Bound to the Extracellular Domains
of Two EPO Receptors
Paolo DaSilva and David Marcey
CLU Biology Department
II. Erythropoietin Structure
III. EPO Receptor Structure and EPO Binding
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(EPO), a cytokine hormone, is responsible for the regulation of red
blood cell (erythrocyte) production. EPO is a glycoprotein produced
by cells of the peritubular capillary endothelium of the kidney and,
to a lesser extent, by liver hepatocytes. EPO production is stimulated
by reduced oxygen content in the renal arterial circulation, mediated
by a transcription factor that is oxygen-sensitive.
EPO (165 amino acids) binds to EPO receptors on the surface of bone
marrow erythroid precursors, resulting in their rapid replication
and maturation to functional red blood cells. This stimulation results
in a rapid rise in erythrocyte counts and a consequent rise in blood
oxygen. Altered levels of EPO or mutations in EPO receptors are linked
to changes in the hematocrit (% of red blood cells in blood). For
example, kidney failure leads directly to severe anemia due to low
EPO levels and hence reduced hematopoiesis.
a bizarre case of benign erythrocytosis (overproduction of red cells),
the"disease" was linked to a heterozygous mutation
in the EPO receptor
gene of a three-time Olympic gold medalist in cross country skiing.
His hematocrit was ~60%, well above the standard 45%, due to loss
of a negative control region in the EPO receptor.
at left is the structure of the cytokine Erythropoietin
(EPO), bound by the extracellular domains
of two identical EPO receptors, designated here
as EPObp2 and EPObp1
(PDB ID 1cn4, Syed, et al., 1998). Several structural features
of this cytokine and its receptors are illustrated by clicking on
, in order.
contains a four-helical bundle with a
topology shared with other cytokines. The four helices of this bundle
are termed A, B,
C, and D.
The A and D
helices are linked by a disulphide
bridge. The B
helices are linked by a short
In addition to
helices, EPO contains two short helices, B'
The structure of
EPO is further stabilized by numerous hydrophobic interactions. For
example, aromatic and hydrophobic amino acids
of the D-helix pack against hydrophobic
residues of helices A,
and C, helping
to form the hydrophobic core of EPO.
Receptor Structure and EPO Binding
of both of the extracellular domains contains two, seven-stranded
beta sandwich subdomains, like other cytokine receptors. Each extracellular
domain has an N-terminal alpha helix
positioned in the "elbow" between the beta sandwich subdomains.
This helix has sidechains that interact with residues of two conserved
regions, and this is thought to be instrumental
in stabilizing the folded EPO receptor.
imposes a distinct orientation of the two identical EPO receptors,
providing for optimal signal transduction. Each of two opposite faces
of EPO interacts with one binding site on each EPO receptor. Interestingly,
these binding sites are non identical, even though the receptors are
identical. Site 1 involves complementary interactions between
six loops (L1-L6) of
EPObp1 and EPO helices A,
part of the loop between the A
helices. Site 2, a lower affinity
binding site, involves interaction between five
L5, L6) of EPObp2
and residues on helices A
la Chapelle A, Sistonen P, Lehväslaiho H, Ikkala E, Juvonen E.
1993. Familial erythrocytosis genetically linked to the erythropoietin
receptor gene. Lancet 341:82-84.
R. S., Reid, S. W., Li, C., Cheetham, J. C., Aoki, K. H., Liu, B.,
Zhan, H., Osslund, T. D., Chirino, A. J., Zhang, J., Finer-Moore,
J., Elliott, S., Sitney, K., Katz, B. A., Matthews, D. J., Wendoloski,
J. J., Egrie, J., Stroud, R. M.. 1998. Efficiency of Signalling Through
Cytokine Receptors Depends Critically on Receptor Orientation. Nature