Dimerization of the Extracellular Domain of the Erythropoietin (EPO) Receptor by EPO:  One High-Affinity and One Low-Affinity Interaction

Philo, John S.; Aoki, Kenneth H.; Arakawa, Tsutomu; Narhi, Linda O.; Wen, Jie

doi:10.1021/bi9524272

Cited by 185 publications

(144 citation statements)

References 35 publications

Supporting

Mentioning

134

Contrasting

Unclassified

Order By: Relevance

“…Various mechanisms are employed for growth factor signaling upon interaction of each ligand with its receptor. These mechanisms include the 2: 1 receptorligand homodimer seen in the EPO and hGH systems (Cunningham et al, 1991;DeVos et al, 1992;Philo et al, 1996), the 2 1 receptor-ligand heterodimer complex seen for many cytokines including GM-CSF (Altman & Kastelein, 1995), and the 2:2 homodimer complex of interferon gamma with its receptor (Walter et al, 1995;Greenlund et al, 1996). We mutated surface-exposed residues on G-CSF to alanine in order to determine which regions of the four-helix bundle were important for receptor binding and activation.…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, the lack of self antagonism and the location of the receptor binding site suggest that a purely 2:1 receptorligand complex is unlikely to be the only active species. Hiraoka et al (1996) have shown that a single G-CSF molecule can form complexes with the extracellular domains of two receptor molecules, and other studies (Hiraoka et al, 1994;Philo et al, 1996) have suggested that 2:2 or even 4:4 ligand-receptor complexes may form at high ligand concentrations. Thus, studies to date are consistent with a receptor activation model whereby 2: 1 receptorligand complexes are formed at low concentrations of ligand and converted to 2:2 or higher order complexes at higher ligand concentrations.…”

Section: T" Lmentioning

confidence: 99%

“…hGH signaling involves initial binding of hGH to one receptor molecule followed by recruitment of a second receptor to form a 2:l receptor-ligand homodimer (Cunningham et al, 1991;DeVos et al, 1992). Recent evidence suggests that EPO mediates proliferative effects through a similar sequential dimerization process to hGH (Matthews et al ., 1996;Philo et al, 1996). G-CSF signaling through the G-CSF receptor is also likely to occur by homodimerization or homo-oligomerization (IshizakaIkeda et al, 1993).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Characterization of the receptor binding determinants of granulocyte colony stimulating factor

et al. 1997

View full text Add to dashboard Cite

We performed a series of experiments using alanine-scanning mutagenesis to locate side chains within human granulocyte colony-stimulating factor (G-CSF) that are involved in human G-CSF receptor binding. We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G-CSF. The G-CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method. We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay. These studies show that there is a region of five charged residues on helices A and C employed by G-CSF in binding its receptor, with the most important residue in this binding patch being Glu 19. Both wild-type G-CSF and the E19A mutant were expressed in E. coli. The re-folded proteins were found to have proliferative activities similar to the analagous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G-CSF. Although G-CSF is likely to signal through receptor homo-dimerization, we found no compelling evidence for a second receptor binding region. We also found no evidence of self-antagonism at high G-CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G-CSF probably does not signal via a pure 2.1 receptor:ligand complex. Thus, G-CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high-affinity interaction with its receptor.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: T" Lmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of the receptor binding determinants of granulocyte colony stimulating factor

et al. 1997

View full text Add to dashboard Cite

show abstract

“…The penalty term can be continuously made more stringent or be relaxed, even abandoned completely when the parameters have converged to a minimum with physically reasonable values (17). This method was used extensively for receptor-ligand interactions (29)(30)(31). Recently, the strategy to use the degree of violations of MC for selecting among alternative models for SE data was also used by Arkin and Lear in a separate post-fitting step (32).…”

Section: Introductionmentioning

confidence: 99%

Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition

Vistica¹,

Dam²,

Balbo³

et al. 2004

Analytical Biochemistry

335

396

View full text Add to dashboard Cite

Sedimentation equilibrium is a powerful tool for the characterization of protein self-association and heterogeneous protein interactions. Frequently, it is applied in a configuration with relatively long solution columns and with equilibrium profiles being acquired sequentially at several rotor speeds.The present study proposes computational tools, implemented in the software SEDPHAT, for the global analysis of equilibrium data at multiple rotor speeds, multiple concentrations, and multiple optical detection methods. The detailed global modeling of such equilibrium data can be a non-trivial computational problem. It was shown previously that mass conservation constraints can significantly improve and extend the analysis of heterogeneous protein interactions. Here, a method for using conservation of mass constraints for the macromolecular redistribution is proposed in which the effective loading concentrations is being calculated from the sedimentation equilibrium profiles. The approach is similar to that described by Roark (Biophys. Chem. 5 (1976) [185][186][187][188][189][190][191][192][193][194][195][196], but its utility is extended by determining the bottom position of the solution columns from the macromolecular redistribution. For analyzing heterogeneous associations at multiple protein concentrations, additional constraints are introduced that relate the effective loading concentrations of the different components or their molar ratio in the global analysis. Equilibrium profiles at multiple rotor speeds also permit the algebraic determination of radial-dependent baseline profiles, which can govern interference optical ultracentrifugation data, but usually also occur, to a smaller extent, in absorbance optical data. Finally, the global analysis of equilibrium profiles at multiple rotor speeds with implicit mass conservation and computation of the bottom of the solution column provides an unbiased scale for determining molar mass distributions of non-interacting species. The properties of these tools are studied with theoretical and experimental data sets.

show abstract

“…The crystal structure of the EPO-EPOR complex has been determined at a resolution of 1.9 Å [Protein Data Bank (PDB) ID code 1EER] (22). This structure demonstrates that Phe-48, Asn-147, Arg-143, and Arg-150 of EPO are among the key residues for binding to EPOR (20)(21)(22).We have taken the primary EPO-EPOR binding site as our target of protein-protein interface redesign. After scanning the PDB, rat pleckstrin homology (PH) domain of phospholipase C-␦1 (PLC␦ 1 -PH) was found to accommodate well the key interaction residues in EPO and at the same time can form a good interface with EPOR.…”

mentioning

confidence: 99%

Nonnatural protein–protein interaction-pair design by key residues grafting

Liu

Zhu

et al. 2007

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

View full text Add to dashboard Cite

Protein-protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein-protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein-protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat PLC␦ 1-PH (pleckstrin homology domain of phospholipase C-␦1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat PLC␦ 1-PH were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of PLC␦ 1-PH (ERPH1) was found to bind EPOR with high affinity (K D of 24 nM and an IC50 of 5.7 M) both in vitro and in a cell-based assay, respectively, although the WT PLC␦ 1-PH did not show any detectable binding under the assay conditions. The in vitro binding affinities of the PLC␦ 1-PH mutants correlate qualitatively to the computational binding affinities, validating the design and the protein-protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting proteinprotein interfaces.de novo design of protein-protein interaction pair ͉ erythropoietin ͉ functional site grafting ͉ key residue at interface P rotein-protein interaction is critical in many biological processes ranging from cell differentiation to apoptosis; however, little is known about the general principles governing the specificity and binding affinity of protein-protein interactions. Recent developments in structural bioinformatics have contributed greatly to our understanding of protein-protein interactions (1-6). Several groups have addressed the feasibility of computational redesign of protein-protein interactions by using native or homologous protein-protein interfaces (7-10). Because different protein backbones can perform similar functions (11), in the current study we explored the feasibility of designing nonnatural protein-protein interaction pairs using known protein scaffolds.To reconstruct the function of a protein, one of the common strategies is grafting, i.e., transferring the functional epitopes from one protein to another. The critical step for protein grafting is to find suitable sites for functional epitope transfer. To this purpose, several computational methods have been developed including a geometric hashing paradigm (12), FITSITE (13), GRAFTER (14), and DEZYMER (15, 16). We have developed a strategy for protein-protein interface redesign by grafting discontinuous interaction epitopes to nonhomologous proteins (17-19). The erythropoietin (EPO)-EPO receptor (EPOR) system was used as an example for nonnatural protein-protein interaction-...

show abstract

Dimerization of the Extracellular Domain of the Erythropoietin (EPO) Receptor by EPO: One High-Affinity and One Low-Affinity Interaction

Cited by 185 publications

References 35 publications

Characterization of the receptor binding determinants of granulocyte colony stimulating factor

Characterization of the receptor binding determinants of granulocyte colony stimulating factor

Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition

Nonnatural protein–protein interaction-pair design by key residues grafting

Contact Info

Product

Resources

About