receptor is glycosylaion-dependent [6,7]. N-Glycosylation at a specific site of the nsulin receptor fl subunit appears to be important for the eceptor activation and transmembrane signaling [4,5]. Degly-:osylation of the basic fibroblast growth factor receptor results n the loss of the ligand-binding [8] and sialylation of the Ninked sugars in the somatostatin receptor is required for mainenance of the high-affinity binding state [9].EPO transduces the proliferation and differentiation signal hrough its receptor on erythroid precursor cells and some )ther lineage cells [16,17]. By molecular cloning of murine 18,19] and human [20,21] EPOR, the mature 53-kDa EPOR ms been predicted to consist of an extracellular 225-226 amino lcid domain, 22 amino acid transmembrane region, and an ntracellular 236 amino acid domain. A single N-glycosylation ;ite is present in the extracellular domain in EPOR. Studies on "Corresponding author. Fax: (81) (75) 753-6274.4bbreviations: EPO, erythropoietin; EPOR, erythropoietin receptor; BHK, baby hamster kidney; IL-3, interleukin 3; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. the IL-3-dependent Ba/F3 cells that were stably transfected with the wild-type EPOR and became EPO-responsive indicated that the EPOR with 66 kDa was the mature glycosylated form exposed on the cell surface [22] and that the 72-kDa EPOR could be an EPO-induced phosphorylated form of 66-kDa EPOR [23,24]. BHK cells stably transfected with the wildtype EPOR cDNA and the N-glycosylation-defective mutant expressed 68-kDa and 66-kDa EPOR, respectively and the two forms of EPOR were not different in the ligand-binding assay [14]. More recently, Sawyer and Hankins [25] reported that an EPO-dependent murine erythroleukemia cell line, which was highly sensitive to EPO, expressed the 78-kDa high molecular mass form of EPOR and that the 78-kDa EPOR was a functional form of EPOR because it was correlated well with cell surface expression, endocytosis and EPO-induced phosphorylation. They also indicated that the 78-kDa EPOR resulted from high N-glycosylation rather than phosphorylation, suggesting the importance of N-glycosylation for expression of the functional EPOR or the ligand-sensitive EPOR. These findings of EPOR prompted us to examine the role of the receptor glycosylation using the N-glycosylation-defective EPOR. Herein, we prepared EPO-dependent cell lines expressing wild-type or mutant EPOR, and compared the ligand-saturation curves with respect to EPO binding and EPO-dependent cell proliferation. Materials and methods I. EPO-dependent cells expressing mouse wild-o,pe EPOR and Nglveosylation-defective EPORThe plasmid for expression of N-glycosylation-defective EPOR (Asn~Gln) was prepared [14] from the plasmid for expression of wild-type EPOR (pXM 190) [18]. IL-3-dependent BaF-BO3 cells, a subclone of the mouse pro-B cell line (Ba/F3), were maintained in the medium consisting of RPMI1640, 10% fetal calf serum and 10% WEHI3B conditioned medium as a sourc...

An extracellular region of the erythropoietin receptor of the subterranean blind mole rat Spalax enhances receptor maturation

“…The N-glycosylation site per se is not sufficient to provide this advantage, because residues s46 -57 containing the Nglycosylation site did not improve EPO-R maturation to the same extent. In that respect, impaired N-glycosylation of EPO-R (46,47) had no measurable effect on cell-surface EPO-R levels, EPO binding kinetics, and EPO-induced cell proliferation. Carbohydrates attached to proteins play a role in multiple biochemical pathways including folding, stability, targeting, and clearance of proteins (48).…”

The role of mevalonate pathway intermediates in erythropoietin receptor signal transduction and surface expression

“…Glycosylation is thought to play a role in controlling EpoR cell surface expression. N-glycosylation-defective EpoR can still transduce the ligand-induced cell proliferation signal, but the level of expression of this mutant EpoR on the cell surface is significantly reduced [35]. Similar findings were obtained when the N-glycosylation inhibitor tunicamycin was used [36].…”

“…Previous studies have shown that inhibition of glycosylation by tunicamycin treatment leads to a decrease in the total number of Epo binding sites [36]. However, other studies suggested that glycosylation is not required for EpoR surface expression and signal transduction [35,36]. These studies used transfected forms of EpoR and not endogenous EpoR.…”

Second-generation non-hematopoietic erythropoietin-derived peptide for neuroprotection

“…First, since our in silico geometrical prediction and in vitro SPR analysis were based on only extracellular domains of EPORs, it is unclear how peptide binding induces the geometric event of membrane-spanned full-length EPOR. Second, endogenous EPOR, which would respond to our peptides, could be extracellularly glycosylated [ [61] , [62] , [63] ]. Although this glycosylation has been known to enhance the extracellular trafficking of EPOR rather than the binding affinity of EPO, its effect on binding with our peptides still remains in question.…”