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associated with several aminoacyl-tRNA synthetases [35]. This protein has many
effects on endothelial cells, including the induction of von Willebrand factor release
and tissue factor activation, as well as the upregulation of E-selectin and P-selectin
[21]. Additionally, it has been shown to have anti-angiogenic effects. EMAP II
induces endothelial cell apoptosis and inhibits endothelial cell proliferation, tube
formation, and angiogenesis in the rat aortic ring assay [5]. One group has demon-
strated that in vivo injection of EMAP II in a flank tumor model results in decreased
tumor vascularization, increased rate of vascular thrombosis, and delayed tumor
growth [39]. The mechanisms by which EMAP II carries out these functions are still
being elucidated, however work by the Kang laboratory indicates that cell surface
ATP synthase is involved in the anti-angiogenic effects. His research group identi-
fied the
subunit of ATP synthase as one of the receptors for EMAP II. Incubation
of endothelial cells with EMAP II decreases proliferation by 40%, and this growth
suppression can be inhibited by approximately 30% via addition of soluble
α
subunit
of ATP synthase. The anti-proliferative activity can also be achieved by incubation
with anti-
α
-ATP synthase antibody in a dose-dependent manner [12].
Under acidic conditions, the interaction between EMAP II and the
α
subunit
is regulated by cell surface heparan sulfate, which binds directly to both proteins
via heparin binding motifs. Heparan sulfate does not appear to affect the interac-
tion at neutral pH, however at pH 6.5 increased binding occurs between EMAP II
and
α
-ATP synthase. This enhanced binding can be inhibited by exogenous heparin,
indicating that the effect is likely due to interaction with heparan sulfate. The authors
suggest that at low pH, cross-linking by heparan sulfate enhances EMAP II-
α
sub-
unit binding. This idea is supported by the increased inhibitory effect of EMAP II
on endothelial cell proliferation at pH 6.5 relative to 7.5, which can be prevented by
disrupting interactions with heparan sulfate by the addition of exogenous heparin or
heparinase [11].
Binding of EMAP II to the
α
subunit of ATP synthase accounts for only part
of the anti-angiogenic effects of this protein. In the initial study demonstrating
EMAP II binding to
α
α
subunit only partly reverses EMAP II-induced inhibition of endothelial cell pro-
liferation indicating that significant anti-proliferative effects are also transmitted
through other receptors [12]. EMAP II also disrupts fibronectin matrix assembly
by endothelial cells via a direct interaction with the integrins
α
-ATP synthase, incubation of cells with excess soluble
α
β
α
β
3, likely
playing a role in its effects on angiogenesis [38]. Vascular endothelial growth fac-
tor (VEGF) receptors I and II are a third target of EMAP II on the endothelial cell
surface. EMAP II inhibits VEGF binding to these receptors, preventing downstream
signaling in the VEGF pathway and abrogating VEGF-induced endothelial cell pro-
liferation and migration [3]. At this time, it is not known whether any of the other
effects of EMAP II on endothelial cells are also mediated in part by the interaction
with
5
1 and
V
α
-ATP synthase.
9.6 Remaining Questions
Although many functions for cell surface ATP synthase have been identified,
the precise mechanism by which it reaches the plasma membrane remains to be
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