INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The heterodimeric  and  subunits of the integrin family of adhesion proteins have no intrinsic signaling capacity. Therefore, transduction of information into cells, after engagement of ligands by integrins, is dependent on the dynamic assembly of signaling complexes around their transmembrane and cytoplasmic integrin tails (Diamond and Springer, 1994; Schwartz et al., 1995; Burridge and Chrzanowska-Wodnicka, 1996). These dynamic aspects of integrin function are regulated in part by the interaction of integrins with neighboring nonintegrin membrane-associated proteins, including tetraspan-4-superfamily 1 members (CD9, CD81, CD151, and others) (Berditchevski et al., 1996; Maecker et al., 1997), integrin-associated protein (CD47) (Cooper et al., 1995), caveolin (Wei et al., 1999), and the glycosylphosphatidylinositol-anchored urokinase receptor (uPAR, CD87) (Wei et al., 1996). In all cases reported to date integrin-associated proteins in some way promote integrin signaling, although there is considerable mechanistic diversity. CD47 associates preferentially with v3, promoting signaling through a heterotrimeric G protein-coupled pathway (Frazier et al., 1999). CD151, in contrast, preferentially associates with the 1 integrin partners 3 and 6 and promotes association of a cytoplasmic lipid kinase with these integrins (Berditchevski et al., 1996). Caveolin also associates with a set of 1 integrins, promoting their association with Src family kinases, probably by concentrating cholesterol-rich membrane "rafts" containing these kinases around integrins (Wary et al., 1998; Wei et al., 1999).

The influence of uPAR on integrin function appears complex. In experimental models either high levels of expressed recombinant uPAR or soluble uPAR have been reported to impair ligand binding by integrins and their adhesive functions (Wei et al., 1996). On the other hand, in most cells bearing endogenously expressed uPAR, uPAR, like other integrin-associated proteins, promotes integrin function. For example, we and others have recently reported evidence that signaling through the Fn receptor 51, and cell migration on Fn, was promoted by the association of this integrin with uPAR (Aguirre Ghiso et al., 1999; Wei et al., 1999). In one study soluble uPAR was found to promote signaling through 51 (Aguirre Ghiso et al., 1999). This is consonant with abundant, more circumstantial observations linking the expression of uPAR with cell migration important to inflammation and tumor metastasis (Bianchi et al., 1996; Andreasen et al., 1997; Ferrero et al., 2000; Huang et al., 2000). Whether the association of uPAR and 51 is direct or indirect is unclear because there has been no structural evidence to explain how uPAR might affect ligand engagement or signaling through integrins.

On the basis of homology with G protein-coupled receptors, Springer (1997) has proposed that the N-terminal region (~450 amino acids) of integrin  subunits folds into a seven-bladed -propeller. In this model repeating units (W1-W7) of antiparallel  sheets connected by surface loops (~60 aa/unit) arrange into a torus around a small central cavity. The upper surface loops are thought to contain the major ligand-binding sites, which synergize with binding sites on the  chain to define the specificity and affinity of interactions of integrins with their ligands. We have recently identified a linear sequence within the  chain of CD11b (Mac-1) (M424-440) that is a critical site for direct interaction between Mac-1 and uPAR (Simon et al., 2000). In the -propeller model, this sequence comprises the entire upper loop sequence of the W4 repeat and extends into the third  strand of this repeat, indicating that uPAR is an atypical integrin ligand, at least for CD11b. We now extend these findings to  chain partners of 1 integrins, identifying 31 as a preferential uPAR-binding integrin.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents

Prourokinase was a kind gift of Dr. Jack Henkin (Abbott Laboratories, Abbott Park, IL). Human soluble uPAR with or without biotinylation and murine uPA were kindly provided by Dr. Steven Rosenberg (Chiron Corporation, Emeryville, CA). Integrin 31 was purified as described (Eble et al., 1998). Purified integrin 51 was a gift from Dr. Sarah C. Bodary (Genentech, South San Francisco, CA). Human fibronectin (Fn), collagen type I (Col), pertussis toxin, and goat anti-mouse IgG secondary antibody were purchased from Sigma (St. Louis, MO) and vitronectin (Vn) was from BD Biosciences (San Jose, CA). Monoclonal antibodies to integrin 2 (P1E6), 3 (P1B5), 5 (P1D6), and 51 (HA5), and polyclonal anti-1 (AB1937) were obtained from Chemicon (Temecula, CA). A monoclonal antibody (mAb) against integrin 1 (JB1A) was a kind gift from Dr. John Wilkins (University of Manitoba, Winnipeg, MB, Canada). The polyclonal antibody to a Gi subunit of heterotrimeric G proteins (Gi-3) and the polyclonal antibody to Src family kinases (Src2) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The mAb to focal adhesion kinase (FAK) and the mAb to phospho-FAK were obtained from Transduction Laboratories (Lexington, KY). Rabbit anti-uPAR polyclonal antibody was purchased from American Diagnostica (Greenwich, CT). Purified mouse anti-human human leukocyte antigen-A,B,C mAb was obtained from BD PharMingen (San Diego, CA). Cy3 conjugated goat anti-rabbit IgG secondary antibody was from Zymed Laboratories (South San Francisco, CA). Monoclonal antibodies to integrin 2 (A2IIE10) and 3 (A3x8) and polyclonal antibody to integrin 3 were raised in Dr. Martin E. Hemler's lab, and the first two were conjugated with fluorescein isothiocyanate (FITC) with the use of a kit from Molecular Probes (Madison, WI). Peptides 325 (PRHRHMGAVFLLSQEAG), sc325 (HQLPGAHRGVEARFSML), 525 (PKGNLTYGYVTILNGSD), 625 (PRANHSGAVVLLKRDMK), v25 (PRA-ARTL GMVYIYDGKN), 25, and M25 were synthesized and purified by Quality Controlled Biochemicals (Framingham, MA).

Cell Lines and Culture Conditions

Human embryonic kidney cell line 293 and human carcinoma cell line MDA-MB-231 were obtained from American Type Culture Collection (Rockville, MD). All these cells were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). Urokinase receptor transfected 293 cells were cultured in DMEM complete medium containing 0.9 mg/ml geneticin (G418) (Invitrogen). Immortalized epithelial cells from 31 integrin-deficient kidney (B12) and human 3-transfected B12 cells (R10) were obtained and cultured as described (Wang et al., 1999).

Adhesion and Spreading Assays

Cells were seeded in fibronectin- (5 µg/ml), collagen type I- (5 µg/ml), or vitronectin (1 µg/ml)-coated 96-well tissue culture plates to assess adhesion or spreading to these matrix proteins. The cell adhesion assays were performed as previously described (Wei et al., 1996). Briefly, 5 × 10⁴/ml cells suspended in 100 µl of DMEM/0.1% bovine serum albumin (BSA) were seeded in triplicate on protein-coated 96-well plates and incubated for 1 h at 37°C, followed by three washes of phosphate-buffered saline (PBS). When performing inhibition assays, integrin 3 mAb (P1B5) and 5 mAb (P1D6) (5 µg/ml), pertussis toxin (100 ng/ml), or peptide 325 or scrambled peptide 325 (10-200 µM) were used. In some experiments, human pro-uPA (1 nM) was added to MDA-MB-231 cells or murine uPA (10 nM) to R10 and B12 cells. Cells attached to each plate were fixed with methanol and then stained with Giemsa. The data were quantified by measuring absorbance at a wavelength of 550 nm. When performing spreading assays, round and spread cells visualized by phase microscopy were counted from three different areas in each of triplicate wells after incubating with various peptides, antibodies, or urokinase.

Flow Cytometry

Wild-type or uPAR-transfected 293 cells were detached and incubated with PBS containing 0.1% BSA and primary antibodies to integrins 3 (P1B5) or 51 (HA5) on ice for 30 min. After washing, cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Sigma) and analyzed on a flow cytometer (FACScan; BD Biosciences).

Immunoprecipitation and Blotting

Cells (5 × 10⁶) expressing uPAR were lysed on ice for 30 min in 1.5 ml of RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% deoxycholate, 0.1% SDS, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin) or Triton lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100), supplemented with protease inhibitors. After preclearing with protein A agarose, lysates were incubated with antibodies to integrins 1 (JB1A), 3 (P1B5), or 51 (HA5) at 4°C overnight. In some experiments, 100 µM peptides 325, sc325, or 25 was added to the lysates. The immunoprecipitates were blotted for uPAR (399R) or Gi. In some cases, the membranes were stripped and reblotted for Src family kinases or 1 integrins. Initial experiments indicated that >95% of the total uPAR was solubilized by both 1% Triton and RIPA buffer. However, ~10% of the total cellular uPAR was coimmunoprecipitated with 31 in either 1% Triton or RIPA buffer.

Purified Protein Binding Assay

Nunc high-binding microtiter plates were coated with purified 31 or 51 (2 µg/ml) and blocked with 10 mg/ml BSA. Biotinylated soluble uPAR (suPAR) (1-200 nM) with or without equimolar amounts of pro-uPA was added to each well in PBS/1 mg/ml BSA, and the plates were incubated for 1 h at 25°C. After washing, bound suPAR was quantified with avidin-peroxidase as described (Wei et al., 1994). To test specificity of binding, 100-fold molar excess nonbiotinylated suPAR was added. Data were expressed as specific binding, i.e., total binding minus the binding observed in the presence of excess unlabeled suPAR, which accounted for <20% of the total. Binding to wells coated with BSA alone accounted for <10% of the total.

The binding of biotinylated suPAR to peptide 325 was performed as described (Simon et al., 2000). In brief, Nunc microtiter plates were coated with 325 (20 µg/ml) in PBS overnight at 37°C and blocked with 1% BSA. Biotinylated suPAR (100 nM) without or with 325, sc325, 525, or v25 (1-50 µM) was then added to each well for 1.5 h at 25°C. After washing, avidin peroxidase was added and biotinylated suPAR was quantified as described above. Relative binding was calculated as the ratio of binding in the presence of peptide to binding in the absence of peptide.

Immunofluorescence and Confocal Fluorescence Microscopy

To visualize integrin and uPAR clustering, human breast cancer cells (MDA-MB-231) were trypsinized, recovered in suspension at 37°C for 1 h to allow reexpression of surface proteins, washed with serum-free DMEM, and incubated with antibodies to 3 (P1B5) and control HLA or FITC-conjugated monoclonal antibodies to integrins 2 (A2IIE10) and 3 (A3x8) at 4°C for 30 min. After washing, cells in suspension were incubated without or with goat anti-mouse secondary antibodies for 1 h at 37°C, immobilized on 50 µg/ml polylysine-coated glass coverslips for 30 min, and then fixed 20 min in 3.7% paraformaldehyde. Fixed cells were blocked in 10% goat serum for 1 h and incubated with rabbit polyclonal antibody to uPAR for 1 h at room temperature then incubated with Cy3-conjugated secondary antibodies and coverslips mounted in Prolong (Molecular Probes). Fluorescence staining was analyzed by Zeiss microscope or confocal laser (model MRC1024; Bio-Rad Laboratories, Hercules, CA) attached to a Zeiss microscope (model Axiovert S100) with the use of separate filters for each fluorochrome. Ventral planes were imported into Adobe Photoshop (Adobe Systems, Mountain View, CA) and processed.

FAK Kinase Assay

To analyze FAK activity, cells were seeded on fibronectin- or collagen type I-coated 24-well plates. After incubating with peptides 325 or sc325 and antibodies to integrins 2 or 3, cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% deoxycholate, 0.1% SDS, 1% Triton X-100) supplemented with protease inhibitors. Lysates were immunoblotted for phospho-FAK and total FAK.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

3 and 6 Contain Sequences Most Homologous to M25

The putative structural organization of integrin  chains is indicated in Figure 1. Although no crystal structure for an integrin  chain has been reported, several lines of evidence favor a seven-bladed -propeller folding pattern for their amino-terminal, ligand-binding region (Irie et al., 1997; Springer, 1997). The position and sequence of an uPAR/CD11b (M) interaction site within the fourth blade (W4 repeat) of the -propeller is also shown in Figure 1 (Simon et al., 2000). Surprisingly, comparison of the Mac-1 sequence with sequences of all other integrin  chains in the GenBank database reveals the two integrin  chains with the closest homology to Mac-1 at this site are 3 and 6 (each 40% identical), two integrin chains not previously recognized to be physically associated with uPAR. Figure 1 shows the aligned sequences of the W4 repeat region of 3 and 6 along with the that of two integrins for which indirect evidence has favored a physical association with uPAR (Xue et al., 1997; Aguirre Ghiso et al., 1999). As is evident, the primary sequences of 5 and v in this region are less homologous than either 3 or 6. Based on this information, we initiated a series of experiments to determine whether the 3 and 6 sequences, termed 325 and 625, respectively, are functionally analogous to the previously reported M25 and whether 31 is a major signaling partner of uPAR.

View larger version (25K): [in this window] [in a new window]   Figure 1.   Sequence homologies between an M interaction site with uPAR and 1 integrin-coupled  chains. The N-terminal region (~450 amino acids) of integrin  subunits has been proposed to fold into a -propeller (Springer, 1997). In this model repeating units (W1-W7) of antiparallel  sheets connected by surface loops (~60 aa/unit) arrange into a torus. A peptide sequence of M spanning W4 was found to mediate binding of M to uPAR (Simon et al., 2000). Homologous amino acid residues in the most similar  chains associating with 1 integrins are indicated in the figure.

uPAR Preferentially Associates with 31 in 293 Cells

To explore whether uPAR physically associates with 31 as predicted by sequence homologies (Figure 1), coprecipitation experiments were performed in uPAR-transfected 293 cells. Previous studies have shown that 293 cells only express uPAR after expression by transfection (Wei et al., 1996). We verified that 293 cells express more 51 than 31 by fluorescence-activated cell sorter (FACS) analysis (Figure 2A). Lysates of uPAR/293 cells were immunoprecipitated sequentially with 3, 5, and 1 antibodies and the precipitates immunoblotted for uPAR. As is evident in Figure 2B, the bulk of 1-associated uPAR coprecipitates with 3, ~90% by densitometric analysis. A small but consistent fraction of uPAR (~10%) was not removed with 3 antibodies but was precipitated with 5 antibodies. After sequential 3 and 5 immunoprecipitations, antibodies to 1 integrin chains recovered little or no uPAR, indicating little uPAR associated with other 1 integrins. Reversing the order of sequential immunoprecipitations (5 then 3) verified the finding that uPAR preferentially associates with 31 in these cells.

View larger version (42K): [in this window] [in a new window]   Figure 2.   uPAR preferentially associates with 31 in 293 cells. (A) FACS analysis of 3 and 51 integrin expression on 293 and uPAR/293 cells. (B) Depletion of uPAR from 1 integrin/uPAR complexes with antibodies to integrin 3 but not 5. uPAR/293 were lysed in RIPA buffer and sequentially immunoprecipitated with antibodies to integrins 3-5-1, 5-3-1, or nonimmune IgG-IgG-1. All precipitates were subjected to immunoblot analysis with antibodies to uPAR. Both experiments were performed three times with similar results.

Consistent with prior studies, 293 cells were found to adhere avidly to Fn with the use of the classic Fn receptor 51. Antibodies to 5 but not 3 completely block adhesion (Figure 3). However, 293 cells expressing high levels of uPAR adhere poorly to fibronectin and instead adhere avidly to vitronectin, with the use of the vitronectin-binding site on uPAR. This adhesion is not blocked by EDTA or Arg-Gly-Asp (RGD) peptides (Wei et al., 1994) or enhanced by urokinase (Wei, unpublished observation). Given the finding that uPAR predominantly associates with 31 in these cells, we asked whether uPAR-dependent adhesion was blocked by antibodies to 3 (P1B5). These antibodies are reported to inhibit 31 function, although they do not block association of uPAR with 3 because P1B5 was used to coimmunoprecipitate uPAR and 3 (Figure 2). Antibodies to 3, but not 5, completely blocked uPAR-dependent adhesion to vitronectin, consistent with the finding that uPAR requires an associated integrin to mediate adhesion and that in 293 cells at least this integrin is predominantly 31.

View larger version (90K): [in this window] [in a new window]   Figure 3.   Association of integrin 3 subunit with uPAR is required for adhesion to vitronectin. Antibodies to integrin 3 (P1B5) completely inhibited uPAR-dependent adhesion to vitronectin, whereas antibodies to integrin 5 (P1D6) blocked fibronectin receptor-mediated adhesion of 293 cells to Fn. 293 and uPAR/293 cells were seeded to Fn- or Vn-coated wells with or without 2 µg/ml monoclonal antibodies to integrin 3 or 5, or nonimmune IgG. After 1-h incubation, cells were rinsed, and adherent cells were stained with Giemsa. The adhesion assay shown is representative of three independent experiments.

uPAR Binds to Immobilized, Recombinant 31

Although a loop sequence in 3 (Figure 1) is most homologous to the previously identified interaction site for uPAR in CD11b (M25), the 3 sequence is not very homologous to the original phage display peptide sequence, peptide 25, used to identify M25 in the first place (Simon et al., 2000). This raises questions as to whether the 3 sequence, termed 325, is really involved in uPAR/integrin interactions and whether the association of uPAR with 31 (Figure 2) is even direct. To address these issues, we examined binding between purified, soluble 31 and purified, suPAR under defined conditions in vitro. In this assay 31 was immobilized on plastic and the binding of biotinylated, soluble uPAR was measured. As indicated in Figure 4A, suPAR binding to 31 was dependent upon uPA. In the presence of uPA, suPAR bound to 31 in a dose-dependent, saturable manner and with high affinity (K_d < 20 nM) (Figure 4B). The uPA/suPAR binding to 31 was almost completely abrogated by 325 but not by scrambled 325 or homologous peptides from either 5 (Figure 4A) or v. To determine whether 325 itself affects uPA binding to uPAR we measured the capacity of 325 and scrambled 325 to inhibit binding of suPAR to immobilized uPA (residues 1-48, the receptor binding domain of uPA). In concentrations up to 50 µM, the highest concentration tested, neither 325 nor scrambled 325 affected binding of suPAR to uPA (Wei, unpublished observation).

View larger version (34K): [in this window] [in a new window]   Figure 4.   Soluble uPAR binds to immobilized, recombinant 31 in a uPA-dependent manner. Binding is blocked by a peptide homologous to the CD11b site. (A) Binding of biotin-suPAR to immobilized 31. 31 (2 µg/ml)-coated wells were incubated with biotin-suPAR (20 nM) with or without pro-uPA (20 nM) in addition to 100 µM peptide 325 or control peptides 525 and sc325. Bound suPAR was detected with avidin-horseradish peroxidase and absorbance measured. Mean ± SD (n = 3). (B) Saturation binding of biotinylated suPAR to immobilized 31 in the presence of pro-uPA. 31-coated wells were incubated with increasing concentration of pro-uPA bound biotin-suPAR (0-200 nM) with or without 100-fold excess unlabeled suPAR to determine specific binding. After washing, bound suPAR was quantified as described above. Mean value of triplicate determinations are given from a representative experiment (n = 3). (C) Binding of biotin-suPAR to immobilized peptide 325. Biotinylated suPAR (100 nM) preincubated without or with peptide 325 (), sc325 (), 525 (), or v25 () (1-50 µM) was added to microtiter wells coated with 325 (20 µg/ml)). Binding of suPAR in the presence of peptides is expressed as percentage of suPAR binding in the absence of peptides (% control). Values represent averages of triplicate determinations of three separate experiments. (D) Biotin-suPAR binds integrin 31 (squares) with higher affinity than integrin 51 (triangles). 31- or 51-coated wells were incubated with pro-uPA bound biotin-suPAR (0-200 nM) (closed symbols). In some experiments peptide 325 (100 µM) was added (open symbols). Bound suPAR was quantified as described above. Mean ± SD (n = 3).

We also tested whether suPAR interacts directly with the 325 peptide. The 325 peptide was immobilized on plastic and binding of suPAR to the peptide measured in the presence of increasing concentrations of 325, scrambled 325, or additional peptides as indicated in Figure 4C. suPAR binding to peptide was detectable and blocked only by 325 (IC₅₀ = ~5 µM) and not the other peptides tested. Interestingly, unlike suPAR binding to intact 31 (Figure 4A), the binding of suPAR to the 325 peptide alone was not influenced by the presence or absence of uPA.

To explore further the direct interaction of uPAR with 1 integrins, the binding of suPAR to immobilized 31 was compared with 51. suPAR/uPA binding was greater to immobilized 31 than to 51 (Figure 4D), and only binding to 31 was blocked by 325. A limitation of this analysis is that, although as judged by micro bicinchoninic acid protein assay (Pierce, Rockford, IL) equivalent integrins bound to the plastic, 51 was purified from tissues, whereas purified 31 had been expressed in soluble form. The folding on plastic of these two proteins could be different. Nonetheless, together these data indicate that uPAR directly binds 31 in a uPA-dependent manner and that this binding, like that to CD11b, involves a W4 loop peptide (325) in the -propeller region. The lack of effect of uPA on the direct interaction between uPAR and the 325 peptide suggests that this loop sequence is only a part of the overall interaction between uPAR and 31.

Consistent with these in vitro data, 325 was found to have similar functional and biochemical properties to those previously described for 25 and M25, the homologous sequence in CD11b (Simon et al., 2000). The 17-mer 3 peptide (325) blocked uPAR-dependent adhesion to Vn in uPAR-transfected 293 cells in a dose-dependent manner, with and IC₅₀ value of ~25 µM (Figure 5A) and at 100 µM blocked coprecipitation of Src family kinases with 31 integrins in these cells (Figure 5B). Neither peptides from 5 or v nor scrambled versions of 3 had any activities in these assays. Thus, the physical and functional effects of 325 appear nearly identical to that of M25 and peptide 25, confirming that the W4 repeat of 3 is probably an interaction site for uPAR paralleling that described for Mac-1.

View larger version (55K): [in this window] [in a new window]   Figure 5.   Peptides containing integrin  chain W4 loop sequences block uPAR-dependent adhesion to Vn and disrupt physical association between uPAR and 1 integrins. (A) Dose effect of  subunit peptides on uPAR-dependent adhesion to Vn. uPAR/293 cells were added to Vn-coated wells in the presence of various peptides (0-200 µM) and the adhesion was performed as described above. Peptides 325 and M25 were from W4 loop of integrin subunits 3 and M, respectively. Peptide sc 325 is a scrambled version of 325. Peptide 25 has been reported to disrupt uPAR/1 integrin association (Wei et al., 1996). (B) Effect of peptide 325 on complex formation between uPAR and integrin 31. uPAR/293 cells were lysed in RIPA buffer and incubated with anti-3 mAb (P1B5) in the presence or absence of peptide 325, 25, or sc325 (100 µM). The immunoprecipitates were blotted with polyclonal anti-uPAR (399R), stripped, and reblotted for Src family kinases (Src2) and 1 (AB1937). Data shown are representative of three independent experiments.

MDA-MB-231 Cell Spreading on Fn and Col Is Regulated by 31 and uPA/uPAR

We next asked whether uPAR/31 interactions regulate integrin function in nontransfected cells. MDA-MB-231 cells are known to express uPAR (Solberg et al., 1994) and a set of 1 integrins, including 2, 3, 5, 6, and v (Morini et al., 2000; Gui et al., 1995; Lundstrom et al., 1998; Meyer et al., 1998). These cells migrate in vitro on the expected extracellular matrix proteins and grow and metastasize in vivo (Holst-Hansen et al., 1999; Kruger et al., 2000). Because the level of expression of uPAR in these cells is much lower than that of transfected 293 cells, we explored a possible interaction between uPAR and 31 functionally rather than biochemically. The 31 on MDA-MB-231 cells was clustered with secondary antibodies and then the distribution of uPAR determined by confocal microscopy. Clustering of 21 and MHC class I molecules served as controls. Clustering of 31 but not 21 resulted in dramatic coclustering of uPAR (Figure 6). Clustered HLA class I molecules also had no effect on uPAR distribution. These data confirm that uPAR associates preferentially with 31 in MDA-MB-231 cells.

View larger version (20K): [in this window] [in a new window]   Figure 6.   Integrin 31 and uPAR cocluster. Human breast cancer MDA-MB-231 cells were stained in suspension with FITC-A3x8 antibodies to integrin 3 or FITC-A2IIE10 antibodies to integrin 2. Cells were then clustered (+) with goat anti-mouse secondary antibodies or left nonclustered (). After immobilization to polylysine-coated coverslips, cells were fixed and stained with polyclonal uPAR antibodies, and Cy3-conjugated anti-rabbit secondary antibodies were used to visualize uPAR. Substitution of FITC-conjugated control antibodies to 4 and rabbit IgG for primary antibodies resulted in much less or no immunostaining. Integrin (green), uPAR (red), and sites of colocalization (yellow).

The influence of uPAR and 31 on spreading of MDA-MB-231 cells on various extracellular matrix-coated surfaces was next tested. Although MDA-MB-231 cells attach and spread on Fn and Col with the use of 51 and 21, respectively, spreading on Fn occurs relatively slowly >2 h at 37°C. The addition of recombinant human urokinase (pro-urokinase), enhanced spreading of MDA-MB-231 cells on both Fn and Col (Figure 7A). uPA-stimulated spreading was blocked by 325 but not controls. As expected, antibodies to 5 and 2 caused cellular detachment from Fn and Col, respectively. When cells were plated on vitronectin, uPA did not enhance spreading. Remarkably, antibodies (P1B5) to 3 but not antibodies to 2 or 5 were also found to enhance the rate of spreading of MDA-MB-231 cells on Fn or Col (Figure 7A). The enhancing effect of 3 ligation with antibodies was again abrogated by the addition of 325 in a dose dependent manner. These functional effects of 325 were mirrored by biochemical effects on FAK phosphorylation (Figure 7B). The addition of uPA (Figure 7B) or 3 antibodies caused enhanced tyrosine phosphorylation of FAK as measured 30 or 15 min after addition of uPA or antibodies to MDA-MB-231 cells plated on either Fn or Col at 37°C. Again 325, but not scrambled 325, abrogated increased FAK phosphorylation on either surface, suggesting the enhancing effects of uPA or 31 ligation on Fn and Col spreading require association of uPAR and indicating that 31 regulates the spreading response to engagement of 51 and 21 by their cognate ligands. Consistent with these observations, MDA-MB-231 cells exposed to uPA in suspension, after plating on poly-lysine-coated surfaces, or after plating on vitronectin failed to increase FAK phosphorylation (Wei, observation). In MDA-MB-231 cells the major vitronectin adhesion receptor appears to be v5 rather than a 1 integrin (Meyer et al., 1998).

View larger version (28K): [in this window] [in a new window]   Figure 7.   Urokinase promotes 1 integrin function of MDA-MB-231 cells. (A) Cell spreading. MDA-MB-231 cells were seeded into Fn- or Col-coated wells and human urokinase, various antibodies, and/or peptides (50-100 µM) added as indicated in the figure. Cells were incubated for 30 min on Fn and 15 min on Col and spread cells were then counted. Data are expressed as percentage of cells spread; mean ± SD, n = 3. (B) FAK phosphorylation. MDA-MB-231 cells were incubated without or with pro-uPA (1 nM) and peptides (100 µM) on Fn for 30 min or on Col for 15 min. Cell lysates were analyzed for FAK phosphorylation by monoclonal anti-phospho-FAK antibody (top). The same membrane was stripped and blotted for total FAK (bottom). This experiment has been conducted three times with similar results. P1B5-induced FAK phosphorylation was also blocked by peptide 325.

Murine Kidney Epithelial Cell Spreading in Response to uPA Requires Presence of 3

To test further whether uPAR interactions with 31 regulate the function of other 1 integrins, immortalized kidney epithelial cells derived from 3-deficient mice were examined (Wang et al., 1999). The influence of uPA on the Fn spreading response of both 3/ cells (B12) and 3/ cells reconstituted with human 3 (R10) was tested. Of note, the baseline spreading response of the 3/ cells to Fn was at least twofold greater than that of 3-reconstituted cells, consistent with prior studies (Lichtner et al., 1998). The addition of 10 nM uPA clearly induced spreading of the 31-reconstituted cells, whereas uPA had no effect on spreading of 3/ cells. Murine uPA enhanced spreading of the 31-reconstituted cells 80-160% on Fn and 50-140% on Col within 120 min of plating (Figure 8A). Accordingly, uPA increased FAK phosphorylation >2-fold in R10 cells at 120 min and this effect was blocked by the 325 peptide but not control (Figure 8B). The addition of uPA had no effect on the FAK phosphorylation state of B12 cells, which consistently had higher baseline phosphorylated FAK. Comparable amounts of uPAR were detected in B12 and R10 cells by semiquantitative PCR and by FACs analysis with the use of murine uPA-FITC.

View larger version (25K): [in this window] [in a new window]   Figure 8.   Urokinase-induced, 1 integrin-dependent spreading and FAK phosphorylation on Fn and Col requires 3-reconstitution of 3 knockout cells. (A) Cell spreading. Epithelial kidney cells derived from integrin 3-deficient mouse (B12) and human 3-transfected R10 cells were plated on Fn (5 µg/ml) (circles) or Col (5 µg/ml) (squares) in the presence or absence of murine uPA (10 nM). Spread cells were counted at different time point within 120 min. Data are expressed as percentage of increase of cell spreading by uPA. Value represent mean ± SD (n = 3). (B) FAK phosphorylation. B12 and R10 cells were incubated with or without murine uPA (10 nM) and peptides (100 µM) on Fn and Col for 2 h. Cell lysates were analyzed for phospho-FAK and total FAK. This experiment was performed three times with similar results.

Coprecipitation of Gi and Src family Kinases with 31 Is Blocked by 325

The enhancing effect of urokinase on Fn or Col spreading, but not basal adhesion, is completely blocked by the addition of pertussis toxin (Figure 9A). Spreading induced by 31 ligation was also pertussis toxin sensitive (Wei, observation). These results suggests that a heterotrimeric G protein is required for "cross talk" between 31/uPAR complexes and 51 or 21. This is not completely unexpected because prior studies have indicated that signaling through 1 integrins is promoted by the presence of caveolin-1 (Wary et al., 1998; Wei et al., 1999). Caveolin-1 localizes to cholesterol-rich regions of cell membranes and has been demonstrated to associate with heterotrimeric G proteins such as Gi. Gi proteins, like Src family kinases, are both myristylated and palmitoylated near their N terminus, providing a driving force for localization to cholesterol-rich membrane rafts (Li et al., 1996; Harder and Simons, 1997). uPAR also localizes to membrane rafts via its glycolipid membrane anchor, the integrin/uPAR protein interaction then promoting accumulation of rafts around integrins (Wei et al., 1999). To determine whether Gi is in fact associated with 31, coprecipitation experiments were again performed. In both uPAR/293 cells and MDA-MB-231 cells (Figure 9B), antibodies to 31 precipitated not only the integrin but also both Gi and Src family kinases. Similar results were obtained whether coimmunoprecipitation was done with cells lysed in either 1% Triton or RIPA buffer. The coprecipitation of both sets of these signaling molecules was blocked by the addition of 100 µM 325 but not scrambled 325 to the cell lysates. Treating intact cells with peptide 325 before lysis gave similar results. Identical experiments in nontransfected 293 cells revealed detectable src family kinases coprecipitating with 3 antibodies but little or no Gi. In the absence of uPAR, the 325 peptide had no effect on association of src family kinases with 31 (Figure 9B). Similar expression of Gi and Src family kinases in nontransfected and uPAR transfected 293 cells was detected by Western blotting of cell lysates. Although these results cannot be viewed as quantitative, the findings indicate that the presence of uPAR alters qualitatively the complement of signaling partners associated with 31.

View larger version (33K): [in this window] [in a new window]   Figure 9.   Heterotrimeric G protein is required for cross talk between 31/uPAR and 51 or 21. (A) uPA-enhanced spreading of MDA-MB-231 cells on Fn or Col is blocked by the addition of pertussis toxin (PTX, 100 ng/ml). Cells were seeded on Fn- (5 µg/ml), Vn- (1 µg/ml), or Col (5 µg/ml)-coated wells with or without pro-uPA (1 nM) or pertussis toxin and spreading assessed as described in the text. Values represent mean ± SD, n = 3. (B) Nontransfected 293 cells, uPAR/293 cells or MDA-MB-231 cells were lysed in RIPA buffer. The lysates were incubated with 3 mAb (P1B5) or 1 mAb (JB1A) in the presence or absence of peptide 325 or sc325 (100 µM). The immunoprecipitates were blotted with polyclonal anti-Gi-3, stripped, and reblotted for Src family kinases (Src2) and 3. The experiment was performed three times with similar results. Intact cells treated with peptide 325 before lysis gave identical results.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Coupling of cellular adhesiveness with proteolytic cascades is an increasingly recognized paradigm for coordinating focal attachment and detachment important to cell migration (Werb, 1997). The uPAR is a prototypical example of this strategy (Chapman, 1997). By localizing with integrins and binding urokinase, uPAR focuses plasmin activation at or near sites of focal contact between the cell surface and extracellular matrix proteins (Blasi et al., 1987). Plasmin activates cascades involving both matrix metalloproteases and growth factors in the pericellular milieu (Carmeliet et al., 1997). Prior studies have also shown that the complexes uPAR forms with integrins are important to binding and adhesion of hematopoietic cells to matrix vitronectin, plasmin cleavage of vitronectin reversing this attachment (Wei et al., 1996; Waltz et al., 1997). Results reported here further develop this paradigm by elucidating the specificity of interaction between uPAR and 1 integrins. Our results indicate that uPAR preferentially interacts with 31 and that this interaction has two important functional consequences: 1) uPAR/31 complexes enable a pathway of cellular adhesion to Vn, especially in cells with little or no v3; and 2) these complexes initiate a signaling pathway promoting the function of 51 and 21. Both pathways of signaling and enhanced adhesion are activated by concurrent binding of urokinase to uPAR. The pathways are nonetheless distinct because pertussis toxin only blocks the cross talk between 31/uPAR and other 1 integrins and not 31/uPAR-dependent Vn adhesion. The observation that urokinase signals through uPAR/31 complex formation is consistent with a recent report that urokinase induces metalloproteinases in oral keratinocytes through an 31-dependent mechanism (Ghosh et al., 2000). Thus, the intricate connections between expression of proteases and function of the adhesive machinery of cells is epitomized by the reorganization of membrane partners induced by uPAR expression and its association with 31.

Our current findings may help clarify previously reported, apparently contradictory observations regarding the influence of uPAR on the function of the Fn receptor 51. In 293 cells, high levels of uPAR expression impair the function of Fn receptors (Wei et al., 1996). Yet our data (Figure 2) indicate that the majority uPAR in these cells is associated with 31 and not the Fn receptor. This finding suggests that the inhibition of Fn receptor function by uPAR is probably indirect. Because caveolin-1 is important to 1 integrin signaling and preferentially associates with uPAR/integrin complexes, and because 293 cells express relatively low levels of caveolin-1, overexpression of uPAR may enrich 31 complexes with caveolin and at the same time deplete Fn receptors of caveolin. This may explain why impaired Fn receptor function in uPAR-transfected 293 cells is reversed by overexpression of caveolin-1 (Wei et al., 1999). In contrast, physiological levels of uPAR expression in most cells appear to promote rather than impair Fn receptor function. Our data suggest that this operates, at least in part, through signals derived from uPAR/31 complexes. Although Fn receptors do not require such signals for adhesion, the presence of these signals accelerates FAK phosphorylation and cell spreading on Fn, and therefore may promote Fn receptor-dependent cell migration. We have previously reported that peptides that disrupt uPAR/integrin association impairs smooth muscle cell migration on Fn (Wei et al., 1999). We postulate that this may explain the recently observed requirement for uPAR expression in Fn receptor-dependent tumor invasion (Aguirre Ghiso et al., 1999). This pathway may also underlie the requirement of uPAR for v5-dependent migration of pancreatic carcinoma cells on vitronectin even though uPAR was not required for vitronectin adhesion of these cells (Yebra et al., 1996).

A series of recent studies by Blasi and colleagues have defined a pathway of urokinase- and uPAR-mediated chemotaxis (Fazioli et al., 1997; Degryse et al., 1999). Urokinase stimulates chemotaxis of uPAR-bearing cells in a pathway involving Src kinase activation and sensitive to heterotrimeric G protein inactivation with pertussis toxin. The requirements for FAK and Src kinase activation for this migration favor integrin activation as a critical feature of urokinase-dependent chemotaxis. Data reported here may shed light on these observations. We find urokinase, by promoting uPAR/31 interactions, promotes FAK activation and spreading of MDA-MB-231 cells on either fibronectin or collagen in a G protein-dependent manner (Figures 7 and 9). This signaling is blocked by peptides that dissociate uPAR and Gi-3 from 31, increasing the possibility that urokinase is chemotactic for cells because urokinase enables ligand-dependent G protein activation through an integrin. It remains to be determined how conformational changes in uPAR or 31 induced by urokinase could mediate G or G activation. Although the mechanism is not defined, our observations are conceptually similar to recent reports that the integrin-associated protein CD47 promotes association of v3 with heterotrimeric G proteins and that this is important to spreading mediated by this integrin (Frazier et al., 1999; Green et al., 1999). The finding of two distinct examples of coupling of integrins to heterotrimeric G proteins by integrin-associated proteins suggest this may be a common adaptive mechanism of cells to link matrix attachment to cell migration.

Prior studies have indicated that in addition to binding laminin-5, the integrin 31 regulates the function of other 1 integrins (DiPersio et al., 1995; Fukushima et al., 1998; Hodivala-Dilke et al., 1998). Antibodies (P1B5) to 31 that block laminin-5 attachment promote spreading and migration of cells on Col and/or Fn (Kubota et al., 1997; Lichtner et al., 1998), consistent with findings reported here (Figure 7A). Furthermore, epithelial cells from mice deficient in 3 show altered organization of integrin focal contacts and enhanced spreading on Fn, suggesting an inhibitory role for 31 on Fn and Col integrin receptors (Lichtner et al., 1998). Our finding that urokinase, mimicking P1B5, evokes 31-dependent signals promoting activation of several 1 integrins indicates uPA-dependent association of uPAR with 31 attenuates and even reverses the dominant negative function of 31 on other 1 integrins. This observation raises the possibility that prior observations of "integrin activation" by soluble uPAR may operate through its association with 31 (Aguirre Ghiso et al., 1999). In addition, our observations may also shed some light on the possible molecular basis for such cross talk. The association of 31 with uPAR appears to be required for coprecipitation of G and Src family kinases with this integrin. Such complexes may complement the binding of the same integrin to CD151, a tetraspan family member that associates specifically with 31 and that has been recently linked to signaling and migration of cells via this integrin (Yauch et al., 1998; Berditchevski and Odinstova, 1999). Antibodies to CD151 coprecipitate uPAR in uPAR-transfected 293 cells. However, peptides 325 and M25, which disrupt uPAR/integrin interactions, have no effect on 31/CD151 complexes (Wei and Hemler, unpublished observations), consistent with the mapping of the interaction site between CD151 and 31 to the membrane proximal region of the  chain and the mapping of the uPAR/integrin interaction site to the -propeller (Simon et al., 2000; Yauch et al., 2000). We postulate that multimeric complexes involving CD151, uPA/uPAR, and 31 have distinct signaling capacity promoting integrin signaling and migration on multiple matrix ligands for 1 integrins. How these complexes organize and whether other membrane adaptor proteins contribute importantly to their signaling function remains to be determined. It is important to reiterate that the discovery that uPAR associates preferentially with 31 is based on sequence homology with a previously defined integrin interaction site on CD11b/CD18 (Mac-1). The 6 amino acid sequence in the same region is also quite homologous and we show here that a 6 peptide based on this sequence also disrupts uPAR/integrin coprecipitation and uPAR-dependent adhesion, whereas peptides of the identical region of 5 (Figure 5A) or v were inactive. The possible functional significance of uPAR/61 complexes in cells expressing both of these receptors remains to be defined.

Finally, our observations that uPAR associates preferentially with integrin  chains mediating laminin-5 binding may provide an explanation for prior findings that uPAR colocalizes with the distribution of laminin-5 in vivo at sites of tumor cell invasion (Pike et al., 1995). Laminin-5 is a major basement membrane matrix protein that is breeched during the invasion of metastatic cells into or out of blood vessels. The finding that uPAR associates preferentially with the laminin-5 binding 1 integrins supports the hypothesis that invasive tumor cells have exploited the advantage of coordinate signaling of integrins, proteases, and protease receptors embodied by uPAR/integrin interactions to promote invasion and metastasis. This is also supported by studies correlating uPAR expression with metastatic capacity and poor prognosis of breast cancer patients (Solberg et al., 1994). If so, our studies identifying a critical site for interaction between uPAR and laminin-5 binding integrins may be a site for intervention in the invasive process.