1. A Signaling Adapter Function for α6β4 Integrin in the Control of HGF-Dependent Invasive Growth 
Livio Trusolino, Andrea Bertotti, Paolo M Comoglio 
Cell 
Volume 107, Issue 5, Pages (November 2001)
DOI: /S (01)

2. Figure 1 Met Associates Selectively and Constitutively with α6β4
(A) Specific interaction between Met and α6β4. Left panels: Extracts were immunoprecipitated with antibodies against the β4 and β1 integrins or with preimmune serum (p.i.), and Western blots probed with anti-Met (upper panel) or anti-integrin antibodies (lower panels). Right panels: Reverse experiment.
(B) Association is specified by both the extra- and intracellular domains of Met. COS cells were cotransfected with vectors encoding β4 along with Met, Ron, or the indicated mutants. Lysates were immunoprecipitated with antibodies against the cytoplasmic domain of Met (to isolate Met, Ron-Met, Tpr-Met, and Tpr-jMet) and Ron (to isolate Ron and Met-Ron), or with an anti-Myc mAb (to isolate the Met extracellular fragment). Western blots were probed with antibodies against β4 (upper panels) or with the same antibodies used for immunoprecipitation (lower panels).
(C) Constitutive interaction between Met and α6β4. Adherent cells were left untreated, detached, or incubated with cytochalasin-D. Extracts were immunoprecipitated with anti-β4 antibodies or preimmune serum (p.i.). Western blots were probed with anti-Met (left panel) or anti-β4 (right panel) antibodies.
(D) Met and α6β4 colocalize in intact A431 cells. Cells were subjected to double immunostaining with anti-β4 (upper panels) or anti-β1 (lower panels) mAbs together with an anti-Met polyclonal antibody. Scale bar: 10 μm.
Cell  , DOI: ( /S (01) )

3. Figure 2 HGF Requires the Met-α6β4 Complex to Elicit Cell Invasion
(A) HGF induces invasion in cells expressing the Met-α6β4 complex, but not in cells expressing Met alone. Invasion assay of MDA-MB-435 mock and β4 transfectants through Matrigel, laminin-5, and fibronectin. Numbers on the y axes of the graphs indicate the increase, in folds, of invasion compared to nonstimulated mock transfectants.
(B–D) The activity of α6β4 is independent of the integrin adhesive function. Invasion assay of MDA-MB-435 β4 transfectants in the presence or absence of anti-β4 (B) or anti-β1 (C) antibodies. (D) Invasion assay using MDA-MB-435 mock and β4-Δextra transfectants.
(E–G) α6β4 is necessary for HGF-dependent cell invasion in other cell systems. (E) Invasion assay using RKO mock, β4, and β4-Δextra transfectants. (F) Invasion assay using A431 mock and β4-AS transfectants. (G) Western blot analysis of β4 expression in A431 mock (−) and β4-AS (+) transfectants.
All records expressed in this figure are means ± SD of three separate experiments performed in triplicate.
Cell  , DOI: ( /S (01) )

4. Figure 3
HGF Elicits the Whole Program of Invasive Growth in MDA-MB-435 Cells Expressing the Met-α6β4 Complex, but Not in Cells Expressing Met Alone
(A) Proliferation assays. Stimulation with 10% FCS was used as a control. Numbers on the y axis of the graphs indicate fold-increase of proliferation.
(B) Survival assays. Numbers on the y axis indicate percentage of survival.
(C) Soft agar assays. Values on the y axis indicate fold-increase of the number of colonies. All records expressed in this figure are means ± SD of three separate experiments performed in duplicate.
(D) Formation of lung metastases after subcutaneous inoculation of MDA-MB-435 mock and β4 transfectants in nude mice. A representative image of lungs from a mouse injected with mock cells (left) or with β4 transfectants (right) is shown.
Cell  , DOI: ( /S (01) )

5. Figure 4 Met Activation Results in Tyrosine Phosphorylation of α6β4
(A) HGF stimulation. A431 cell extracts were immunoprecipitated with anti-β4 antibodies and Western blots were probed with antibodies against phosphotyrosine (upper panel) or β4 (lower panel).
(B) Protein overexpression. COS cells were cotransfected with vectors encoding β4, MetWT, MetD, and MetK−. Lysates were immunoprecipitated with antibodies against Met (left panels) or β4 (right panels). Western blots were probed with an anti-phosphotyrosine antibody (upper panels) or with antibodies against the immunoprecipitated proteins (lower panels).
(C) Cell attachment. Serum-starved A431 cells were detached and either kept in suspension or plated for the indicated times on poly-L-lysine or fibronectin. Extracts were immunoprecipitated with anti-Met (upper two panels) or anti-β4 (lower two panels) antibodies. Western blots were probed with antibodies against phosphotyrosine, and reprobed with antibodies against the immunoprecipitated proteins.
Cell  , DOI: ( /S (01) )

6. Figure 5
Tyrosine phosphorylated α6β4 Recruits Shc and PI3K and Amplifies HGF-Dependent Signals
(A) COS cells were cotransfected with vectors encoding β4 and either MetK− or MetD. Extracts were incubated with agarose-immobilized GST fusion proteins corresponding to the SH2 domain of Shc or the carboxy-terminal SH2 domain of PI3K. Bound proteins were analyzed by probing with antibodies against β4. GST alone was used as a control. One-tenth of the total lysates were loaded to verify equal input of β4.
(B and C) MDA-MB-435 mock and β4 transfectants were stimulated with 100 ng/ml HGF for the indicated times. Cell extracts were immunoprecipitated with antibodies against Shc (B) or PI3K (C). Western blots were probed with an anti-phosphotyrosine antibody (B and C, upper panels) or with antibodies against Shc (B, lower panel) or PI3K (C, lower panel).
(D and E) MDA-MB-435 mock and β4 transfectants were stimulated with 100 ng/ml HGF for the indicated times. Total lysates were resolved by SDS-PAGE, Western blotted, and probed with antibodies against active and total MAPK (D) or against active and total PKB/Akt (E).
(F and G) MDA-MB-435 mock and β4-Δextra transfectants were stimulated with 100 ng/ml HGF for the indicated times. Total lysates were resolved by SDS-PAGE, Western blotted, and probed with antibodies against active and total MAPK (F) or against active and total PKB/Akt (G).
Cell  , DOI: ( /S (01) )

7. Figure 6
An α6β4 Mutant Unable to Bind Shc Prevents HGF-Dependent Signals and Cell Responses
(A and B) The β4-ΔShc mutant cannot restore HGF-dependent invasion. Invasion assay using MDA-MB-435 (A) or RKO (B) cells.
(C) Dominant-negative effect of the β4-ΔShc mutant in A431 cells expressing endogenously the Met-α6β4 complex. Invasion assay using A431 mock and β4-ΔShc transfectants.
(D) β4-ΔShc is unable to amplify Met-dependent signals. MDA-MB-435 β4 and β4-ΔShc transfectants were stimulated with 100 ng/ml HGF for 15 min. Total lysates were resolved by SDS-PAGE, Western blotted, and probed with antibodies against active and total MAPK (left panels) or against active and total PKB/Akt (right panels).
Cell  , DOI: ( /S (01) )