Tethering membrane targeting sequences of Rap1 to RIAM-(1–176)
bypasses Rap1 requirement for integrin activation. A5 cells were
co-transfected with HA-talin and GFP-tagged RIAM-(1–176) constructs with
or without Rap1GAP, as indicated. Integrin activation was assayed by PAC1
binding and quantified using flow cytometry as described in
Fig. 2. A, fusion of
the membrane targeting sequences of Rap1 with RIAM-(1–176) to form
RIAM-(1–176)-CAAX induced integrin activation. B, the
extent of integrin activation (Activation index), calculated as
described in legend to Fig.
2B, was plotted against expression of RIAM-(1–176)
or RIAM-(1–176)-CAAX as assessed by GFP fluorescence intensity.
C, integrin activation induced by RIAM-(1–176)-CAAX
was insensitive to inhibition by Rap1GAP co-expression. D, integrin
activation depends on talin binding to the integrin because it is abolished
with HA-talin (W359A), a mutant talin that is deficient in binding to the
integrin β3 cytoplasmic tail. Integrin activation indices were calculated
as described in Fig. 2 and are
the mean ± S.E. for n ≥ 3(A–D). Transfected
protein expression levels were assessed by Western blotting (right
panels) with antibodies against GFP or HA for detection of GFP-RIAM,
HA-talin, or HA-Rap1GAP, as indicated (A–D).