Functional analysis of the cytoplasmic domain of the integrin α1 subunit in endothelial cells by Tristin D. Abair, Nada Bulus, Corina Borza, Munirathinam Sundaramoorthy, Roy Zent, and Ambra Pozzi Blood Volume 112(8):3242-3254 October 15, 2008 ©2008 by American Society of Hematology

Amino acids 1146-1148 of the integrin α1 tail control cell spreading, adhesion, and migration. Amino acids 1146-1148 of the integrin α1 tail control cell spreading, adhesion, and migration. (A) Schematic representation of the cytoplasmic truncation mutants of the human integrin α1 cDNA. (B) α1-null endothelial cells were transduced with either empty vector (α1KO) or the human integrin α1 mutant cDNAs indicated in panel A, and cell populations with equal levels of expression of the integrin α1 subunits were sorted by FACS using anti–human integrin α1 antibodies. (C) One millligram of total cell lysates of the cell populations indicated was immunoprecipitated with anti–human integrin α1 antibodies, subjected to SDS-PAGE, and immunoblotted with antibodies to either human integrin α1 or mouse β1 subunits. Equal loading was confirmed by analyzing the levels of total ERK in 40 μg total cell lysates. (D,E) Integrin α1KO endothelial cells expressing either the empty vector or cytoplasmic tail deletion mutants were plated in serum-free medium on 10 μg/mL collagen IV (D) or fibronectin (E). After 4 hours, the cells were fixed and stained with rhodamine-phalloidin. A representative cell is shown for each cell population. Bar represents 10 μm. (F,G) The cell populations were plated in serum-free medium on collagen IV at the concentrations indicated (F) or 10 μg/mL collagen IV with or without anti–human integrin α1 antibodies (10 μg/mL) (G) for 1 hour and their adhesion determined as described in “Cell adhesion.” Values are mean plus or minus SD of one representative experiment performed in quadruplicate. (H,I) The cell populations were plated on serum-free medium transwells coated with 10 μg/mL collagen IV with (▬) or without (▭) anti–human integrin α1 antibodies (10 μg/mL), and migration was evaluated 16 hours after plating. Values are mean plus or minus SD of one representative experiment performed in duplicate (5 fields/transwell were analyzed). Differences between α1KO and α1 mutant–expressing cells (*) and antibody-untreated versus-treated α1 mutant–expressing cells (**) were significant with P < .05. Tristin D. Abair et al. Blood 2008;112:3242-3254 ©2008 by American Society of Hematology

Integrin α1 cytoplasmic tail mutants activate distinct signaling pathways in endothelial cells. Integrin α1 cytoplasmic tail mutants activate distinct signaling pathways in endothelial cells. (A) The cell populations were plated in serum-free medium on solidified collagen I + IV gels. Tubulogenesis was quantified 16 hours after plating as described in “Tubulogenesis.” Values are mean plus or minus SD of 4 independent experiments. Gels were viewed with a Nikon Diaphot inverted research microscope (Nikon, Tokyo, Japan) using a lens at 20×/0.2 Ph2 LD 0.4. Images were acquired using a Canon PowerShot S5 IS camera (Canon USA, Lake Success, NY) and were processed with Adobe Photoshop version 9.0 software (Adobe Systems, San Jose, CA). (B) The cell populations were plated in 96-well plates coated with 10 μg/mL collagen IV. Four hours later, the cells were incubated with serum-free medium containing 3H-thymidine (0.5 μCi/well) for a further 48 hours, and proliferation was then evaluated as described in “Cell proliferation.” Values are mean plus or minus SD of one representative experiment performed in quadruplicate. *Statistically significant differences (P < .05) between the α1KO cells and α1 mutant–expressing cells. (C) The cell populations were serum-starved for 24 hours and embedded in collagen I + IV gels for the time indicated. The gels were sonicated and run on an SDS-PAGE gel to detect levels of activated and total p38 MAPK, ERK, and Akt. Images are representative of 3 independent experiments. Vertical line(s) have been inserted to indicate a repositioned gel lane. (D) Phosphorylated and total kinase bands were quantified by densitometry analysis, and the phosphorylated signal was expressed as phosphorylated kinase/total kinase ratio. Values are the mean plus orf minus SD of 3 independent experiments. * indicates significant differences (P < .05) relative to α1KO cells. (E-G) The cell populations indicated were subjected to migration (E), tubulogenesis (F), and proliferation (G) assays on collagen IV or fibronectin in the presence or absence of 10 μM PD169316, 10 μM PD98059, or 5 μM LY294002. Values are the mean plus or minus SD of one representative experiment. Differences between untreated α1KO and α1 mutant–expressing cells (*) and inhibitor untreated versus treated α1 mutant–expressing cells (#) were significant with P < .05. Tristin D. Abair et al. Blood 2008;112:3242-3254 ©2008 by American Society of Hematology

K1146A point mutation of the integrin α1 cytoplasmic tail leads to decreased endothelial cell adhesion and migration. K1146A point mutation of the integrin α1 cytoplasmic tail leads to decreased endothelial cell adhesion and migration. (A) Schematic representation of single point mutants (K/A) generated from the full-length integrin α1 subunit. (B) α1KO endothelial cells were transduced with either empty vector or the integrin α1 mutant cDNAs indicated in panel A, and cell populations were sorted by FACS using antihuman integrin α1 antibodies. (C) Total cell lysates of the cell populations were used to detect the levels of full-length and mutant human integrin α1 as well as mouse β1 subunits as described in detail in Figure 1C. (D-G) Cell adhesion on different concentrations of collagen IV (D) or in the presence of anti–human integrin α1 antibodies (E) as well as migration in the absence (F) and presence (G) of anti–human integrin α1 antibodies were determined as described in Figure 1. Note that only cells expressing K1146A mutant fail to adhere and migrate on collagen IV. * indicates statistically significant differences (P < .05) between the α1KO and the α1 mutant–expressing endothelial cells; * indicates statistically significant differences (P < .05) between untreated and antibody-treated α1 mutant–expressing cells. (H,I) The endothelial cells indicated were plated on 10 μg/mL collagen IV or fibronectin. After 4 hours, the cells were fixed and stained with rhodamine-phalloidin. A representative cell is shown for each cell population. Bar represents 10 μm. Tristin D. Abair et al. Blood 2008;112:3242-3254 ©2008 by American Society of Hematology

K1146A and K1147A point mutations of the integrin α1 cytoplasmic tail lead to decreased tubulogenesis. K1146A and K1147A point mutations of the integrin α1 cytoplasmic tail lead to decreased tubulogenesis. Tubulogenesis (A), cell proliferation (B), cell signaling (C), and densitometry analysis (D) were determined as described in Figure 2. Images of cells undergoing tubulogenesis were taken as described in Figure 2A. *Statistically significant differences (P < .05) between the α1KO and the α1 mutant–expressing endothelial cells. Vertical line(s) in panel C have been inserted to indicate a repositioned gel lane. Tristin D. Abair et al. Blood 2008;112:3242-3254 ©2008 by American Society of Hematology

Generation of integrin α1-null endothelial cells expressing deletion mutants lacking Glu1150 and/or Lys1151 of the integrin α1 cytoplasmic tail. Generation of integrin α1-null endothelial cells expressing deletion mutants lacking Glu1150 and/or Lys1151 of the integrin α1 cytoplasmic tail. (A) Schematic representation of the cytoplasmic truncation mutants of the human integrin α1 cDNA. (B) α1KO endothelial cells were transduced with either empty vector or the integrin α1 mutant cDNAs indicated in panel A, and cell populations expressing the integrin α1 constructs were sorted by FACS using anti–human integrin α1 antibodies. (C) Total cell lysates of the cell populations were used to detect the levels of full-length and mutated human integrin α1 as well as mouse β1 subunits as described in detail in Figure 1C. (D,E) Integrin α1KO endothelial cells expressing vector alone or the truncation mutants were plated on 10 μg/mL collagen IV or fibronectin. After 4 hours, the cells were fixed and stained with rhodamine-phalloidin. A representative cell is shown for each cell population. Bar represents 10 μm. Tristin D. Abair et al. Blood 2008;112:3242-3254 ©2008 by American Society of Hematology

Lys1151 within the integrin α1 tail is required for endothelial cell adhesion, migration, and tubulogenesis, but not proliferation. Lys1151 within the integrin α1 tail is required for endothelial cell adhesion, migration, and tubulogenesis, but not proliferation. Cell adhesion (A), migration (B), tubulogenesis (C), and proliferation (D) were determined as described in Figures 1 and 2. (E,F) Integrin α1β1–dependent signaling and densitometry analysis were determined as described in Figure 2C,D. * indicates statistically significant differences (P < .05) between the α1KO and the α1 mutant–expressing endothelial cells. Tristin D. Abair et al. Blood 2008;112:3242-3254 ©2008 by American Society of Hematology

Negatively charged amino acids at the COOH-terminus of the integrin α1 tail inhibit cell adhesion and migration, but not proliferation. Negatively charged amino acids at the COOH-terminus of the integrin α1 tail inhibit cell adhesion and migration, but not proliferation. (A) Schematic representation of mutants generated from the full-length integrin α1 subunit. (B) CHO cells were transfected with either empty vector or the integrin α1 mutant cDNAs indicated in panel A, and cell populations were sorted by FACS using anti–human integrin α1 antibodies. (C) The CHO cell populations indicated were plated on 10 μg/mL collagen IV and after 1 hour they were fixed and stained with rhodamine-phalloidin. A representative cell is shown for each cell population. Bar represents 10 μm. (D-F) Cell adhesion (D), migration (E), and proliferation (F) were determined as described in Figures 1 and 2. * indicates statistically significant differences (P < .05) between the vector transfected and α1 mutant–expressing CHO cells. Tristin D. Abair et al. Blood 2008;112:3242-3254 ©2008 by American Society of Hematology