Biology Reference
In-Depth Information
Processing of EPOR by JAK-2
Only low numbers of EPOR (200 to 3,000 receptors/cell) are expressed on the
surface of primary erythroid cells or on cells engineered to overexpress the
receptor [14, 35-47]. Most newly synthesized molecules are retained in the
endoplasmic reticulum and appear to be degraded without further processing
[14, 15].
In vitro
experiments have suggested that unliganded EPOR normally
is tethered to JAK-2 [23], and it was thought that their interaction occurs only
at the plasma membrane. Our results showed that JAK-2 assembles with newly
synthesized EPOR in the endoplasmic reticulum, and that this assembly results
in efficient expression of the receptors at the cell surface [48]. This interaction
is specific to the
N
-terminal domain of JAK-2, as JAK1 has no effect on EPOR
N
surface expression. Therefore, in addition to its signaling ability, JAK-2 is
essential for optimal expression of the signaling EPOR/JAK-2 complex at the
cell surface.
JAK-2 activation in the EPOR/JAK-2 complex
JAK-2 contains an
N
-terminal domain followed by a pseudokinase domain,
N
and a kinase domain at its
C
-terminus. The
N
-terminal domain is responsible
N
for functional EPOR interaction and EPOR surface expression [48]. JAK-2
physically associates with the membrane-proximal Box1/Box2 region of
EPOR cytoplasmic domain [49]. EPO induces auto- or transphosphorylation
of a tyrosine in the kinase activation loop and triggers JAK-2 activation [50].
Figure 2 illustrates our current model for EPOR induction of JAK-2 activa-
tion.
In light of a preformed dimeric EPOR/JAK-2 complex in the absence of
EPO on the cell surface, JAK-2 activation in this complex may involve the
transmission of the presumed conformational change in the extracellular
domain of EPOR upon EPO binding to the intracellular activation of pre-asso-
ciated JAK-2 and the subsequent phosphorylation of EPOR cytoplasmic
domain. Three hydrophobic residues, Leu253, Ile257, and Trp258, in the jux-
tamembrane region of EPOR constitute a hydrophobic motif critical for JAK-2
activation and EPOR signaling [48]. Mutations of the individual residues in
this motif generated EPOR that form cell surface EPOR/JAK-2 complexes,
however, these mutant EPOR are unable to activate JAK-2 upon EPO stimula-
tion. We propose that this motif acts as a molecular switch to turn on JAK-2
tyrosine kinase activity upon EPO binding to the EPOR/JAK-2 complex. Since
this motif is highly conserved among cytokine receptors, this mechanism may
be used by other cytokine receptors and their cognate JAK. Further studies are
required to test this hypothesis.
Results from studies using knock-in and transgenic mice showed that a trun-
cated EPOR without any tyrosine residues can support erythropoiesis
in vivo
,
although much poorer than the wild-type receptor [51, 52]. These receptors
retain the Box 1/Box 2 region that binds and activates JAK-2, highlighting the
essential role of JAK-2 in EPOR signal transduction in erythropoiesis.
Furthermore, these experiments and others [53] underscore the contribution of
