Oligomerization and Scaffolding Functions of the Erythropoietin Receptor Cytoplasmic Tail

Abstract

Signal transduction by the erythropoietin receptor (EPOR) is activated by ligand-mediated receptor homodimerization. However, the relationship between extracellular and intracellular domain oligomerization remains poorly understood. To assess the requirements for dimerization of receptor cytoplasmic sequences for signaling, we overexpressed mutant EPORs in combination with wild-type (WT) EPOR to drive formation of heterodimeric (i.e. WT-mutant) receptor complexes. Dimerization of the membrane-proximal portion of the EPOR cytoplasmic region was found to be critical for the initiation of mitogenic signaling. However, dimerization of the entire EPOR cytoplasmic region was not required. To examine this process more closely, we generated chimeras between the intracellular and transmembrane portions of the EPOR and the extracellular domains of the interleukin-2 receptor ␤ and ␥ c chains. These chimeras allowed us to assess more precisely the signaling role of each receptor chain because only heterodimers of WT and mutant receptor chimeras form in the presence of interleukin-2. Coexpression studies demonstrated that a functional receptor complex requires the membrane-proximal region of each receptor subunit in the oligomer to permit activation of JAK2 but only one membrane-distal tail to activate STAT5 and to support cell proliferation. Thus, this study defines key relationships involved in the assembly and activation of the EPOR signal transduction complex which may be applicable to other homodimeric cytokine receptors. Erythropoietin (EPO)1 is essential for the survival, proliferation, and terminal differentiation of erythroid progenitor cells (1-3). The erythropoietin receptor (EPOR) belongs to the cytokine receptor superfamily and as such contains the structural motifs of four cysteine residues with canonical spacing and the sequence WSXWS in the extracellular domain (4, 5). Although protein tyrosine phosphorylation is required for cytokine signaling (6), the cytoplasmic regions of these receptors lack intrinsic enzymatic activity. Signal transduction instead is mediated by cytoplasmic protein tyrosine kinases of the JAK family (7). These kinases are constitutively associated with receptor chains; residues in the membrane-proximal region of the EPOR cytoplasmic tail are required for this association. Ligand binding results in homodimerization of EPOR chains, which is critical for the initiation of intracellular signaling (8 -12).The role of homodimerization in the initiation of signaling is highlighted by constitutively active variants of the EPOR chain (10, 13). In the three mutants described to date, R129C, E132C, and E133C, amino acids in the extracellular domain are replaced by cysteines, leading to the formation of intermolecular disulfide bridges and constitutive signaling. In contrast, a number of other cytokine receptors signal through the heterodimerization of asymmetric receptor chains. For example, interleukin-2 (IL-2) binds and heterodimerizes the IL-2R␤ and ␥ c receptor chains (for review, see Refs. 14...

Structural requirements of the interleukin-6 signal transducer gp130 for its interaction with Janus kinase 1: the receptor is crucial for kinase activation

“…Moreover, it is known that mutation of a tryptophan residue in the interbox1\box2 region of the EPOR also abrogated signal transduction, although binding to Jak2 was maintained [12,27] or, conversely, could not be detected [29].…”

“…In such a receptor complex, both chains associate with Jaks, which is a prerequisite for signal transduction to occur [18,27]. However, only the wild-type cytoplasmic part of gp130 sustains Jak activation.…”

Dominant action of mutated erythropoietin receptors on differentiation in vitro and erythroleukemia development in vivo

“…Structural and biochemical analyses have demonstrated that epo induces receptor dimerization (Barber et al, 1994;Livnah et al, 1996;Matthews et al, 1996;Watowich et al, 1992Watowich et al, , 1994. The dominant action of mutant receptors in J2E cells suggests that they heterodimerize with wild type receptors upon ligand binding, thereby altering normal receptor function.…”

“…Mutant epo receptors D257 (Watowich et al, 1994), D321 (Watowich et al, 1999) and W282R , together with wild type epo receptor tagged at the carboxy terminus with HA (wt-HA), were subcloned into the retroviral vector MSCV 2.2 (Hawley et al, 1994). Plasmids were transfected into PA317 amphotropic packaging cells and virus-containing supernatants added to J2E or J2E-NR cultures before selection in Geneticin (Sigma, St. Louis, MO, USA) as described previously .…”

“…Upon hormone binding the epo receptor dimerizes, Janus kinase 2 (JAK2) is activated and tyrosine residues in the cytoplasmic domain of the receptor phosphorylated (Matthews et al, 1996;Watowich et al, 1992;Witthuhn et al, 1993). These phosphorylated tyrosines act as docking sites for signaling proteins including Signal Transducer and Activator of Transcription 5 (STAT 5), the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) and phosphatases SHP-1 and SHP-2 (Damen et al, 1995a,b;Gobert et al, 1996;Klingmuller et al, 1995Klingmuller et al, , 1996Klingmuller et al, , 1997Penta and Sawyer, 1995;Quelle et al, 1996;Sharlow et al, 1997;Tauchi et al, 1995Tauchi et al, , 1996Yi et al, 1995).…”