A minimized Rap-RIAM module is sufficient for integrin activation.
A, minimal talin binding sequence of RIAM is in the N-terminal 30
residues. Purified His6-talin was incubated with
GST-RIAM-(1–103), -(1–43), or -(1–30) or GST immobilized on
glutathione-Sepharose. Bound proteins were fractionated by SDS-PAGE and
analyzed by Coomassie staining. B, talin binding fragment of
RIAM-(1–30) contains a putative amphipathicα-helix.
RIAM-(1–30) was analyzed using a secondary structure prediction program
(PBIL) and predicted helical residues (h) were identified with DSC,
MLRC, and PHD programs. Symbols represent α-helix (h), random
coil (c), and extended strand (e). Sec. Cons.,
secondary consensus residues. Helical wheel analysis was performed on
RIAM-(11–24) containing the predicted α-helical region, and the
asterisk denotes hydrophobic amino acid residues that were mutated to
charged Glu residues (see Fig. 5C). C, disruption of
hydrophobic face of the predicted helix abolishes talin binding. Hydrophobic
amino acid residues aligned along one side of the predicted amphipathic
α-helix in RIAM-(1–30) were mutated to glutamic acids and
expressed as GST-tagged proteins immobilized on glutathione-Sepharose beads.
Purified recombinant His6-talin was incubated with
GST-RIAM-(1–30) and the corresponding mutants M11E,F12E,L15E,L16E
(RIAM-(6–30)-4E) or L15E,L16E,L21E,L22E. Bound proteins were
fractionated by SDS-PAGE followed by Coomassie Blue staining for detection.
D, interaction of RIAM with talin is inhibited by a short RIAM wild
type peptide but not by mutant peptide. The complex of GST-RIAM-(1–301)
was incubated with full-length recombinant talin, and binding was performed as
described in Fig. 1B
in the presence of increasing amounts of peptides containing sequences from
RIAM; that is, wild type peptides spanning (6–30) or a mutant peptide
(6–30)-4E. 4E denotes the M11E,F12E,L15E,L16E mutant. Bound talin was
quantified by densitometry of Coomassie Blue-stained bands, and percent
inhibition was calculated as 100 × (B0 -
B)/B0, where B0 = binding in
the absence of peptide, and B = binding in the presence of peptide.
E, Rap1 membrane targeting sequence fused to RIAM-(1–30) induce
integrin activation that is abrogated by mutations that abolish talin binding.
A5 cells expressing HA-talin in combination with the indicated
GFP-RIAM-(1–30) proteins or GFP control were assessed for PAC1 binding
using flow cytometry to measure activation of αIIbβ3 (data are the
mean ± S.E. of independent experiments; n ≥ 3). Transfected
protein expression was verified by Western blotting. F, mutations
that perturb talin binding abolishes integrin activation induced by
RIAM-(1–301). A5 cells expressing HA-talin and GFP-tagged
RIAM-(1–301), RIAM-(1–301)-4E. or GFP vector control were assessed
for their ability to bind PAC1 using flow cytometry (data are the mean
± S.E. of independent experiments; n ≥ 3). 4E
denotes the M11E,F12E,L15E,L16E mutant. Transfected protein expression was
assessed by Western blotting. G, RIAM-(1–30)-CAAX
recruits talin to clusters at the membrane. A5 cells expressing mCherry-talin
and GFP-tagged RIAM-(1–30)-CAAX,
RIAM-(1–30)-4E-CAAX, or RIAM-(1–30) were adhered to
fibrinogen-coated coverslips and imaged to show talin (red) and RIAM
(green). Epifluorescent images as shown are maximal projections of
deconvoluted 0.1-μm z-section images of the entire cell volume. The
right panels show a two-color component scatter plot comparing the
distribution of correlated pixels for the indicated labeled proteins and the
calculated Pearson's correlation coefficient.