Regulation of alpha5beta1 integrin conformation and function by urokinase receptor binding

Abstract

Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with beta1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the beta-propeller of alpha3beta1 empowers vitronectin adhesion by this integrin. How uPAR modifies other beta1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds alpha5beta1 and rather than blocking, renders fibronectin (Fn) binding by alpha5beta1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of alpha5beta1-uPAR to Fn type III repeats 12-15 in addition to type III repeats 9-11 bound by alpha5beta1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall alpha5beta1 conformation. A beta1 peptide (res 224NLDSPEGGF232) that models near the known alpha-chain uPAR-binding region, or a beta1-chain Ser227Ala point mutation, abrogated effects of uPAR on alpha5beta1. Direct binding and regulation of alpha5beta1 by uPAR implies a modified "bent" integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.

Figure 1.

uPAR and ligand-engaged integrin are required for uPA–PAI-1 mediated matrix detachment. (A) Cell detachment from Ln-5 and Fn. 96-well plates were coated with Fn or Ln-5 and blocked with BSA. Wt α3, wt α3/U, mut α3, or mut α3/U cells were allowed to attach for 1.5 h, were acid washed, and then were incubated with uPA, PAI-1, or PAI-1 followed by uPA. The wells were washed as described in Materials and methods, and the number of remaining attached cells was determined. Each column is expressed as a percentage of the control (acid washed only; 100%). n = 3. (B) Biotin-III 9-11 Fn fragment binding to HT1080 cells. HT1080 cells were acid washed and incubated with or without uPA, or with uPA followed by PAI-1. The cells were then incubated with biotinylated III 9-11. After washing, the cells were lysed and the bound biotin III 9-11 was detected by avidin-HRP. The densitometry of the bands was quantified and graphed. All the steps were done at 4°C. Each column is expressed as a percentage of the control (100%). Data shown are representative from three independent experiments with similar results. (C) Effect of uPA–PAI-1 on purified α5β1 binding to Fn. Recombinant α5β1-Fc integrin was analyzed by Western blotting with peroxidase-conjugated anti–human Fc antibody. The α5-Fc and β1-Fc proteins are shown as an inset. Nunc high binding plates precoated with Fn were incubated with α5β1-Fc with or without suPAR in the presence of 1 mM Mn²⁺. Increasing amounts of uPA–PAI-1 mixture were then added. Bound α5β1-Fc was detected by protein A–HRP and quantified by OD at 490 nm. Data are expressed as specific binding (total binding subtract the binding to BSA-coated wells). n = 3. Each experiment was done with triplicate determinations.

Figure 2.

Model of α5β1 structure and homologous sequence alignment. (A) An energy-minimized model of integrin α5β1 structure was developed based on the atomic coordinates of αvβ3 crystal structure (Xiong et al., 2001). The putative W4 BC loop (NLTY) of the predicted β-propeller structure of the α5-chain (blue) is marked in red. The two β1 peptides β1P1 and β1P2 from β1-chain (gray) are indicated in yellow and purple, respectively. Both side and top view of the model are shown. (B) The sequence alignment of the two β1 peptide regions in β1-chain shows homologies among different β-chains. β1P1 peptide is underlined yellow and β1P2 purple. In the α3β1 model, these are the two β1 sequences closest to the reported α3 interaction site with uPAR (W4 BC loop of the β-propeller structure). Asterisk highlights Ser227 within the β1P1 sequence because only the β1-chain contains this Ser and a Ser→Ala point mutation in the β1P1 peptide was made and used in some experiments.

Figure 3.

uPAR binding to α5β1 changes the Fn-binding mechanism. (A) Adhesion to Fn. mut α3 (−uPAR), or mut α3/U (+uPAR) cells pretreated with different peptides were plated on Fn and cell adhesion was measured as described in Materials and methods. RGD and RAD: 500 μM; β1 peptides: 400 μM. A representative of three independent experiments with triplicate wells is shown. (B) Dose effect of β1P1 peptide on Fn adhesion. Wt α3/U cells were seeded on Fn-coated wells with β1P1 peptide (50–400 μM) and the adhesion was assessed as above. Data are expressed as percentage of control (no peptide added). n = 3. (C) Effect of β1P1 peptide on uPAR–α5β1 complex formation. HT1080 cells were lysed in 1% Triton X-100 lysis buffer and the lysates were incubated with peptide β1P1, its scrambled control (scβ1p1), or left untreated. Lysates were immunoprecipitated (P1D6) and the lysates and immunoprecipitates separated by SDS-PAGE and blotted for uPAR (R2) and integrin α5. Data shown are representative of three independent experiments. White lines indicate that intervening lanes have been spliced out. (D) Biotin-suPAR binding to α5β1–Fn complex. 20 nM biotinylated suPAR was added to Fn-coated wells and incubated with or without α5β1-Fc or α5β1SA-Fc (20 μg/ml). The bound biotin-suPAR was detected by avidin-HRP and Fn-bound integrin was detected by Protein A–HRP. “−” represents the background binding on Fn or BSA. Both are quantified by measuring OD at 490 nm. n = 3.

Figure 4.

uPAR–α5β1 binds to heparin-binding domain II of Fn. (A) Effect of RGD peptides on wt or mutant α5β1 binding to Fn. Purified α5β1-Fc or mutant α5β1SA-Fc (20 μg/ml) was added to Fn-coated wells in the presence or absence of suPAR and incubated with RGD peptides. The bound α5β1 was detected by protein A–HRP. n = 3. (B) Binding of biotin-RGD to uPAR–α5β1–Fn complexes. 20 μg/ml purified α5β1 was allowed to bind immobilized Fn in the presence or absence of 20 nM suPAR. 0.5 mM biotinylated RGD peptides or buffer were then added and incubated without or with excess unlabeled peptides (5 mM). The bound biotin was detected by avidin-HRP and bound α5β1 in parallel plates was detected by protein A–HRP. The data are expressed as absorbance at 490 nm. n = 3. (C) Cell adhesion to Fn fragments. Mut α3 (−uPAR) and mut α3/U (+uPAR) cells were plated in Fn or Fn fragment (III 9-11 or III 12-15)–coated wells and incubated without or with different peptides: RGD or control RAD peptides (500 μM); β1P1 or control scβ1P1 peptide (400 μM). The attached cells were quantified and the data from a representative experiment are shown. n = 3.

Figure 5.

Suppression of uPAR expression induces LIBS epitope and changes α5β1-mediated Fn binding in tumor cells. (A) FACS analysis of HT1080, MDA-MB-231, and Skov-3 cells with uPAR (siRNA uPAR) or control siRNA (control) transfection. Cells were harvested 48 h after transfection and incubated with antibodies against uPAR (uPAR), total β1 (JB1A), or conformation-sensitive β1 integrin antibodies (HUTS-21, 9EG7), followed by FITC-conjugated secondary antibodies. (B) HT1080 adhesion to Fn. siRNA uPAR or control cells were seeded to Fn-coated wells and incubated with different peptides. All the above experiments were performed at least three times with similar results.

Figure 6.

uPAR overexpression enhances Fn fibril formation. (A) HT1080 adhesion to low concentration of Fn. siRNA uPAR or control cells were seeded to Fn (0.2–5 μg/ml)-coated wells and incubated for ∼20 min. The adhesion was quantified as described in Materials and methods. (B) Top: FACS analysis of uPAR inducible clone Tet-uPAR cells. Both Tet-treated (2 μg/ml; +Tet) and nontreated cells (−Tet) were stained with FITC-conjugated uPAR antibody. n = 8. Bottom: Fn in Tet-uPAR cells. Cells without (−Tet) or with (+Tet) tetracycline induction were lysed with 3% Triton X-100 and centrifuged. Triton-insoluble pellets were then extracted with 2% deoxycholate (DOC) and centrifuged. The insoluble and soluble fractions on different membranes were analyzed by Western blotting using anti–human Fn antibodies. The same samples were blotted for β-actin to normalize the loading. n = 3. White line indicates that intervening lanes have been spliced out.

Figure 7.

RGD and β1P1 peptides inhibit HT1080 cell wound healing. Serum-starved HT1080 monolayers were wounded and incubated with different peptides (RAD, RGD, scβ1P1, and β1P1; 20 μM) in DME/0.1% BSA. The wounded areas were imaged at 0 and 24 h using a bright-field imaging system (Spot camera). The migration of HT1080 cells was quantified using SimplePCI software. The percent wound closure of each peptide-treated cell is shown on the right. n = 3. Marked pair (*) shows significant difference by t test (P < 0.006).

Figure 8.

Model for regulation of α5β1 integrin conformation and function by uPAR binding. Model proposes three basic forms of α5β1 exist on the cell surface: (1) A bent inactive form in the absence of integrin ligand; (2) an extended, active form induced by Fn binding in the absence of uPAR; and (3) a modified bent active form stabilized by uPAR binding, and potentially other cis-acting membrane proteins. In the case of uPAR, the modified bent form engages Fn differently as judged by Fn fragment III 9-11 and III 12-15 binding, altered β1P1 peptide sensitivity, the reversal of Fn binding by the presence of uPA–PAI-1 complexes, and enhanced Fn matrix assembly.