Relationships between Rap1b, affinity modulation of integrin alpha IIbbeta 3, and the actin cytoskeleton.
The affinity of integrin alpha(IIb)beta(3) for fibrinogen is controlled by inside-out signals that are triggered by agonists like thrombin. Agonist treatment of platelets also activates Rap1b, a small GTPase known to promote integrin-dependent adhesion of other cells. Therefore, we investigated the role of Rap1b in alpha(IIb)beta(3) function by viral transduction of GFP-Rap1 chimeras into murine megakaryocytes, which exhibit inside-out signaling similar to platelets. Expression of constitutively active GFP-Rap1b (V12) had no effect on unstimulated megakaryocytes, but it greatly augmented fibrinogen binding to alpha(IIb)beta(3) induced by a PAR4 thrombin receptor agonist (p < 0.01). The Rap1b effect was cell-autonomous and was prevented by pre-treating cells with cytochalasin D or latrunculin A to inhibit actin polymerization. Rap1b-dependent fibrinogen binding to megakaryocytes was blocked by POW-2, a novel monovalent antibody Fab fragment specific for high affinity murine alpha(IIb)beta(3). In contrast to GFP-Rap1b (V12), expression of GFP-Rap1GAP, which deactivates endogenous Rap1, inhibited agonist-induced fibrinogen binding (p < 0.01), as did dominant-negative GFP-Rap1b (N17) (p < 0.05). None of these treatments affected surface expression of alpha(IIb)beta(3). These studies establish that Rap1b can augment agonist-induced ligand binding to alpha(IIb)beta(3) through effects on integrin affinity, possibly by modulating alpha(IIb)beta(3) interactions with the actin cytoskeleton.
Bertoni
Alessandra
A
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Tadokoro
Seiji
S
Eto
Koji
K
Pampori
Nisar
N
Parise
Leslie V
LV
White
Gilbert C
GC
Shattil
Sanford J
SJ
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011485:Protein Binding; D020668:rap GTP-Binding Proteins / Q000378:metabolism*
D000199:Actins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C116753:RAP1B protein, human; C492136:Rap1b protein, mouse; D020668:rap GTP-Binding Proteins