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News and Publications
Identification of New Integrin Ligands
Y. Takada, T. Tarui, T. Kamata, W. Puzon-McLaughlin, M. Majumdar,
N. Andronicos, J.Q. Yu
Integrins are critically involved in many biological and pathologic
processes. We are interested in identifying potential integrin ligands
and in determining how they are involved in these processes.
ANGIOSTATIN
Angiostatin, the N-terminal 4 kringles (K1-K4) of plasminogen,
blocks tumor-mediated angiogenesis in animal models and has great
therapeutic potential. Angiostatin is now in clinical trials. However,
its mechanism of action is unclear. We found that bovine arterial
endothelial (BAE) cells adhere to angiostatin in an integrin-dependent
manner and that integrins avb3, a9b1,
and, to a lesser extent, a4b1 specifically bind
to angiostatin. The integrin avb3
is a predominant receptor for angiostatin on BAE cells; an antibody
to avb3 that blocks the
function of avb3 effectively blocks
adhesion of BAE cells to angiostatin, but an antibody to a9b1 does not.
*-Aminocaproic acid, a lysine analog, effectively blocks angiostatin
binding to BAE cells, indicating that an unoccupied lysine-binding
site of the kringles may be required for integrin binding. Other
plasminogen fragments containing 3 or 5 kringles (K1-K3 or K1-K5)
have an antiangiogenic effect, but plasminogen itself does not.
We found that K1-K3 and K1-K5 bind to avb3,
but plasminogen does not. These results suggest that the antiangiogenic
action of angiostatin may be mediated via interaction with avb3. Angiostatin binding to avb3
does not strongly induce formation of stress fibers, suggesting
that angiostatin may prevent angiogenesis by perturbing the avb3-mediated signal transduction that may be necessary
for angiogenesis. We hope to identify the potential target for angiostatin.
FUNCTIONAL CLASSIFICATION OF ADAM PROTEINS
ADAM proteins, which have both a disintegrin domain and a metalloprotease
domain, are members of the metzincin superfamily of metalloproteases.
Among integrins binding to disintegrin domains of ADAM proteins
are a9b1 and avb3,
and they bind in an arginine-glycine-aspartic acid (RGD)-independent
and an RGD-dependent manner, respectively. Human ADAM-15 is the
only ADAM protein with the RGD motif in the disintegrin domain.
Thus, both integrins a9b1 and avb3
recognize the disintegrin domain of ADAM-15.
We used mutational analysis to determine how these integrins recognize
the disintegrin domain of ADAM-15. We found that the Arg481 and
the Asp-Leu-Pro-Glu-Phe residues (residues 488-492) were critical
for a9b1 binding, but the
RGD motif (residues 484-486) was not. In contrast, the RGD motif
was critical for avb3 binding, but the
other residues flanking the RGD motif were not. Because the R(X6)DLPEF
a9b1-recognition motif
(residues 481-492) is conserved among ADAM proteins, except for
ADAM-10 and ADAM-17, we hypothesized that a9b1
may recognize disintegrin domains in all ADAM proteins except ADAM-10
and ADAM-17. Indeed, we found that a9b1
bound avidly to the disintegrin domains of ADAM-1, ADAM-2, ADAM-3,
and ADAM-9 but not to the disintegrin domains of ADAM-10 and ADAM-17.
Because several ADAM proteins have been implicated in sperm-oocyte
interaction, we tested whether the functional classification of
the proteins, based on specificity for integrin a9b1,
applies to sperm-egg binding. We found that the disintegrin domains
of ADAM-12 and ADAM-15 bound to oocytes, but the disintegrin domain
of ADAM-17 did not. Furthermore, the disintegrin domains of ADAM-12
and ADAM-15 effectively blocked binding of sperm to oocytes, but
the disintegrin domain of ADAM-17 did not.
These results suggest that oocytes and a9b1
have similar binding specificities for ADAM proteins and that a9b1, or a receptor with similar specificity, may
be involved in sperm-egg interaction during fertilization. Because
a9b1 is a receptor for
many ADAM disintegrins and a9b1
and ADAM proteins are widely expressed, interaction between a9b1 and ADAM proteins may have broad biological
importance.
PUBLICATIONS
Eto, K., Huet, C., Tarui, T., Kupriyanov, S., Liu, H.Z., Puzon-McLaughlin,
W., Zhang, X.P., Sheppard, D., Engvall, E., Takada, Y. Functional
classification of ADAMs based on a conserved motif for binding to
integrin a9b1: implications for
sperm-egg binding and other cell interactions. J. Biol. Chem. 277:17804,
2002.
Kamata, T., Takada, Y. Platelet integrin aIIbb3-ligand
interactions: what can we learn from the structure? Int. J. Hematol.
74:382, 2001.
Kamata, T., Tieu, K.K., Irie, A., Springer, T.A., Takada, Y.
Amino acid residues in the aIIb subunit that are critical for ligand binding
to integrin aIIbb3 are clustered in
the b-propeller model.
J. Biol. Chem. 276:44275, 2001.
Kamata, T., Tieu, K.K., Tarui, T., Puzon-McLaughlin, W., Hogg,
N., Takada, Y. The role of the CPNKEKEC sequence in the b2 subunit I domain
in regulation of integrin aLb2 (LFA-1). J. Immunol.
168:2296, 2002.
Legge, G.B., Morris, G.M., Sanner, M.F., Takada, Y., Olson,
A.J., Grynszpan, F. Model of the aLb2
integrin I-domain/ICAM-1 DI interface suggests that subtle changes
in loop orientation determine ligand specificity. Proteins 48:151,
2002.
Miao, H., Li, S., Hu, Y.L., Yuan, S., Zhao, Y., Chen, B.P.,
Puzon-McLaughlin, W., Tarui, T., Shyy, J.Y., Takada, Y., Usami,
S., Chien, S. Differential regulation of Rho GTPases by b1 and b3 integrins: the role of an extracellular domain
of integrin in intracellular signaling. J. Cell Sci. 115:2199, 2002.
Nakamura, T., Lozano, P.R., Ikeda, Y., Iwanaga, Y., Hinek,
A., Minamisawa, S., Cheng, C.F., Kobuke, K., Dalton, N., Takada,
Y., Tashiro, K., Ross, J., Jr., Honjo, T., Chien, K.R. Fibulin-5/DANCE
is essential for elastogenesis in vivo. Nature 415:171, 2002.
Tani, N., Matsumoto, K., Ota, I., Yoshida, S., Takada, Y.,
Shiosaka, S., Matsuura, N. Effects of fibronectin cleaved by
neuropsin on cell adhesion and migration. Neurosci. Res. 39:247,
2001.
Tarui, T., Miles, L.A., Takada, Y. Specific interaction
of angiostatin with integrin avb3
in endothelial cells. J. Biol. Chem. 276:39562, 2001.
Triantafilou, K., Takada, Y., Triantafilou, M. Mechanisms
of integrin-mediated virus attachment and internalization process.
Crit. Rev. Immunol. 21:311, 2001.
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