Transmembrane Protein Aptamer Induces Cooperative Signaling by the EPO Receptor and the Cytokine Receptor β-Common Subunit

摘要

class="head no_bottom_margin" id="sec1title">IntroductionMany aspects of cell behavior are controlled by cell surface receptors that receive extracellular signals and orchestrate the cellular response. The formation and activation of alternative receptor complexes with different subunits and signaling properties can dictate receptor output (e.g., ). The cytokine erythropoietin (EPO) can activate alternative complexes of the EPO receptor (EPOR), a transmembrane (TM) cell surface protein lacking intrinsic kinase activity. Binding of EPO to the EPOR can trigger the homodimerization of EPORs in a productive orientation, leading to the transphosphorylation of Janus kinase 2 (JAK2), which is constitutively associated with the EPOR (, , ). Activated JAK2 phosphorylates multiple tyrosines in the intracellular domain of EPOR, allowing the recruitment and phosphorylation of downstream signaling proteins, including signal transducer and activator of transcription 5 (STAT5) (, , , ). This signaling pathway is essential for the survival, proliferation, and differentiation of erythroid progenitors.In addition to erythropoiesis, the EPOR can mediate non-erythroid outcomes in response to EPO treatment, including a tissue-protective response that prevents apoptosis and promotes proliferation in non-hematopoietic cells subjected to injury or metabolic stress (, , , , , , ), reviewed in href="#bib28" rid="bib28" class=" bibr popnode">Jelkmann et al. (2009). The protective effect of EPO appears to require the activation of a heteroreceptor composed of EPOR and the cytokine receptor β-common subunit (β-common receptor [βcR] also known as CD131). In addition to constitutively binding EPOR, βcR also binds the α-chain of interleukin (IL)-3 receptor, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-CSFR), and IL-5 receptor (href="#bib4" rid="bib4" class=" bibr popnode">Blake et al., 2002, href="#bib29" rid="bib29" class=" bibr popnode">Jubinsky et al., 1997, href="#bib27" rid="bib27" class=" bibr popnode">Hercus et al., 2013, href="#bib38" rid="bib38" class=" bibr popnode">Lopez et al., 1992). βcR plays an essential role in signaling by these receptors, which lack JAK2 binding or a significant cytoplasmic domain, by providing bound JAK2 and cytoplasmic tyrosines for phosphorylation (href="#bib25" rid="bib25" class=" bibr popnode">Hansen et al., 2008, href="#bib27" rid="bib27" class=" bibr popnode">Hercus et al., 2013). βcR^−/− mice lack EPO-induced tissue protection but retain normal hematopoiesis, showing that βcR is required for tissue protection but not for erythroid differentiation in at least some settings (href="#bib53" rid="bib53" class=" bibr popnode">Weber et al., 2005, href="#bib7" rid="bib7" class=" bibr popnode">Brines et al., 2004). In addition, certain modified versions of EPO, such as lysine-carbamylated EPO, specifically induce the tissue-protective, but not the erythroid, effects of EPO (href="#bib20" rid="bib20" class=" bibr popnode">Erbayraktar et al., 2009, href="#bib33" rid="bib33" class=" bibr popnode">Leist et al., 2004, href="#bib40" rid="bib40" class=" bibr popnode">Murphy and Young, 2006, href="#bib55" rid="bib55" class=" bibr popnode">Yamanaka et al., 2018). These results suggest that the tissue-protective effect of EPO is mediated by an EPOR/βcR heteroreceptor, and not by EPOR homodimerization (href="#bib5" rid="bib5" class=" bibr popnode">Bohr et al., 2015). However, the role of the heteroreceptor in tissue protection remains controversial (see citations in href="#bib11" rid="bib11" class=" bibr popnode">Cheung Tung Shing et al. 2018), and in some situations a classical EPOR homodimer can provide a protective signal (href="#bib51" rid="bib51" class=" bibr popnode">Um et al., 2007). Notably, the elements on the EPOR and βcR required for heteroreceptor formation and the molecular mechanism of signaling by the βcR/EPOR heteroreceptor are unknown.Various receptors, including the EPOR, can be activated through interactions involving their TM domain (TMD). The murine spleen focus-forming virus envelope protein gp55-P specifically binds to the TMD of the mouse EPOR (mEPOR), triggering EPOR activation, erythroid cell proliferation, and polycythemia (href="#bib34" rid="bib34" class=" bibr popnode">Li et al., 1990, href="#bib15" rid="bib15" class=" bibr popnode">Constantinescu et al., 1999b). The platelet-derived growth factor β receptor (PDGFβR) can be activated by the bovine papillomavirus E5 oncoprotein, a 44-residue TM protein that binds specifically to the TMD of the PDGFβR (href="#bib17" rid="bib17" class=" bibr popnode">DiMaio and Petti, 2013, href="#bib41" rid="bib41" class=" bibr popnode">Petti and DiMaio, 1992, href="#bib42" rid="bib42" class=" bibr popnode">Petti et al., 1991). We developed a genetic approach to isolate small biologically active TM proteins in which we construct libraries expressing up to millions of different, small, artificial TM proteins (termed traptamers, for TM aptamers) with randomized, hydrophobic segments. Traptamer libraries are expressed in mammalian cells, and active traptamers are recovered from cells selected for particular biological activities, with the rationale that, by chance, rare traptamers interact with cellular TM proteins and modulate their activity or expression (href="#bib9" rid="bib9" class=" bibr popnode">Cammett et al., 2010, href="#bib21" rid="bib21" class=" bibr popnode">Freeman-Cook et al., 2004, href="#bib22" rid="bib22" class=" bibr popnode">Freeman-Cook and DiMaio, 2005, href="#bib47" rid="bib47" class=" bibr popnode">Scheideman et al., 2012). We have isolated traptamers that specifically activate human EPOR (hEPOR) or mEPOR and cause EPOR-dependent proliferation of murine BaF3 cells (href="#bib9" rid="bib9" class=" bibr popnode">Cammett et al., 2010, href="#bib12" rid="bib12" class=" bibr popnode">Cohen et al., 2014, href="#bib26" rid="bib26" class=" bibr popnode">He et al., 2017). These traptamers bind the TMD of the EPOR and induce hEPOR homodimerization and tyrosine phosphorylation of EPOR and JAK2.Here, we isolate and characterize a new traptamer, ELI-3, that induces proliferation of BaF3 cells that express the EPOR. ELI-3 interacts with the hEPOR and, unlike EPO or previously isolated traptamers that activate the EPOR, does not require intracellular hEPOR tyrosines, but instead requires the endogenously expressed βcR in addition to the EPOR. ELI-3 does not support differentiation in erythroid cells and inhibits serum withdrawal-induced apoptosis in non-hematopoietic cells. These results show that small TM proteins can specifically activate either the EPOR homodimer or the EPOR/βcR heteroreceptor, with distinct biological outcomes. Our results also demonstrate that the EPOR in the EPOR/βcR heteroreceptor uses a non-canonical mechanism to generate a proliferative signal.