An antagonist peptide–EPO receptor complex suggests that receptor dimerization is not sufficient for activation

Livnah, Oded; Johnson, Dana L.; Stura, E.A.; Farrell, Francis X.; Barbone, Francis P.; You, Yun; Liu, Kathleen D.; Goldsmith, Mark A.; He, Wen; Krause, Christopher D.; Pestka, Sidney; Jolliffe, Linda K.; Wilson, Ian A.

doi:10.1038/2965

Cited by 212 publications

(154 citation statements)

References 45 publications

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“…It may also be possible to mutagenize TC2-3 to identify essential structural features; optimize its affinity or specificity; or fine-tune its activity. It may also be possible to isolate inhibitory derivatives of TC2-3 that force the hEPOR dimer to adopt a non-productive orientation, as has been reported for a peptide that binds to the extracellular domain of the hEPOR (24). Finally, traptamers may also serve as structurally simple templates that can inform the design of peptide or peptidomimetic reagents and drugs that specifically modulate a wide variety of cellular and viral transmembrane protein targets.…”

Section: Discussionmentioning

confidence: 99%

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

Cammett¹,

Jun²,

Cohen³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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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 β receptor (PDGFβR). We used the E5 protein as a scaffold to construct a retrovirus library expressing ∼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 PDGFβR. 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.bovine papilloma virus | E5 protein | expression libraries | protein engineering | traptamers I t is thought that up to 30% of all proteins span cell membranes (1). Most membrane-spanning protein segments adopt an α-helical conformation that is largely independent of their amino acid sequence and minimally influenced by extramembraneous protein domains. Although transmembrane (TM) domains were once thought to be rather nonspecific protein segments whose primary function was to anchor proteins in membranes, they can participate in highly-specific, inter-and intramolecular side-by-side interactions that control important biological processes (2).The 44-amino acid bovine papillomavirus type 1 (BPV) E5 protein is essentially an isolated TM domain. It contains a hydrophobic central segment and exists in the intracellular membranes of transformed cells as a type II TM homodimer stabilized by disulfide bonds in its carboxy-terminus (3-5). The E5 dimer binds to the TM domains of two molecules of the platelet-derived growthfactor β receptor (PDGFβR) tyrosine kinase, resulting in receptor dimerization and activation, leading to cell transformation (6-10). The E5 protein does not bind to other TM domains or to certain PDGFβR TM mutants (11).Based on our analysis of the E5 protein, we proposed that it might be possible to construct small, artificial TM proteins that regulate a variety of viral and cellular TM proteins in trans (11, 12). To identify small proteins with different TM sequences that transform cells, we constructed expression libraries in which the TM domain of the E5 protein was replaced with randomized sequences of primarily hydrophobic amino acids and isolated clones from these libraries that induced focus formation or growth-factor independe...

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Section: Discussionmentioning

confidence: 99%

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

Cammett¹,

Jun²,

Cohen³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…These findings imply that the nonfunctional W282R mutant must alter either the relative orientations of the receptor cytoplasmic regions or the alignment of JAK2 molecules in the receptor dimer. In addition, EPO-mimetic peptides that bind to and dimerize the EPOR extracellular domain are not always sufficient to activate intracellular signal transduction (53). Thus, it appears that JAK2 molecules and/or the cytoplasmic regions of the EPOR must be brought into the correct orientation in the receptor dimer to create a competent, activated receptor complex.…”

Section: Discussionmentioning

confidence: 99%

Oligomerization and Scaffolding Functions of the Erythropoietin Receptor Cytoplasmic Tail

Watowich¹,

Liu²,

Xie³

et al. 1999

Journal of Biological Chemistry

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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...

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“…For instance, if we take at face value what we have already learned about the EPO antibody that has full agonist activity, it appears to require cooperativity of two different binding specificities whose agonist mechanism is similar to the asymmetric interaction of EPO with the EpoR (14). Thus, the fact that antibodies with either specificity can bind to EpoR, but only act as potent agonists when they are combined in the same molecule, suggests that simple dimerization of the EpoR is not sufficient for activity, or their dimer configurations are more similar to partial agonists (16), antagonists (19), or unliganded dimers (17,18) (Fig. 6).…”

Section: Discussionmentioning

confidence: 99%

“…One homodimeric antibody is only a partial agonist, which suggests a more symmetric interaction with less activation as for a peptide agonist (16). Antibodies that bind EpoR, but do not activate, likely bind to only one arm of the EpoR unliganded dimer (17)(18)(19) in an orientation that precludes bivalent association of the antibody. dimers, they could not replicate the mechanism of authentic EPO that requires asymmetric binding and, as seen here for homodimeric molecules, their best antibody from the screening had only about 60% of the activity of authentic EPO.…”

Section: Discussionmentioning

confidence: 99%

“…Remarkably, we selected a mechanistically unique antibody whose agonist function depends on the obligate synergy between two different antigen-binding specificities that must be contained within the same antibody molecule. This asymmetric binding of the heterodimeric antibody replicates the mechanism of authentic EPO whose full agonist activity depends on activation of the JAK2/Stat5 pathway by its asymmetric binding to two nearly identical sites on the EPO receptor (EpoR) monomers (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

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Selection of antibodies that regulate phenotype from intracellular combinatorial antibody libraries

Zhang

Wilson

Lerner

2012

Proc. Natl. Acad. Sci. U.S.A.

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A method is presented that uses combinatorial antibody libraries to endow cells with new binding energy landscapes for the purpose of regulating their phenotypes. Antibodies that are expressed in cells infected with a lentiviral combinatorial antibody library are selected directly for function rather than only for binding. The potential diversity space can be very large because more than one lentivirus can infect a single cell. Thus, the initial combinatorial diversity of ∼1.0 × 10 11 members generated by the random association of antibody heavy and light chains is greatly increased by the reassortment of the antibody Fv domains themselves inside cells. The power of the system is illustrated by its ability to select unusual antibodies. Here, the selected antibodies are potent erythropoietin agonists whose ontogeny depends on recombination at the protein level of pairs of antibodies expressed in the same cell to generate heterodimeric bispecific antibodies. The obligate synergy between the different binding specificities of the antibody's monomeric subunits appears to replicate the asymmetric binding mechanism of authentic erythropoietin.agonist antibodies | phenotypic perturbation | erythropoietin phenocopy

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An antagonist peptide–EPO receptor complex suggests that receptor dimerization is not sufficient for activation

Cited by 212 publications

References 45 publications

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

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

Oligomerization and Scaffolding Functions of the Erythropoietin Receptor Cytoplasmic Tail

Selection of antibodies that regulate phenotype from intracellular combinatorial antibody libraries

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