Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor

Abstract

This work describes a genetic approach to isolate small, artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric, 44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). We used the E5 protein as a scaffold to construct a retrovirus library expressing approximately 500,000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small, dimeric TM proteins that were structurally unrelated to erythropoietin (EPO), but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFbetaR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore, this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus, we have changed the specificity of a protein so that it no longer recognizes its natural target but, instead, modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word "traptamer" for these transmembrane aptamers.

Fig. 1.

Construction and use of small TM protein libraries. (A) Amino acid sequence of the E5 protein (Top Line), the TJC-5 library with randomized positions indicated by Xs (Second Line), and five unselected clones from the library. Randomized amino acids shown in gray. (B) Diagram of the scheme to select small TM activators of the hEPOR. The Round Objects represent cells, those with Black Nuclei represent dying cells. The Dark Lines represent the hEPOR and the Gray X’s represent small TM proteins encoded by the library. See text for details.

Fig. 2.

Small TM proteins induce growth-factor independence in cells expressing the hEPOR. (A) Amino acid sequence of the TM domains of the E5 protein (positions 12–33) (Top Line), four clones recovered from growth-factor independent BaF3/hEPOR cells, and gp55P (Bottom Line). Sequence differences from TC2-3 in the recovered clones shown in Gray. The TM domains of the E5 protein and TC2-3 are thought to adopt a type II transmembrane orientation and are shown with their N termini to the Left, whereas gp55P, which adopts the opposite orientation, is shown with its N terminus to the Right. (B) Small TM proteins or empty CMMP vector were expressed in parental BaF3 cells (Striped Bars) or BaF3 cells expressing mPR (Gray Bars), hEPOR (White Bars), or mEPOR (Black Bars). After 2 d, IL-3 was removed, and viable cells were counted 6 d later. Cells expressing TC2-3 grew the fastest and at 6 d were dying due to overcrowding.

Fig. 3.

Dimerization of TC2-3. (A) RIPA extracts were prepared from BaF3/hEPOR cells expressing the empty CMMP vector (V), TC2-3, or BPV E5. After immunoprecipitation with αE5, samples were subjected to gel electrophoresis in the presence or absence of reducing agent and detected by immunoblotting with the same antibody. Size of protein markers (in kDa) is shown on Left. (B) BaF3/hEPOR cells expressing the empty T2H-F13 (Vector), TC2-3, or TC2-3CCSS were cultured in the absence of IL-3 and EPO. Viable cells were counted after 7 d.

Fig. 4.

Activation of the hEPOR. RIPA extracts were prepared from BaF3 cells or BaF3/HA-hEPOR cells expressing empty T2H-F13 vector (V) or TC2-3. Where indicated, cells were acutely stimulated with EPO (Plus Sign). Samples were immunoprecipitated and immunoblotted with C20 α-EPO receptor antibody (Left) or immunoprecipitated with anti-HA antibody and immunoblotted with 4G10 anti-phosphotyrosine antibody (Right). Half as much protein was immunoprecipitated for the EPO-stimulated lanes on the Right. Size of protein markers is shown on Left.

Fig. 5.

The TM domain of the hEPOR is required for TC2-3 activity. (Top) Sequences of the TM domains and flanking sequences of the HA-hEPOR, HA-hEPOR(5A), and HA-hEPOR(mPR). PDGF receptor amino acids shown in Grey. (Bottom) BaF3 cells expressing HA-hEPOR (Solid Black Line), HA-hEPOR(5A) (Dashed Line), or HA-hEPOR(mPR) (Dotted Line) or parental BaF3 cells (Solid Gray Line) were infected with MSCV expressing TC2-3 and selected for hygromycin resistance. Drug-resistant cells were cultured in the absence of growth factors, and viable cells counted on the indicated days. Similar results were obtained in two independent experiments.

Fig. 6.

TC2-3 stimulates EPO-independent proliferation and differentiation of HPCs. (A) CD34⁺ cells were infected with the CMMP-IRES-GFP vector or the same vector expressing TC2-3, sorted for GFP expression, and transferred to differentiation medium in the absence or presence of EPO. After 4 d in differentiation medium, expression of cell-surface GpA was assessed by immunostaining non-permeabilized cells and flow cytometry. Green, vector-infected cells minus EPO; Lavender, vector-infected cells plus EPO; and Red, TC2-3-expressing cells minus EPO. (B) Cells were handled as in part A. The total number of viable cells expressing cell-surface GpA (> 50 fluorescence units) was determined by immunostaining and flow cytometry at various times. The ability of TC2-3 to stimulate erythroid differentiation was observed in four independent experiments, although the magnitude of the effect was somewhat variable in these primary cells. (C) Cells were infected with CMMP-IRES-GFP (Top) or the same vector expressing TC2-3 (Bottom), sorted for GFP expression, and plated in replicate in methylcellulose. Cells infected with empty vector were also treated with EPO. After 14 d, colonies were visualized by brightfield microscopy (Left) or after staining with benzidine (Right). (D) Table showing the generation of dense erythroid colonies in methylcellulose. P-value for the difference between untreated cells infected with vector and TC2-3, < 0.001 (binomial test P-value).