Like TC2-3, EBC5-16 is dimeric, can serve as a transmembrane domain, and functionally interacts with the transmembrane domain of the hEPOR. The high activity of EBC5-16 in inducing erythroid differentiation is particularly striking because it is so dissimilar to EPO,Puerarin which is monomeric, soluble, and binds the extracellular domain of the EPOR. We used a similar directed evolution strategy to optimize traptamers that down-regulate CCR5. These results demonstrate the utility of random mutagenesis and selection to optimize artificial transmembrane domains that target single-pass and multi-pass transmembrane proteins. Several lines of evidence suggest that the enhanced activity of EBC5-16 is due to increased homodimerization caused by the substitution of a serine for an isoleucine. First, EBC5-16 exists in cells as a disulfide bond-linked homodimer. Second, mutation of the cysteines that mediate covalent dimerization abolishes activity. Third, the serine substitution increases the fraction of EBC5-16 in the dimeric form, as assessed by non-reducing gel electrophoresis and TOXCAT experiments. Although the TOXCAT result indicated the transmembrane domain of EBC5-16 is sufficient for dimerization in bacterial membranes,Dipsacoside B the defect caused by the cysteine mutations implies that in mammalian cells the dimer is stabilized by disulfide bonds. Similarly, the transmembrane domain of BPV E5 lacking the C-terminal cysteines has intrinsic dimerization potential, but the presence of the cysteines or fusion to a heterologous dimerization domain is required for high-level activity in mammalian cells. Finally, we identified Gly11, Gly15, Ile18, Pro22, Ser25, and Phe29 as the residues constituting the homodimer interface of EBC5-16. Importantly, insertion of these interfacial residues into an inactive variant of EBC5-16 containing a monomeric poly-leucine transmembrane domain was sufficient to reconstitute a dimeric protein that activates the hEPOR. Although our results show unequivocally that dimerization of EBC5-16 is required for activity, it remains possible that alterations in amino acid side-chain orientation caused by the I25S substitution has a direct effect on the increased activity of EBC5-16 compared to TC2-3.