Recombinant αvβ3 integrin binds directly to the rα4LN fragment. Wells of a 96-well plate were coated with either fibronectin (FN) or the rα4LN fragment. Recombinant ΔTMαvβ3 integrin (5 ng/μl) was diluted in physiologic cation binding buffer (1 mm MgCl₂ and 1 mm CaCl₂) and then added to the wells and allowed to bind for 1 h at 37 °C in the absence or presence of 10 mm EDTA. Integrin binding was evaluated by ELISA using an antibody against αvβ3 integrin, followed by a secondary antibody conjugated to alkaline phosphatase. The absorbance was measured at 405 nm. Data are expressed as specific binding (±S.D.).

Endothelial cell adhesion is regulated byαvβ3 andβ1 integrin. A, endothelial cells in suspension were incubated with control IgG, function-blocking antibodies against αvβ3 integrin, function-blocking antibodies against β1 integrin, or a combination of both at 25 ng/μl for 10 min at 37 °C. Cells were then added to wells of a 96-well plate coated with the rα4LN fragment at 5 ng/μl. Adherent cells were fixed and stained with crystal violet. Cell adhesion was determined by measuring absorbance at 595 nm in triplicate in at least three separate studies. Data are the average from three separate trials. 100% cell adhesion represents adhesion obtained in cells treated with control IgG. Data are presented as percent of adhesion of cells treated with control IgG (±S.D.) B, endothelial cells in suspension were incubated with function-blocking antibodies as above in the presence of 0.5 mm MnCl₂. Cell adhesion to the rα4LN fragment was determined as above. Note cell adhesion to uncoated wells was minimal under these conditions. Data are presented as percent of adhesion of cells treated with control IgG (±S.D.).

Overexpression of talin does not rescue cell adhesion in endothelial cells. A, endothelial cells were mock-transfected (mock) or were co-transfected with a construct expressing HA-tagged talin (F2/F3 domain) (HA-talin F2/F3). Talin expression was determined by Western blot analysis 48 h after transfection. Immunoblots were incubated with anti-HA antibodies to detect HA-tagged talin protein. The same blots were incubated with anti-actin antibodies. Actin was used as a loading control. B, endothelial cells were transfected with the GFP construct alone or co-transfected with talin (F2/F3 domain) and the GFP expressing construct to identify transfected cells. Transfected cells were trypsinized, and the cell suspension was treated with IgG or function-blocking antibodies against β1 integrin at 25 ng/μl for 10 min at 37 °C. Cells were then added to non-tissue cultured dishes coated with the rα4LN fragment at 5 ng/μl. Adherent cells were fixed, and GFP-positive cells were counted. Data are presented as GFP-positive cells per 4× field ± S.D. Data are from three independent trials.

Serine residue 752 regulates αvβ3-mediated cell adhesion to the rα4LN fragment. A, sequence of the cytoplasmic domain of wild type β3 integrin and β3 integrin bearing point mutations at residue 752. B, GFP-tagged wild type β3 integrin or β3 integrin protein bearing point mutations at residue 752 were expressed in CHO cells, and stably transfected cells were selected and gated by flow cytometry. Surface expression of β3 integrin and point mutants in CHO cells was assessed. Surface proteins in CHO cells were biotinylated and precipitated with streptavidin-conjugated beads. Immunoprecipitates (IP) were subjected to SDS-PAGE and analyzed by Western blot analysis using an antibody against GFP. Mock-transfected cells (mock), β3 integrin wild type (β3WT), β3 integrin S752A (β3S752A), β3 integrin S752D (β3S752D) are shown. C, mock-transfected CHO cells and CHO cells stably expressing wild type or cytoplasmic point mutants of β3 integrin were plated on rα4LN fragment-coated surfaces for 1 h. Nonadherent cells were washed off the wells, and the remaining cells were fixed and stained with crystal violet. Absorbance was read at 570 nm. Adhesion assays were undertaken in triplicate, and results are presented as percent of adhesion of CHO cells expressing wild type β3 integrin (±S.D.).

Protein kinase A regulates αvβ3 integrin ligand binding in endothelial cells. A, endothelial cells were preincubated with 10 μm H-89, a PKA inhibitor for 1 h. Cells were trypsinized and resuspended in complete media ± PKA inhibitor in the presence of individual or combinations of the following antibodies: IgG, β1 function-blocking antibody, or αvβ3 integrin function-blocking antibody for 10 min as indicated. Cells were added to wells coated with the rα4LN fragment, and cell adhesion was performed as above. All antibodies were used at 25 ng/μl. Data are from triplicate experiments presented as percent of adhesion shown by IgG-treated cells (±S.D.). 100% cell adhesion is adhesion measured in endothelial cells treated with IgG control in the absence of H-89 inhibitor. B, endothelial cells were preincubated with 10 μm H-89 for 1 h at 37 °C. Cells were trypsinized, and suspended cells were treated with IgG or anti-β1 integrin function-blocking antibody for 10 min at 37 °C. β3 integrin was immunoprecipitated (IP) using antibodies against αvβ3 integrin and analyzed by Western blot using phosphoserine antibodies, followed by antibodies against β3 integrin as indicated. C, endothelial cells were treated with pharmacologic inhibitors against serine/threonine kinases, calmodulin kinase II, ROCK, and PKC (2.5 μm KN-93, 5 μm Y-27632, 5 μm GF109203X, respectively) for 1 h. Cell adhesion was determined as above in the presence or absence of the indicated integrin function-blocking antibodies. Data are presented as percent of endothelial cells treated with IgG control. 100% cell adhesion of cells were treated with IgG control in the absence of pharmacologic inhibitors.

Schematic showing a model that depicts the way β1 subunit-containing integrins regulate ligand binding of αvβ3 integrin in endothelial cells. In the basal state, endothelial cells adhere to the rα4LN fragment in a β1 integrin-dependent manner. Clustering of β1 integrin with ligand (or following incubation of cells with β1 integrin function-blocking antibody) leads to activation of PKA. PKA phosphorylates inhibitor-1 which when phosphorylated is a potent inhibitor of serine/threonine PP1. PP1 regulates the activation state of αvβ3 integrin by maintaining the β3 integrin cytoplasmic tail in an unphosphorylated state. Under conditions when PKA activity is blocked, αvβ3 integrin-ligand binding is no longer inhibited by clustered β1 integrin, and cell adhesion increases 2-fold. In this instance, both αvβ3 and α3β1 integrin contribute to overall cell adhesion, and their effect is additive.

αvβ3 and α3β1 integrins bind to distinct sites of the rα4LN fragment. A, purified, soluble αvβ3 (5 ng/μl) was added to wells coated with the rα4LN fragment in the presence of increasing concentrations of purified, soluble α3β1 at 37 °C. αvβ3 integrin binding was assayed by ELISA using an antibody against the αvβ3 integrin. The absorbance was measured at 405 nm. B, purified, soluble α3β1 (5 ng/μl) binding to the rα4LN fragment-coated wells was assessed as above in the presence of increasing concentrations of αvβ3 integrin. Studies were undertaken in triplicate. Data are presented as % of binding in the absence of “competitor” (±S.D.).

β1 integrin regulates αvβ3 integrin binding to vitronectin in endothelial cells. A, endothelial cells in suspension were incubated with control IgG, function-blocking antibodies against αvβ5, αvβ3 integrin, β1 integrin, or a combination of αvβ3 and β1 integrin at 25 ng/μl for 10 min at 37 °C. Cells were then added to wells of 96-well plate coated with vitronectin at 2 ng/μl. Data are the average from three separate trials and are presented as percent of binding to cells treated with control IgG (±S.D.). 100% cell adhesion is adhesion obtained in endothelial cells treated with control IgG. B, endothelial cells in suspension were incubated with function-blocking antibodies against αvβ3, β1 integrin, or the combination of both in the presence of 0.5 mm MnCl₂. Cell adhesion to vitronectin was determined as above. Note cell adhesion to uncoated wells was minimal under these conditions. Data are presented as percent of adhesion of cells treated with control IgG (±S.D.).

β3 integrin is serine-phosphorylated in endothelial cells treated with β1 integrin function-blocking antibody. A and B, endothelial cells were trypsinized, and the cell suspension was treated with IgG or with a β1 integrin function-blocking antibody for 30 min at 37 °C. Cells lysates were prepared as described under “Experimental Procedures,” and β3 integrin was immunoprecipitated (IP) using an antibody against αvβ3 integrin. Immunoprecipitates were analyzed by Western blotting using an antibody against phosphotyrosine (pTyr) (A) or phosphoserine (pSer) (B). A and B, blots were stripped and reprobed with an antibody that recognizes β3 integrin. Bands corresponding to β3 integrin were quantified, and values were normalized to total β3 integrin. Data are expressed as fold phosphorylation over control. Representative of three independent trials.

Treatment of endothelial cells with β1 integrin antibodies results in protein kinase activity activation. A, endothelial cells were serum-starved and plated in serum-free media on tissue cultured plates coated with a β1 function-blocking antibody or control IgG. After 10 min, cells were washed once with cold PBS and lysed with hypotonic extraction buffer. PKA activity was determined by the incorporation of phosphate in Leu-Arg-Arg-Ser-Leu-Gly (Kemptide) using [γ-³²P]ATP. Samples were assayed in duplicate for each condition and quantified on a scintillation counter. Protein kinase A inhibitor-inhibitable kinase activity was calculated, and the data from triplicate samples are expressed as percent of total PKA activity ± S.E. B, endothelial cells were pretreated with control IgG or anti-β1 integrin antibodies for 30 min at 37 °C and plated onto vitronectin-coated plates. After 1 h, cells were processed for immunoblotting using antibodies against phospho-VASP. Immunoblots were stripped and reprobed with actin antibodies. Bands corresponding to phospho-VASP were quantified, and values were normalized to total actin protein. Data are expressed as fold phosphorylation over endothelial cell-treated IgG control. C, endothelial cells were pretreated with 10 μm H-89, a PKA inhibitor for 1 h at 37 °C. Endothelial cells in suspension were then treated with function-blocking antibodies as above and plated onto vitronectin-coated plates. The level of phospho-VASP was assessed as described above. Representative of three independent trials.

Treatment of endothelial cells with calyculin A blocks αvβ3-mediated cell adhesion to the rα4LN fragment. A, endothelial cells were left untreated by drug or preincubated with individual or combinations of drugs (10 μm H89, 1 nm calyculin A (Cal A)) as indicated for 1 h. Cells were trypsinized, and cell suspension was treated with IgG or function-blocking anti-β1 integrin antibodies for 10 min at 37 °C. Cells were added to wells coated with the rα4LN fragment, and cell adhesion was measured as above. Data are from three experiments presented as percent of adhesion of IgG-treated cells (±S.D.). 100% cell adhesion is adhesion measured in endothelial cells treated with IgG control in the absence of pharmacologic inhibitors. B, endothelial cells were preincubated with 1 nm calyculin A (CalA) for 1 h at 37 °C. Cells were trypsinized, and cell suspension was treated with IgG or anti-β1 integrin function-blocking antibody for 10 min at 37 °C. β3 integrin was immunoprecipitated (IP) using antibodies againstαvβ3 integrin and analyzed by Western blot using phosphoserine (pSer) antibodies, followed by antibodies against β3 integrin as indicated. C, endothelial cells were left untreated or preincubated with individual or combinations of drugs (10 μm H-89, 100 μm NaVO₄) as indicated for 1 h. Cells were trypsinized, and the cell suspension was treated with IgG or function-blocking anti-β1 integrin antibody for 10 min at 37 °C. Cell adhesion assay was performed as above. Adhesion assays were undertaken in triplicate, and results are presented as percent of adhesion of control IgG-treated cells (±S.D.). D, endothelial cells were infected with an adenovirus encoding inhibitor-1 for 48 h. Infected cells were trypsinized and preincubated with IgG control or anti-β1 integrin function-blocking antibodies for 30 min at 37 °C. Protein extracts of cells were collected and processed for immunoblotting using antibodies specific for phospho-inhibitor-1. The same blots were probed with anti-actin antibodies to determine loading. Representative blot of three independent experiments is shown. E, endothelial cells were incubated with 10 μm H-89 for 1 h. Endothelial cells were trypsinized and preincubated with function-blocking antibodies as above. Protein extracts of cells were collected and processed as above for phospho-inhibitor-1 and actin.