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than EPO itself (
13
): asialoEPO does not trigger erythropoiesis
in vivo because its serum half-life is only several minutes. In con-
trast, asialoEPO is more active in vitro than EPO as an erythropoi-
etic agent. Therefore, asialoEPO is not a specific ligand for the
tissue protective receptor.
In the late 1990s, there was intense interest in the structure of
the hematopoietic receptor and the corresponding structure-activ-
ity relationships of EPO. Elegant mutagenesis studies coupled with
detailed NMR and X-ray receptor structure studies defined which
regions of EPO bound to the receptor and further, which amino
acids were key for binding (
14
). It was determined that similar to
the growth hormone receptor homodimer that had been elucidated
previously, (EPOR)
2
formed a dimeric structure which subsequently
bound different parts of EPO sequentially to each subunit (
8
). With
the knowledge of specific essential regions of EPO needed to bind
to the assembled homodimer, specific changes to these regions
could be accomplished (e.g., by chemical or molecular biological
methods) that would abolish binding to (EPOR)
2
. Such engineered
molecules might still bind to the hypothesized tissue protective
receptor isoform. A number of modified EPO molecules were con-
structed which as expected, did not bind to the hematopoietic
receptor and therefore were not erythropoietic (
15
). Excitingly,
many of these molecules retained tissue protective activity similar to
EPO in a wide variety of animal models, thereby confirming the
existence of an alternative receptor system (
15
).
4
The Injury-Induced Cytoprotective Receptor
The identity of this alternate EPO receptor was discovered to consist
of a heteromeric structure (
16
). As background, EPO is a member
of the type I cytokine family that includes a host of other cytokines,
including GM-CSF, IL-3, IL-5, prolactin, growth hormone, ciliary
neurotrophic factor, and leukemia inhibitory factor, among others.
In contrast to the homodimeric hematopoietic receptor for EPO,
other members of this family typically form heteromers. Additionally,
receptor subunits that assemble to form an active receptor can share
subunits. For example, GM-CSF, IL-3, and IL-5 all share a receptor
subunit, termed the beta common receptor (
CR)—the specificity
of the receptor is conferred by an additional unique alpha receptor
subunit (
17
). It was particularly notable that the
β
CR had been
reported to associate with EPOR in vitro (
18
). Based on this knowl-
edge, one possible structure for the tissue protective receptor could
be EPOR complexed with
β
CR.
Search for the alternative receptor system was initiated by con-
structing an affinity column from carbamylated EPO (CEPO), a
non-erythropoietic, tissue protective molecule in which lysines
had been chemically converted to homocitrulline (
15
). Membrane
β
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