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Erythropoietin

Diana Spahlinger, '07 and Lisa King , '07


Contents:


I. Introduction

Erythropoietin, shown here complexed with the extracellular domains of its two receptor molecules (EPObp 1 and EPObp2), is the "incredible hulk" of hormone- receptor complexes. . Erythropoietin (EPO) resembles a classic endocrine hormone in that it exerts its effect on target cells in bone marrow through interactions with specific cell-surface receptors. The role of erythropoietin is to control red blood cell production by regulating the differentiation and proliferation of erythroid progenitor cells in the bone marrow. Produced primarily in the kidney, erythropoietin circulates in the plasma and acts on target cells in the bone marrow.

Erythropoietin activates target erythroid colony-forming cells by binding and orienting two cell surface erythropoietin receptors. The orientation of the EPO receptors is critical for efficient signalling. Proper binding triggers an intracellular phosphorylation cascade, which activates both the Ras/MAP kinase pathway and the STAT (signal transducer and activator of transcription) pathway. These pathways play a major role in cytokine-induced signalling and are involved in increased cell proliferation in response to EPO. Erythropoeitin participates in a classic feedback control system, as production of erythropoietin is regulated by impaired oxygen delivery to the kidney. Hypoxia causes an increase in erythropoietin gene transcription, enhancing red blood cell production.

Synthetic erythropoietin (Epoetin) is prescribed for the treatment of anemia, especially for individuals with cancer, AIDS, or kidney problems. However, because it stimulates the production of red blood cells, EPO hormone is also used illegally by athletes to increase their aerobic capacity and muscle endurance, making it worthy of the title "incredible hulk".


II. General Structure

Erythropoietin (EPO) is a 30.4 kD glycoprotein hormone, 166 amino acids in length. It is shown here complexed with its receptors, EPObp1 and EPObp2 . Each molecule of EPO activates cells by binding two identical cell-surface receptors. The EPO molecule is composed of 4 helices:αA , αB , αC , and αD , which display an up-up-down-down four-helical bundle topology. αA and αD are held together by a disulfide bond between Cys 7 and Cys 161 , and αB and αC are held together by a short loop .

Each EPO receptor each has a ~200 amino acid cytokine binding domain containing 2 β sandwich domains, D1( N-terminal) and D2 (carboxy-terminal) (Sayed et al 1998) . The distinctive structural features of the N-terminal and C-terminal domains place the EPObp in a novel class of receptors termed the hematopoietin receptor superfamily, which includes human growth hormone and prolactin receptors (Krantz 91).


III. Receptor Binding

Erythropoietin has two receptor binding sites, located on opposite faces of the molecule: site 1: EPObp EPO and site 2:EPObp EPO . These binding sites have been identified as high affinity (site 1 ) and low affinity (site 2). Site 1 includes segments of αA , αB' , αD , and part of the AB loop and contains a central hydrophobic binding pocket surrounded by hydrophilic interactions. This hydrophobic binding pocket interacts with EPObp loops L1 , L5 , and L6 . Phe 93 on the receptor is critically important for ligand binding, participating in hydrogen bonding with erythropoietin residues Thr 44 and Asn 147 .

Interactions at site 2 are less extensive, and primarily involve αC of EPO and loop L3 of the EPObp. The hydrophobic surface on EPObp is created by Phe 93 , Phe 205 , and Met 150 . At site, 2 Met 150 is more buried than at site 1, making van der Waals contacts with Arg 10 , Val 11 , and Arg 14 of EPO. Hydrophilic interactions are comprised of 11 hydrogen bonds between the αA and αC helices of erythropoietin and loops L1, L2, L3, and L5 of EPObp .

IV. EPO/ EPObp binding orientation

EPO orientation

Syed, et al (1998) found that when EPO binds to its two receptors it imposes a unique 120° angular relationship between them . This is in contrast to the orientation of the human growth hormone complex, which has a similar structure but demonstrates a 160° angle between its receptors. The unique angular orientation of the EPO-(EPObp)2 complex appears to be a critical factor in determining its biological activity. The intracellular surfaces created by this 120° orientation are responsible for optimal EPO-induced signalling through intracellular kinase pathways.


V. Epo-induced intracellular signaling

phosphorylation

EPO activates target cells by binding and orienting two identical cell-surface receptors, EPObp1 and EPObp2, triggering an intracellular phosphorylation cascade. Protein phosphorylation is an important early step in signal transduction by many growth factor receptors, including EPObp (Youssoufian et al). The first step of intracellular signalling is the activation of the Jak2 tyrosine kinase, which is constitutively associated with the EPO receptor. Jak2 activation leads to the phosphorylation of several proteins, including the EPO receptor itself, which undergoes phosphorylation of 8 tyrosines located within the intracellular domain. These phosphorylated tyrosines become docking sites for other various intracellular proteins, which then can become tyrosine phosphorylated and activated. This phosphorylation cascade activates several intracellular pathways, including the Ras/MAP kinase pathway, which is involved in cell proliferation in response to EPO (Lacombe 1999).


VI. References

Adamson, John W. 1996. Regulation of Red Blood Cell Production. The American Journal of Medicine. 101: 4S-6S.

Krantz, Sanford B. 1991. Erythropoietin review article. The Journal of the American Society of Hematology 77 (3): 419-434.

Lacombe, C. and P. Mayeux. 1999. The molecular biology of erythropoietin. Nephrology Dialysis Transplantation 14: 22-28.

Syed, Rashid S., Scott W. Reid, Cuiwei Li, Janet C. Cheetham, Kenneth H. Aoki, Beishan Liu, Hangjun Zhan, Timothy D. Osslund, Arthur J. Chirino, Jiandong Zhang, Janet Finer-Moore, Steven Elliott, Karen Sitney, Bradley A. Katz, David J. Matthews, John J. Wendoloski, Joan Egrie, and Robert M. Stroud. 1998. Efficiency of Signalling through Cytokine Receptors Depends Critically on Receptor Orientation. Nature 395: 511-516.