Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor.
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.
Cammett
Tobin J
TJ
Department of Genetics, Yale University School of Medicine, New Haven, CT 06520-8005, USA.
Jun
Susan J
SJ
Cohen
Emily B
EB
Barrera
Francisco N
FN
Engelman
Donald M
DM
Dimaio
Daniel
D
D000595:Amino Acid Sequence; D049109:Cell Proliferation; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D039902:Multipotent Stem Cells / Q000187:drug effects / Q000502:physiology; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D019151:Peptide Library; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000819:agonists; D017467:Receptors, Erythropoietin / Q000819:agonists*; D012190:Retroviridae; D012641:Selection, Genetic; D054852:Small Molecule Libraries
D008565:Membrane Proteins; D009856:Oncogene Proteins, Viral; D019151:Peptide Library; D017467:Receptors, Erythropoietin; D054852:Small Molecule Libraries; C054304:oncogene protein E5, Bovine papillomavirus type 1; D020797:Receptor, Platelet-Derived Growth Factor beta