3Aβ1 integrin localizes to focal contacts in response to ... · INTRODUCTION The integrin family...

2321Journal of Cell Science 108, 2321-2336 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

α3Aβ1 integrin localizes to focal contacts in response to diverse extracellular

matrix proteins

C. Michael DiPersio, Sejal Shah and Richard O. Hynes*

Howard Hughes Medical Institute, Center for Cancer Research, and Department of Biology, Massachusetts Institute ofTechnology, Cambridge, MA 02139, USA

*Author for correspondence

In vitro binding assays and inhibition of cell adhesion withmonoclonal antibodies have implicated the integrin α3β1as a receptor for a variety of extracellular ligands.However, reports of α3β1-ligand interactions are inconsis-tent, and transfection studies have suggested that α3β1 isnot sufficient for cell attachment to ligands other thankalinin/laminin 5. We used immunofluorescence to studysubcellular localization of the α3A cytoplasmic domainvariant in different cultured cell types. Using standardfixation and permeabilization methods, antibodies specificfor α3A stained most cell types in a diffuse pattern, con-sistent with previous reports. Surprisingly, however,chemical cross-linking of integrins to the extracellularmatrix and extraction of the cytoskeleton prior to immuno-fluorescence revealed α3A in focal contacts of most cellstested, suggesting that the cytoplasmic domain wasconcealed in intact focal contacts by cytoskeletal or othercytoplasmic proteins. The α3A subunit localized to focalcontacts in several cell types cultured on fibronectin,

kalinin/laminin 5, EHS-laminin/laminin 1, type IV col-lagen, or vitronectin. In contrast, α5 and αV integrins weredetected in focal contacts only in cells grown on theirknown ligands (fibronectin, and fibronectin or vitronectin,respectively). Therefore, our results show that α3Aβ1responds to a broad spectrum of extracellular ligands.Time course comparisons of the recruitment of α subunitsfrom different fibronectin receptors suggested that local-ization of α3Aβ1 to fibronectin-induced focal contacts wasindependent of the recruitment of α5 and α4 integrins.However, other studies have shown that α3Aβ1 does notmediate initial cell adhesion to many of the ligands thatinduced its focal contact localization, including fibronectin.Therefore, we suggest that α3Aβ1 may be a secondaryreceptor with post-cell-adhesion functions for a broadspectrum of extracellular matrices.

Key words: integrin α3β1, cell adhesion, focal contact, fibronectin

SUMMARY

INTRODUCTION

The integrin family of cell adhesion receptors includes morethan twenty distinct heterodimers, each consisting of an α anda β subunit (for a review, see Hynes, 1992). Each subunit hasa large, globular extracellular domain, a single transmembranedomain, and a short cytoplasmic domain that is usually lessthan 50 amino acids long. The β1 subunit can dimerize with αsubunits 1 through 9 and αV. The ligand specificities of β1integrins are determined by the particular α subunit with whichthe β1 subunit is paired, since each of the resulting integrinsrecognizes a distinct, but often overlapping set of extracellularligands. For example, α5β1 binds to fibronectin (Pytela et al.,1985), while α4β1 binds to both fibronectin (Wayner et al.,1989; Mould et al., 1990; Guan and Hynes, 1990) and vascularcell adhesion molecule 1 (VCAM-1) (Elices et al., 1990; Chanet al., 1992a). Cell type-specific factors also appear to play arole in determining ligand specificities of integrins. Forexample, α2β1 is a receptor for collagen and not laminin onplatelets (Staatz et al., 1989), but is a receptor for both ligandson other cell types (Elices and Hemler, 1989; Kirchhofer et al.,

1990). An additional level of integrin diversity is generated bythe alternative splicing of certain β and α subunits to produceisoforms that differ in their cytoplasmic or extracellulardomains (see Hynes, 1992, and references therein).

Many adhesion structures involved in cell-cell and cell-sub-stratum attachment are dependent on integrins. For example,focal contacts are formed by the accumulation of integrins atpoints of cell adhesion to the extracellular matrix, where theintegrins bind through their extracellular domains to the matrixand through their cytoplasmic domains to components of theactin cytoskeleton (Burridge et al., 1988); however, not allintegrins form focal contacts (Wayner et al., 1991; Hynes,1992). The β1 cytoplasmic domain can bind to certaincytoskeletal proteins in vitro, including talin (Horwitz et al.,1986) and α-actinin (Otey et al., 1990). Furthermore, certainmolecules that appear important in integrin-mediated signaltransduction localize to focal contacts (Juliano and Haskill,1993), including focal adhesion kinase, or FAK (Guan et al.,1991; Schaller et al., 1992; Zachary and Rozengurt, 1992).

The integrin α3β1 is present in most adherent cell cultures(Fradet et al., 1984; Rettig et al., 1986; Hemler et al., 1987;

2322 C. M. DiPersio, S. Shah and R. O. Hynes

Wayner et al., 1988). The transcript for the α3 subunit is alter-natively spliced to encode one of two isoforms, α3A and α3B,which differ in their cytoplasmic domains, sharing homologyonly within the membrane proximal GFFKR motif that isconserved in all α subunits (Tamura et al., 1991; C. M.DiPersio et al., unpublished). Published studies in which theα3 isoform was identified have involved α3A (Takada et al.,1991), since they employed antibodies that are specific for theα3A cytoplasmic domain (Hynes et al., 1989; Takada et al.,1991; Enomoto et al., 1993; Delwel et al., 1994). Other studiesutilized antibodies raised against either the intact α3 subunitor the α3 extracellular domain that cannot distinguish betweenthe cytoplasmic domain variants (Fradet et al., 1984; Waynerand Carter, 1987; Carter et al., 1990a; Takada et al., 1991).Detection of α3Bβ1 with α3B-specific antibodies has not yetbeen reported. In this report, we will specify an α3 isoformonly when it has been determined using appropriate antibodiesor cDNAs.

Immunofluorescence experiments suggest that α3β1localizes to adhesion structures in only a few cell types. Forexample, in cultured keratinocytes, α3β1 can localize to bothcell-cell contacts and focal contacts (Carter et al., 1990b;Enomoto et al., 1993). α3β1 was also detected in focal contactsof kidney mesangial cells (Grenz et al., 1993). In markedcontrast, standard immunofluorescence of fibroblasts and othercells suggests that α3β1 is not present in focal contacts, butinstead is distributed in a diffuse pattern over the cell surface(Carter et al., 1991; Elices et al., 1991; Enomoto et al., 1993).Therefore, α3β1 has been considered to be an integrin that doesnot generally localize to focal contacts.

Although α3Aβ1 is clearly a strong receptor forkalinin/laminin 5/epiligrin (Rousselle et al., 1991; Carter et al.,1991; Wayner et al., 1993) and a weak receptor for a novellaminin isoform(s) from bovine kidney (Delwel et al., 1994),the full repertoire of extracellular ligands for α3β1 is contro-versial. Indeed, biochemical assays or inhibition of celladhesion with anti-α3 monoclonal antibodies suggest a role forα3β1 in some cells as a receptor for fibronectin (Wayner andCarter, 1987; Wayner et al., 1988; Staquet et al., 1990; Eliceset al., 1991), collagens (Wayner and Carter, 1987; Elices et al.,1991), EHS laminin/laminin 1 (Wayner and Carter, 1987;Gehlsen et al., 1988; Tomaselli et al., 1990; Elices et al., 1991;Adams and Watt, 1991), and entactin/nidogen (Dedhar et al.,1992). In addition, α3β1 has been implicated in both het-erophilic interactions with α2β1 (Symington et al., 1993) andhomophilic interactions in cell-cell adhesion (Sriramarao et al.,1993). Interestingly, some of these reported interactions are notreproducible in different in vitro or cell culture systems. Forexample, several groups have shown that α3β1 in cell extractscan bind to fibronectin affinity columns under certain con-ditions, such as low salt (Wayner and Carter, 1987; Elices etal., 1991; Clyman et al., 1992). However, in other studies,α3β1 did not bind to fibronectin affinity columns under con-ditions where α5β1 or other fibronectin receptors did bind,such as physiological salt conditions (Plantefaber and Hynes,1989; Hynes et al., 1989). Furthermore, adhesion assays withtransfected cells failed to support a role for α3β1 as afibronectin receptor (Weitzman et al., 1993; Delwel et al.,1994). Finally, monoclonal antibodies to α3 can inhibitadhesion to fibronectin in some cell types (Wayner and Carter,1987; Staquet et al., 1990; Elices et al., 1991) but not in most

cells (Adams and Watt, 1991; Elices et al., 1991). Conflictingresults have also been reported for α3β1 binding to collagensor EHS-laminin/laminin 1 in vitro (Wayner and Carter, 1987;Gehlsen et al., 1988, 1989; Elices et al., 1991; Clyman et al.,1992) or in cell adhesion and antibody inhibition assays(Wayner and Carter, 1987; Staquet et al., 1990; Elices et al.,1991; Adams and Watt, 1991; Wayner et al., 1993; Weitzmanet al., 1993; Delwel et al., 1994). Taken together, theseseemingly incompatible results suggest that binding of α3β1to a particular ligand may be dependent on cell type or exper-imental conditions, or influenced by other integrin receptors forthe ligand (Elices et al., 1991; Tomaselli et al., 1990), and thephysiological significance of these interactions has beenunclear.

To understand better the functions of α3Aβ1 in living cells,we have conducted a careful study of α3A subunit localizationin several cultured cell types. We used a previously describedimmunofluorescence technique (Enomoto-Iwamoto et al.,1993) to show that, in contrast with earlier reports, α3Aβ1 canbe readily detected in focal contacts in a number of cell types.Staining of focal contacts with antibodies to the α3A cyto-plasmic domain required chemical cross-linking of the integrinto the extracellular matrix followed by extraction of thecytoskeleton, suggesting that the α3A cytoplasmic domaininteracts with components of the cytoskeleton. We have usedthis method to identify specific extracellular matrix proteinsthat promote α3Aβ1 localization to focal contacts.

MATERIALS AND METHODS

Cell lines and reagentsChicken embryo fibroblasts (CEFs) were prepared as described pre-viously (Hynes et al., 1989), and grown in Dulbecco’s modifiedEagle’s medium (DME; GibcoBRL, Grand Island, NY) plus 5% fetalbovine serum (FBS; HyClone, Logan, UT). Human foreskin fibrob-lasts (HFFs) were purchased from the American Type Culture Col-lection (Rockville, MD). HFFs, MG63 human osteosarcoma cells, andMadin-Darby canine kidney (MDCK) cells were grown in DME plus10% FBS. Chinese hamster ovary (CHO) cells were grown in MEMα(GibcoBRL) plus 10% FBS. COS monkey kidney cells and normalrat kidney (NRK) cells were grown in DME plus 5% FBS.

Extracellular matrix proteinsHuman kalinin/laminin 5 was kindly provided by R. Burgeson (Mass-achusetts General Hospital, Charlestown, MA). Mouse EHSlaminin/laminin 1, human type IV collagen and human vitronectinwere purchased from Collaborative Research, Inc. (Bedford, MA).Human plasma fibronectin was purchased from the New York BloodCenter (New York, NY). Poly-L-lysine was purchased from SigmaChemical Co. (St Louis, MO). Glass coverslips were coated overnightat 4°C with extracellular matrix proteins dissolved in phosphatebuffered saline (PBS) at a concentration of 20 µg/ml in all casesexcept for the following: Fig. 6, vitronectin at 25 µg/ml;kalinin/laminin 5 at 90 µg/ml; EHS laminin/laminin 1, type IVcollagen and poly-L-lysine at 40 µg/ml; and Fig. 7, fibronectin at 40µg/ml. Protein-coated coverslips were blocked with 2 mg/ml heat-denatured bovine serum albumin (BSA) in PBS.

AntibodiesAnti-vinculin monoclonal antibody was purchased from SigmaChemical Co. (St Louis, MO). The monoclonal antibody P1B5,against human α3, was purchased from GibcoBRL (Grand Island,

2323Promiscuous focal contact targetting of α3β1

NY). Rabbit polyclonal antiserum against the cytoplasmic domain ofhuman αV was purchased from Chemicon International, Inc.(Temecula, CA), and antisera against the cytoplasmic domains ofhuman α2 and α4 were kindly provided by M. Hemler (Dana-FarberCancer Institute, Boston, MA). Rabbit antisera against the cytoplas-mic domains of human β1 (antiserum 210), human α5 (antiserum160), and chicken α3A (antisera 8-4 and 207) were prepared asdescribed (Marcantonio and Hynes, 1988; Hynes et al., 1989). Thefollowing synthetic Cys-oligopeptides from the chicken α3A cyto-plasmic domain were used as antigen: 8-4, CYEAKGQKAEM-RIQPSETERLTDDY; 207, CSTRAMYEAKGQKAEMRIQPSET-ERLTDDY. In general, the anti-cytoplasmic domain sera cross-reactwith integrin subunits from different species. Rabbit polyclonalantiserum against the chicken α3 extracellular domain, a gift from L.Reichardt (UCSF School of Medicine, San Francisco, CA), was raisedagainst a fusion protein containing the amino-terminal portion of α3encoded by chicken α3 cDNA (DiPersio et al., in preparation). In ourhands, this antiserum did not cross-react with mammalian α3.

Immunofluorescence staining by standard methodsCells were removed from stock plates with PBS/0.5 mM EDTA,washed with PBS, and plated at subconfluent densities onto matrix-coated coverslips. Cells were cultured on coverslips for 2 hours beforeimmunofluorescence. Cells were rinsed with PBS plus 1 µM each ofcalcium chloride and magnesium chloride (PBS/Ca2+, Mg2+), fixed in3% paraformaldehyde for 10 minutes, permeabilized with 0.5% NP40in PBS/Ca2+, Mg2+ for 10 minutes, then blocked with 10% normalgoat serum (NGS) for at least 30 minutes. Coverslips were stained at37°C for at least 30 minutes with rabbit polyclonal antisera specificfor α or β1 cytoplasmic domains used at 1:100 dilution in 10% NGS,or with mouse monoclonal antibodies used at dilutions of 1:100 to1:500. Fluorescein-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti-mouse IgG (BioSource International, Inc.,Camarillo, CA) were used as secondary antibodies at dilutions of1:500 and 1:1000, respectively. Coverslips were washed extensivelywith PBS and mounted on microscope slides. Representative fieldswere photographed at a magnification of 63× through an oilimmersion lens on a Zeiss Axiophot microscope using Kodak T-MAXP3200 film. Control experiments showed that neither bleed-throughof fluorescein to rhodamine, nor bleed-through in the oppositedirection, occurred using our staining conditions.

In most experiments, synthesis of endogenous matrix proteins wasprevented by pretreatment of cells for 2 to 3 hours with 30-300 µg/mlcycloheximide (Sigma Chemical Co., St Louis, MO) in serum-freemedium, then plating onto matrix-coated coverslips in the samemedium, as indicated in figure legends. Cells were then washed withserum-free medium plus cycloheximide and plated onto matrix-coatedcoverslips in the same medium for an additional 2 hours. Metaboliclabelling of cells with [35S]methionine and assay of secreted proteinsby polyacrylamide gel electrophoresis showed that this treatmentprevented endogenous protein production (data not shown). Further-more, focal contact formation in adherent cells under these conditionswas dependent on the presence of exogenously provided matrixproteins and did not occur on poly-L-lysine (see Fig. 6).

Immunofluorescence staining of integrins cross-linked toextracellular matrix proteinsIntegrins were cross-linked to extracellular matrix proteins and visu-alized by immunofluorescence following cell extraction as describedby Enomoto-Iwamoto et al. (1993). Briefly, cells were washed 3 timeswith PBS and incubated in 0.4 mM BS3 (bis (sulfosuccinimidyl)suberate; Pierce, Rockford, IL) in PBS at room temperature for 10minutes; the cross-linking reaction was stopped by addition of PBSplus 10 mM Tris-HCl (pH 7.5) at room temperature for 2 minutes.Cross-linked cultures were washed 4 times with PBS/Ca2+, Mg2+,extracted with 0.5% NP40 in PBS/Ca2+, Mg2+ plus 2 mM PMSF for10 minutes at room temperature, washed several times with PBS/Ca2+,

Mg2+, then fixed with 3% paraformaldehyde for 10 minutes at roomtemperature. Cells were then blocked with 10% NGS and stained byimmunofluorescence as described above.

In control experiments (Fig. 2), BS3 was omitted from the cross-linking protocol (Fig. 2E,F), or cells were cross-linked with 0.4 mMBS3 prior to standard fixation, permeabilization and immunofluores-cence (Fig. 2G,H).

Time course comparisons of α subunit focal contactlocalizationCells were pretreated with 100 µg/ml cycloheximide and plated ontofibronectin-coated coverslips (20 µg/ml) in the continuous presenceof cycloheximide, as described above. Parallel cultures were thenprocessed by the cross-linking protocol at 15, 30, 60, 90, and 120minutes after plating. Coverslips were stained by double-labelimmunofluorescence with the anti-α3 monoclonal antibody P1B5 andantisera against the cytoplasmic domains of β1 (antiserum 210), α3A(antiserum 8-4), or other α subunits (see Fig. 5), as described above.

RESULTS

Detection of the α3A integrin subunit in focalcontacts of chicken embryo fibroblastsWe used a standard immunofluorescence protocol, in whichcells were first fixed with 3% paraformaldehyde then perme-abilized with 0.5% NP40, to determine the intracellular local-ization of the α3A subunit in chicken embryo fibroblasts(CEFs). A polyclonal antiserum directed against the α3A cyto-plasmic domain variant failed to stain focal contacts in CEFsplated on fibronectin-coated coverslips; instead, α3A appearedto be distributed diffusely (Fig. 1A). This non-localized,diffuse distribution of α3A is typical of that reported for α3by others (Carter et al., 1991; Elices et al., 1991), and is con-sistent with the notion that α3β1 does not localize to focalcontacts in most cells.

We also performed immunofluorescence with a polyclonalantiserum raised against the extracellular domain of chickenα3. Surprisingly, in contrast with the anti-α3A cytoplasmicdomain serum, this antiserum appeared to stain focal contactsin CEFs plated on fibronectin (Fig. 1B). Focal contact stainingwas not due to reactivity of the antiserum with the α3B cyto-plasmic domain variant, since CEFs lack detectable α3B asassayed by immunofluorescence or immunoprecipitation withα3B-specific antisera (C. M. DiPersio et al., unpublished). Analternative explanation for the distinct α3 staining patternsobtained with the anti-extracellular and anti-cytoplasmicdomain antisera is that epitopes recognized by the latter areblocked when α3A is present in a focal contact.

The α3A cytoplasmic domain is masked infibronectin-induced focal contactsTo determine whether the α3A cytoplasmic domain wasmasked in focal contacts, we employed a previously describedtechnique (Enomoto-Iwamoto et al., 1993) in which integrinsare first cross-linked to the extracellular matrix with thechemical cross-linker BS3 (bis (sulfosuccinimidyl) suberate),to which cells are impermeable, and the cytoskeleton is thendetergent-extracted prior to immunofluorescence (seeMaterials and Methods). Using this technique, Enomoto-Iwamoto et al. (1993) demonstrated that the cytoplasmicdomain of α5 could be revealed in focal contacts of CEFs


Fig. 1. α3Aβ1 is localized in focalcontacts. CEFs were cultured onfibronectin for 2 hours in 10% serumand then processed forimmunofluorescence as follows: (Aand B) ‘STANDARD’, cells werefixed with 3% paraformaldehyde thenpermeabilized with 0.5% NP40; (C-F)‘BS3 CROSS-LINKED’, cells werecross-linked to the extracellular matrixwith BS3, extracted with 0.5% NP40,then fixed with 3% paraformaldehyde.Cells were then stained with antiserum207 to the cytoplasmic domain of α3A(A and E) or 207 preimmune serum(F), an antiserum to the extracellulardomain of chicken α3 (B), orantiserum 210 to the cytoplasmicdomain of β1 (C) or 210 preimmuneserum (D), followed by fluorescein-conjugated goat anti-rabbit IgG.

under culture conditions where it was undetectable usingstandard immunofluorescence protocols. When CEFs onfibronectin were cross-linked with BS3 then extracted with0.5% NP40, β1 was readily detected in focal contacts by an

antiserum to the β1 cytoplasmic domain (Fig. 1C), but not bythe preimmune serum (Fig. 1D), demonstrating that β1integrins had been cross-linked to the extracellular matrix.Similarly, antiserum to the α3A cytoplasmic domain was now


Fig. 2. α3Aβ1 is revealed in fibronectin-induced focal contacts following cytoplasmic extraction. HFFs were pretreated with 30 µg/ml (A-F) or100 µg/ml (G and H) cycloheximide in serum-free medium for two hours before plating onto fibronectin in the continuous presence ofcycloheximide for an additional two hours. Cells were then processed for immunofluorescence as follows: (A-D) ‘BS3 CROSS-LINKED’, asdescribed in Fig. 1; (E and F) ‘BS3 OMITTED’, cells were treated as in (A-D), except that BS3 was omitted; (G and H) ‘STANDARD + BS3’,cells were cross-linked with BS3, then immediately fixed with 3% paraformaldehyde and permeabilized with 0.5% NP40. Cells were thenstained with polyclonal antisera to the cytoplasmic domains of β1 (antiserum 210; B, E and G) or α3A (antiserum 8-4; D, F and H), or withtheir respective preimmune sera (A and C), followed by fluorescein-conjugated goat anti-rabbit IgG.

able to detect α3A in focal contacts of the same cells (Fig. 1E),while no staining was seen with the corresponding preimmuneserum (Fig. 1F). These results confirm that α3A is present inCEF focal contacts, in marked contrast with earlier reports.

We wanted to determine whether focal contacts containingα3Aβ1 were formed in response to the exogenously providedfibronectin or to cell-secreted matrix proteins or serum com-ponents. Since a variety of antibodies are available fordetection of human integrins, but not for chicken integrins, weperformed subsequent experiments in human cells. Humanforeskin fibroblasts (HFF) were preincubated for two hours inserum-free medium containing 30 µg/ml cycloheximide toinhibit endogenous matrix production, and then plated ontofibronectin-coated coverslips in the same medium for twohours before cross-linking with BS3, extraction with 0.5%NP40 and immunofluorescence. Exogenous fibronectin wasrequired for adhesion, since cells did not attach to uncoated,BSA-blocked coverslips. Focal contacts were clearly detectedwith an antiserum to the β1 cytoplasmic domain (Fig. 2B), butnot with the preimmune serum (Fig. 2A), showing that β1-con-taining focal contacts did not require cell-secreted proteins orserum. Similarly, focal contacts were detected with theantiserum to the α3A cytoplasmic domain (Fig. 2D), but notwith the preimmune serum (Fig. 2C). These results demon-

strate that α3Aβ1 can localize to fibronectin-induced focalcontacts.

Control experiments, in which BS3 was omitted from thecross-linking protocol, showed that neither β1 nor α3A wasdetected in HFF focal contacts following NP40 extraction (Fig.2E and F, respectively). Therefore, using this protocol, cross-linking of α3A to the matrix with BS3 was required for itsretention in focal contacts during extraction, as described forother β1 integrins (Enomoto-Iwamoto et al., 1993). WhenHFFs were cross-linked with BS3 and then immediately fixedwith 3% paraformaldehyde and permeabilized with NP40, β1was detected in focal contacts (Fig. 2G) but α3A was notdetected (Fig. 2H). Therefore, exposure of the α3A cytoplas-mic domain to antibodies is not simply a consequence of cross-linking the integrin extracellular domain. Instead, it seemslikely that when the α3A cytoplasmic domain is in a focalcontact it is masked from antibodies by cytoskeletal or othercytoplasmic components, as has been suggested for the α5subunit (Enomoto-Iwamoto et al., 1993). Consistent with thisnotion, double-label immunofluorescence showed that at leastone cytoskeletal protein, vinculin, was present in β1-contain-ing focal contacts using the standard protocol (compare Fig.3A,B) but was not detected using the cross-linking protocol(compare Fig. 3C,D). In contrast with our results, Enomoto et


Fig. 3. β1 integrins, but not thecytoskeletal protein vinculin, arecross-linked in HFF focal contacts.HFFs were cultured on fibronectinin 30 µg/ml cycloheximide asdescribed in Fig. 2, then processedby the ‘standard’ (A and B) or‘BS3 cross-linking’ (C and D)methods, as described in Fig. 1.Cells were then assayed bydouble-label immunofluorescencewith rabbit polyclonal antiserum210 to the cytoplasmic domain ofβ1 (A and C) and a mousemonoclonal antibody to vinculin(B and D), followed byfluorescein-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti-mouse IgG.

al. (1993) showed that in chicken muscle cells cross-linked tofibronectin, extraction with 0.5% NP40 was not sufficient toremove vinculin or expose the α5 cytoplasmic domain, butextraction with 0.5% Chaps was sufficient. The reason for thisdiscrepancy is not clear, but it may reflect cell-specific differ-ences in integrin-cytoskeleton interactions that affect resis-tance to detergent-extraction. Indeed, the cross-linking methodrevealed changes in interactions of α5β1 with the cytoskel-eton during myogenesis in the same study (Enomoto et al.,1993).

Detection of focal contacts in HFFs with the monoclonalantibody P1B5 (see Fig. 7), which binds to the human α3 extra-cellular domain and inhibits adhesion of some cells (Waynerand Carter, 1987), was also dependent on cross-linking and cellextraction prior to immunofluorescence (not shown). Thisfinding is in contrast with the efficient detection of focalcontacts in CEFs using the antiserum to the chicken α3 extra-cellular domain (Fig. 1B). It is likely that the epitope for P1B5is obscured when α3 is bound to matrix in focal contacts(Carter et al., 1990a) and is somehow exposed during the cross-linking protocol, while some epitopes for the polyclonal α3antiserum are always exposed. It is also possible that diffusestaining of excess α3 on the cell surface generally contributesto the difficulty of detecting α3 in focal contacts using standardimmunofluorescence.

α3Aβ1 localizes to fibronectin-induced focalcontacts in different cell typesWe wanted to determine whether α3Aβ1 can also localize tofibronectin-induced focal contacts in other cell types. The anti-α3A serum did not stain focal contacts in the kidney-derivedCOS and Madin-Darby canine kidney (MDCK) cell linescultured on fibronectin using the standard protocol (Fig. 4A

and C, respectively), although cell-cell contacts were stainedin MDCK cells. However, α3A was detected in fibronectin-induced focal contacts in both cell types using the BS3 cross-linking protocol (Fig. 4B and D). α3A was also present infibronectin-induced focal contacts of Chinese Hamster Ovary(CHO) cells (Fig. 4E), normal rat kidney (NRK) cells (Fig. 4F),and the osteosarcoma cell line MG63 (see Fig. 7), demon-strating that focal contact localization is a general function ofα3Aβ1. Interestingly, however, neither NIH 3T3 cells norHeLa cells displayed α3A in focal contacts following cross-linking to fibronectin, despite the fact that both of these celllines express α3A (data not shown), and α3A was not revealedin focal contacts of muscle cells cross-linked to fibronectin(Enomoto et al., 1993). Therefore, not all cells recruit α3Aβ1into focal contacts on fibronectin, and it is not yet clear whetherthese differences reflect cell-specific differences in α3Aβ1itself or differences in focal contact composition.

α3Aβ1 is detected in fibronectin-induced focalcontacts before α5β1Since α3Aβ1 is not sufficient to mediate cell attachment tofibronectin (Weitzman et al., 1993; Delwel et al., 1994; ourunpublished results), it seemed possible that recruitment ofα3Aβ1 into fibronectin-induced focal contacts might bedependent on other fibronectin receptors. To begin to addressthis question, the incorporation of α3A into fibronectin-induced focal contacts was compared with that of the αsubunits for several other fibronectin receptors, namely α5, α4and αV (Fig. 5). HFFs that had been pretreated with cyclo-heximide were plated onto coverslips coated with fibronectinin the continuous presence of cycloheximide for up to twohours, cross-linked with BS3, then processed for double-labelimmunofluorescence with the anti-α3 monoclonal antibody


Fig. 4. α3Aβ1 localizes to fibronectin-induced focal contacts in different celltypes. COS cells (A and B), MDCKcells (C and D), CHO cells (E), andnormal rat kidney (NRK) cells (F)were cultured on fibronectin in thepresence of cycloheximide asdescribed in Fig. 2, then processed bythe ‘standard’ (A and C) or ‘BS3 cross-linking’ (B,D,E and F) methods, asdescribed in Fig. 1. Cells were thenstained with antiserum 8-4 to thecytoplasmic domain of α3A, followedby fluorescein-conjugated goat anti-rabbit IgG.

P1B5 and polyclonal antisera against the cytoplasmic domainsof different integrin subunits. In general, relatively few cellsadhered and formed strong focal contacts at earlier time points(15 to 30 minutes). Therefore, we focused on cells that wereable to attach rapidly and form focal contacts in order tocompare recruitment of distinct integrins into focal contactswithin an adherent cell.

In general, staining of focal contacts by P1B5 was weakercompared with that of the different polyclonal antisera andrequired longer photographic exposures for visualization (Fig.5, rows labelled ‘α3’). Nevertheless, as expected, P1B5 andthe anti-α3A serum stained the same fibronectin-induced focalcontacts at all time points (not shown). Similarly, P1B5 stainedthe same focal contacts as did the anti-β1 cytoplasmic domainserum as early as 15 minutes after plating (not shown), andshowed the same focal contact staining patterns for up to two

hours (Fig. 5, compare β1 and α3). Therefore, using this assay,the α3A and β1 subunits are incorporated into fibronectin-induced focal contacts at similar rates. In contrast, α5 was notdetected in α3A-containing focal contacts after 30 minutes andwas only barely detectable in some cells after 60 minutes (Fig.5, compare α5 and α3). The initial appearance of α5 in HFFswas slightly later in a separate experiment, in which α3A wasalso detected by 15 minutes, and similar results were obtainedin MG63 cells (data not shown). These results suggest that therecruitment of significant amounts of α3Aβ1 was independentof the recruitment of detectable amounts of α5β1. α4 was notdetected in HFF focal contacts at any time point (for example,the 60 minute time point for α4 vs α3 is shown in Fig. 5), solocalization of α3Aβ1 to focal contacts must also be indepen-dent of detectable amounts of the fibronectin receptors α4β1(Wayner et al., 1989; Mould et al., 1990; Guan and Hynes,


Fig. 5. Time course comparisons of integrin recruitment intofibronectin-induced focal contacts. HFFs were pretreated with 100µg/ml cycloheximide as described in Fig. 2, then plated ontofibronectin in the presence of cycloheximide. Parallel cultures wereprocessed by the BS3 cross-linking method, as described in Fig. 1, at15 (not shown), 30, 60, 90 (not shown), or 120 minutes after plating,as indicated at the top of each column. Cells were then assayed bydouble-label immunofluorescence with the anti-α3 monoclonalantibody P1B5 (rows labelled ‘α3’), and a polyclonal antiserum tothe cytoplasmic domain of either β1, α5, αV, or α4, as indicated tothe left of each row, followed by rhodamine-conjugated goat anti-mouse IgG and fluorescein-conjugated goat anti-rabbit IgG.Corresponding P1B5 staining is shown directly below staining foreach polyclonal antiserum; only the 30 and 60 minute time points areshown for αV; only the 60 minute time point is shown for α4.

1990) and α4β7 (Rüegg et al., 1992). In contrast to α5 and α4,the time course of αV detection in focal contacts was similarto that of α3A (Fig. 5, compare αV and α3). αVβ1, αVβ3 andαVβ6 have each been reported to bind fibronectin (Charo etal., 1990; Vogel et al., 1990; Busk et al., 1992). Therefore,although we have not identified the αV-containing integrins inthe HFF focal contacts, we cannot rule out the possibility thatlocalization of α3Aβ1 to focal contacts is dependent on an αVintegrin. It also remains possible that undetectable amounts ofother integrins are required for initiation of cytoskeletal inter-actions at sites of focal contact formation before recruitmentof α3Aβ1 (see model in Fig. 8B). Importantly, these experi-ments also do not rule out a requirement for other integrins ininitial cell attachment to fibronectin, an obvious prerequisitefor focal contact formation.

α3Aβ1 localizes to focal contacts on diverseextracellular matrix proteinsSince α3β1 has been implicated in other studies as a receptorfor a variety of ligands, we tested α3A for localization to focalcontacts induced by other extracellular matrix proteins. WhenHFFs were cultured on EHS laminin/laminin 1 and assayed bystandard immunofluorescence, β1 was detected in focalcontacts (Fig. 6A), but α3A was not detected (Fig. 6B).However, the cross-linking protocol revealed α3A in laminin1-induced focal contacts (Fig. 6C). Similarly, α3A wasdetected in vitronectin-induced focal contacts only after usingthe cross-linking protocol (Fig. 6D-F). α3A was also recruitedto focal contacts induced by kalinin/laminin 5 (Fig. 6G) or typeIV collagen (Fig. 6H). Similar results were obtained withMG63 cells on each of these matrix proteins (Fig. 7, and notshown), with COS cells on EHS laminin/laminin 1, type IVcollagen, or vitronectin, and with CHO cells on vitronectin (notshown). Our results were confirmed in most cases by focalcontact staining with P1B5 and/or with two distinct anti-α3Aantisera, but not with preimmune sera (not shown). As anegative control, anti-α3 antibodies did not stain β1-contain-ing focal contacts in HepG2 hepatoma cells following cross-linking to EHS laminin or type IV collagen, consistent with theabsence of α3A from the surface of these cells (not shown). Ingeneral, α3A-containing focal contacts were somewhat lessabundant and less prominent in cells on EHS laminin/laminin1 and type IV collagen than in cells on fibronectin. Also, bothcell adhesion to and focal contact formation on purifiedkalinin/laminin 5 were relatively weak, possibly due to theabsence of other matrix components present in basement

membranes. However, all cell types adhered poorly to and didnot spread on poly-L-lysine, and both β1 and α3A focalcontacts were completely absent (Fig. 6I for HFFs), demon-strating that attachment per se was not sufficient, and matrixproteins were required, for focal contact formation. Therefore,our studies show that α3Aβ1 responds to a broad spectrum ofextracellular matrix proteins by localizing to focal contacts.α3Aβ1 localization to vitronectin-induced focal contacts wasunexpected since, to our knowledge, there have been no reportsof α3β1 binding to vitronectin, and in at least one study α3β1in extracts from muscle cells did not bind to a vitronectinaffinity column (Clyman et al., 1992).

Comparison of α3A focal contact localization withthat of other integrin α subunitsWe wanted to determine whether the broad response to matrixproteins as detected by the BS3 cross-linking method is uniqueto the α3Α subunit. We cultured MG63 cells in cycloheximideon different extracellular matrix proteins for several hours,treated them with the cross-linking protocol, then used double-label immunofluorescence to determine which α subunits weredetected in α3A-containing focal contacts on each matrix. Asexpected, P1B5 and the antiserum to the α3A cytoplasmicdomain co-stained focal contacts on both fibronectin and vit-ronectin (Fig. 7A and B, respectively, columns labelled α3A).In contrast, the antiserum to the α5 cytoplasmic domaindetected α5 in α3A-containing focal contacts on fibronectin(Fig. 7A, α5 column), but not on vitronectin (Fig. 7B, α5column), consistent with the function of α5β1 as a receptor forfibronectin but not for vitronectin (Hynes, 1992, and referencestherein). A polyclonal antiserum to the αV cytoplasmic domaindetected this subunit in α3A-containing focal contacts on bothfibronectin and vitronectin (Fig. 7A and B, respectively, αVcolumns), consistent with known ligand-binding specificitiesof αV integrins (Hynes, 1992, and references therein). Finally,antisera specific for the α cytoplasmic domains of α2 (Fig. 7Aand B, α2 columns), α1, α6A and α6B (not shown) each failedto stain α3A-containing focal contacts on either fibronectin orvitronectin, showing that staining of focal contacts by anti-α3antibodies does not reflect a general staining artifact associatedwith the cross-linking protocol. Similar results were obtainedwith COS cells cultured on fibronectin or vitronectin (data notshown). Therefore, focal contact localization of other αsubunits was consistent with their known ligand specificities.Although α3β1 binds to several of these matrix proteins insome in vitro systems, direct binding of α3Aβ1 to the matrixproteins tested has not been shown for these cells. Therefore,our results reflect either an unusually broad ligand-bindingcapacity for α3Aβ1 or its ability to join focal contacts formedby other integrins specific for different ligands.

DISCUSSION

α3Aβ1 is present in focal contacts of many adherentcell typesWe employed a modified immunofluorescence technique(Enomoto-Iwamoto et al., 1993) to show that the α3A integrinsubunit is revealed in the focal contacts of a number of celltypes following cross-linking of integrins to the extracellularmatrix and NP40 extraction. Our results demonstrate that


Fig. 6. α3A localizes to focal contacts on different extracellular matrix proteins. HFFs were pretreated with 300 µg/ml cycloheximide for threehours before culture on coverslips coated with EHS laminin/laminin 1 (‘LN1’; A-C), vitronectin (‘VN’; D-F), kalinin/laminin 5 (‘LN5’; G),type IV collagen (‘Col IV’; H), or poly-L-lysine (‘Lys’; I) for an additional 2 hours in cycloheximide. Cells were then processed forimmunofluorescence by the ‘standard’ (A,B,D, and E) or ‘BS3 cross-linking’ (C, F and G-I) methods, as described in Fig. 1. Cells were thenstained with antisera to the cytoplasmic domains of β1 (antiserum 210; A and D) or α3A (antiserum 8-4; B, C and E-I), followed byfluorescein-conjugated goat anti-rabbit IgG. Two photographs are shown for (G) and (H).

α3Aβ1, which has generally been considered to be absent fromfocal contacts in most cells, is in fact present in focal contactsof many cell types on a variety of extracellular matrices. Thesefindings raise the possibilty that α3Aβ1 functions as anadhesion receptor and/or a signalling molecule for diversematrices. Our results also suggest that in standard immunoflu-orescence protocols, where cells are fixed before permeabi-lization, the cytoplasmic domain of α3A is masked in mostcells from interaction with antibodies, possibly by cytoskel-

etal proteins that bind integrins at focal contacts (Burridge etal., 1988; Horwitz et al., 1986; Otey et al., 1990). A similarphenomenon has been described for α5β1 (Enomoto-Iwamotoet al., 1993). Despite the wide variety of cell types in whichα3Aβ1 localized to focal contacts, there may be some cell-specific component of this function, since α3A was notdetected using similar antibodies and protocols in focalcontacts of chicken muscle cells (Enomoto et al., 1993), HeLacells or NIH 3T3 cells (data not shown).


Fig. 7. Comparison of α3A focal contact localization with that of other integrin α subunits. MG63 cells were pretreated with 100 µg/mlcycloheximide for three hours before culture on coverslips coated with fibronectin (‘FN’; A) or vitronectin (‘VN’; B) for an additional twohours in cycloheximide. Cells were processed by the BS3 cross-linking method, as described in Fig. 1, then assayed by double-labelimmunofluorescence with the anti-α3 monoclonal antibody P1B5 (A,B; rows labelled ‘P1B5’) and polyclonal antisera to different αcytoplasmic domains (A,B; rows labelled ‘anti-α CYTO’), followed by rhodamine-conjugated goat anti-mouse IgG and fluorescein-conjugatedgoat anti-rabbit IgG. Individual α subunits tested (α3A, α5, αV, and α2) are indicated at the top of each column.

Since detection of α3A in focal contacts of a number of celltypes required cross-linking and extraction prior to immuno-fluorescence, it is interesting to note that α3Aβ1 can be readily

detected by standard immunofluorescence in focal contacts ofkeratinocytes (Carter et al., 1990b; Enomoto et al., 1993; C.M. DiPersio and R. O. Hynes, unpublished) and kidney


mesangial cells (Grenz et al., 1993). Standard protocols canalso detect α3β1 in focal contacts of fibroblasts cultured on akeratinocyte-secreted matrix but not on fibronectin or collagen(Carter et al., 1991; C. M. DiPersio and R. O. Hynes, unpub-lished). These results suggest differences in the nature ofα3β1-containing focal contacts between some cell types and/oron certain extracellular matrices and may reflect differences incytoskeletal interactions. For example, cytoskeletal proteinsthat bind to and mask the α3A cytoplasmic domain in fibro-blasts may be absent from mesangial cells or keratinocytes.Alternatively, α3Aβ1 may occur in focal contacts at muchhigher concentrations in the latter cell types or on complexmatrices that contain kalinin/laminin 5, making it more easilydetectable by standard immunofluorescence. It will be inter-esting to determine whether α3Aβ1 in focal contacts interactswith different subsets of cytoskeletal components in a cell-specific or matrix-specific manner.

Diverse extracellular matrix proteins promotelocalization of α3Aβ1 to focal contactsThe best ligand for α3β1-mediated adhesion described so faris kalinin/laminin 5, a component of the extracellular matrixsecreted by keratinoctyes (Rousselle et al., 1991; Carter et al.,1991). Indeed, K562 cells, which do not express endogenousα3A, acquire the ability to adhere to kalinin/laminin 5 orkalinin-containing matrices upon transfection with human α3A(Weitzman et al., 1993; Delwel et al., 1994). However, aplethora of other potential extracellular ligands has beenreported for α3β1, including fibronectin, EHS-laminin/laminin1, and collagens. Evidence for many of these interactions hascome from in vitro binding to ligand affinity columns (Waynerand Carter, 1987; Gehlsen et al., 1989; Elices et al., 1991;Clyman et al., 1992) and blocking of cell adhesion with anti-bodies against α3β1 (Wayner and Carter, 1987; Elices et al.,1991).

We have shown that α3Aβ1 can localize to focal contactsin several cell types in response to fibronectin, EHS laminin/laminin 1, kalinin/laminin 5, type IV collagen, and vitronectin.Our experiments were performed under conditions that preventendogenous matrix production, and cell adhesion to coverslipsrequired prior to coating with matrix proteins, demonstratingthat focal contacts were formed in response to the exogenouslyprovided ligands. Adhesion per se was not sufficient, sincecells adhered to poly-L-lysine but did not form focal contacts.Finally, other integrin α subunits did not demonstrate the broadrange of responsiveness to extracellular matrix proteins thatα3A did, and α5 and αV localized to focal adhesions only ontheir known ligands. These results are consistent with apossible role for α3Aβ1 as a receptor for diverse matrixproteins. Indeed, as mentioned above, direct binding by α3β1has been demonstrated for all proteins tested except vit-ronectin.

In general, our results confirm those of Grenz et al. (1993),who used immunofluorescence with the monoclonal antibodyP1B5 to show that α3β1 is recruited to focal contacts in kidneymesangial cells cultured on fibronectin, type I collagen, or EHSlaminin/laminin 1. However, while the latter studies showedthat cycloheximide had no effect on mesangial cell attachmentto type I collagen and focal contact formation in general, datawere not presented to rule out secretion of endogenous ligandsfor α3β1 in particular. Therefore, it was not proven that these

ligands were sufficient for recruitment of α3β1 to focalcontacts. In contrast with our results, α3β1 was not detectedin focal contacts of mesangial cells on vitronectin (Grenz et al.,1993). Furthermore, while only a fraction (35% to 55%) offocal-contact-containing mesangial cells on different matricescontained α3, our double-label immunofluoresence experi-ments suggest that most β1-containing focal contacts thatremained on a given matrix after cross-linking and extractionalso contained α3A (Fig. 5, and not shown). These differencesmay reflect cell-specific differences in the degree of α3 focalcontact localization. Alternatively, staining of α3 in mesangialcell focal contacts by the P1B5 monoclonal antibody may beinefficient using standard immunofluorescence. Indeed,adhesion blocking antibodies, such as P1B5, are often ineffec-tive at detecting ligand-occupied integrins (Carter et al., 1990a;Kawaguchi et al., 1994). Consistent with this idea, we did notdetect α3 in focal contacts with P1B5 by standard immuno-fluorescence, suggesting that the epitope for this antibody isless accessible in intact focal contacts.

It seems likely that the α3B cytoplasmic domain variant(Tamura et al., 1991) will display a similarly broad range ofmatrix-responsiveness, since the A and B cytoplasmic domainvariants of the related α6 subunit have the same ligand speci-ficities (Delwel et al., 1993; Shaw et al., 1993), and substitu-tion of cytoplasmic domains in α subunit chimeras does notusually alter ligand specificity (Chan et al., 1992b). However,the α3A and α3B cytoplasmic domains may differentiallyaffect relative affinities or migratory properties of cells, as hasbeen shown for other α cytoplasmic domains (Chan et al.,1992b), or perform different post-adhesion signallingfunctions. We are currently comparing functions of the α3isoforms in transfected cells.

α3Aβ1 may be a secondary receptor for diverseextracellular matricesTransfection of α3A into K562 cells did not cause adhesion toEHS laminin/laminin 1, collagens, entactin/nidogen, orfibronectin (Weitzman et al., 1993; Delwel et al., 1994; C. M.DiPersio and R. O. Hynes, unpublished); therefore, α3Aβ1 isnot sufficient for initial cell adhesion to these matrix proteins.Furthermore, there have been no reports to our knowledge thatα3β1 binds vitronectin. Therefore, except for its role as akalinin receptor (Delwel et al., 1994), it seems possible thatα3Aβ1 is not a primary adhesion receptor for the former matrixproteins, and initial cell adhesion to these ligands may requireother integrins expressed on the cell surface. Since focalcontact formation is a post-cell-adhesion event, we suggest amodel in which focal contact localization on most matricesreflects a post-adhesion role for α3Aβ1. For example, as illus-trated in Fig. 8A, following initial cell adhesion to fibronectinby a primary integrin receptor, α3Aβ1 may bind with lowaffinity to fibronectin and initiate cytoskeletal interactions,resulting in the recruitment of more α3Aβ1 (and possibly otherintegrins) to form focal contacts detectable by immunofluores-cence. Alternatively, as shown in Fig. 8B, a receptor for initialcell adhesion may also initiate cytoskeletal interactions,followed by relatively rapid recruitment of α3Aβ1 into focalcontacts. Importantly, the initial receptor in this case may notbe present in sufficient quantities for detection by immunoflu-orescence. A possible role for α3β1 as a low affinity, sub-sidiary receptor for certain ligands has been suggested by


Fig. 8. A model for α3Aβ1 as a secondary adhesion receptor for diverse extracellular matrices. Cell adhesion to fibronectin (‘FN’) is shown asan example, but the model is applicable to cell adhesion on other matrices that induce α3Aβ1 focal contact localization. (A) Following initialcell adhesion mediated by a primary fibronectin receptor (‘1°FnR’) (step 1), α3Aβ1 may bind with low affinity to the extracellular matrix andinitiate interactions with the cytoskeleton (‘CSK’) (step 2), resulting in the recruitment of more α3Aβ1, and possibly other integrins, to the siteand formation of a focal contact (step 3). In an alternative, but not mutually exclusive, model (B), a primary receptor may mediate both initialcell adhesion (step 1) and initial cytoskeletal interactions (step 2); α3Aβ1 may then be recruited into focal contacts at a relatively rapid rate(step 3).

others (Wayner and Carter, 1987; Elices et al., 1991; Grenz etal., 1993). However, it is also possible that α3Aβ1 is recruitedto focal contacts in a ligand-independent manner on somematrices (see below).

Precedent for post-adhesion integrin function is found inhuman umbilical vein endothelial cells, where antibodyblocking experiments showed that αVβ3 (Cheresh, 1987) wasimportant for maintenance of normal cell adhesion to the extra-cellular matrix but was not required for initial cell attachment(Chen et al., 1987). Some integrin-mediated adhesionprocesses are known to require initial cell attachment mediatedby non-integrin receptors. During inflammation, for example,selectins mediate the initial, weak adhesion of leukocytes toendothelial cells of the vessel wall, allowing them to roll alongthe wall in the proximity of cytokines which eventuallyactivate β2 integrins on the leukocyte surface, leading toleukocyte invasion into adjacent tissues (Mayadas et al., 1993;Springer, 1994).

Following initial cell adhesion, the localization of integrinsto focal contacts on particular extracellular matrices usuallyreflects their known ligand-binding specificities (Grenz et al.,1993). Nevertheless, we do not yet know whether α3Aβ1initiates focal contact formation in cells on different matrices,or is recruited into focal contacts formed by other integrins.Indeed, in kidney mesangial cells, α3β1 moved into focal

contacts on fibronectin or type I collagen more slowly thandid α5β1 or α2β1, respectively, and was detected in a signif-icantly smaller proportion of the cells than the latter twointegrins, suggesting a requirement for pre-existing focalcontacts (Grenz et al., 1993). In contrast, we detected α3A infibronectin-induced focal contacts of HFFs before α5 wasdetected, and where α4 was never detected, suggesting thatfocal contact formation by α5β1 or α4-containing fibronectinreceptors is not a prerequisite for localization of α3Aβ1.However, we cannot rule out the possibility of undetectableamounts of α5 or α4 in α3-containing focal contacts. SinceαV was recruited into fibronectin-induced focal contacts inparallel with α3A, we cannot rule out the possibility that αV-containing fibronectin receptors initially form focal contactsinto which α3Aβ1 is recruited subsequently. We attempted toaddress directly such a requirement for other integrins byinhibiting their localization to focal contacts with monoclonalantibodies known to inhibit their adhesion functions. Unfor-tunately, binding of such function-blocking antibodies wasineffective at preventing focal contact localization of α3, αVor α5 in adherent cells (not shown). In fact, it is possible thatbinding to integrins of certain function-blocking antibodiesmay mimic binding of extracellular ligands and cause a con-formational change that enhances interactions with thecytoskeleton, thereby causing, rather than inhibiting, integrin


recruitment into pre-existing focal contacts (LaFlamme et al.,1992; Takada et al., 1992).

Although published studies support direct, low affinity inter-actions of α3Aβ1 with many extracellular matrix proteins, thepromiscuous recruitment of α3Aβ1 into focal contacts couldbe enhanced by, or in some cases caused by, interactions of thecytoplasmic domain with the cytoskeleton. Indeed, severalexperimental systems have shown that integrins can berecruited into pre-existing focal contacts independently ofbinding to the extracellular matrix. For example, LaFlamme etal. (1992) showed that binding to soluble RGD peptidepromoted recruitment of α5β1 into pre-existing focal contactsindependently of its binding to the extracellular matrix, sug-gesting that ligand-occupancy of the integrin results in a con-formational change that allows the β1 cytoplasmic domain tointeract with cytoskeletal proteins. Similarly, Takada et al.(1992) showed that a point mutation in the β1 extracellulardomain that abrogated RGD-dependent binding of α5β1 tofibronectin was nevertheless recruited into pre-existing focalcontacts, possibly due to a conformational change in thereceptor that mimics the ligand-occupied form. In severalrecent studies, deletion of the α1 (Briesewitz et al., 1993), α2(Kawaguchi et al., 1994), or αIIb (Ylänne et al., 1993) subunitcytoplasmic domains back to the conserved GFFKR motif alsoresulted in their ligand-independent recruitment into pre-existing focal contacts. Therefore, in the absence of an appro-priate extracellular ligand these α cytoplasmic domains mayrepress constitutive binding of the β1 cytoplasmic domain tocytoskeletal components (LaFlamme et al., 1992; Briesewitzet al., 1993; Ylänne et al., 1993; Kawaguchi et al., 1994).Given the broad spectrum of matrix proteins on which α3Aβ1is recruited to focal contacts, it is tempting to speculate that theα3Aβ1 cytoplasmic domains are particularly conducive tointeractions with cytoskeletal complexes at focal contacts. Weare currently testing the α3 cytoplasmic domains for theirabilities to regulate β1 integrin localization to specific adhesionstructures.

Possible roles for α3Aβ1 in vivoImmunological methods have been used to detect α3expression in parietal epithelial, visceral endothelial andmesangial cells of kidney (Adler, 1992; Patey et al., 1994), andin the basal layer of the epidermis throughout development(Fradet et al., 1984; Wayner et al., 1988; Peltonen et al., 1989).Similarly, the α3A subunit is detected by immunofluorescencein epithelial cells of the skin, digestive tract, and kidney duringmouse development (C. M. DiPersio and R. O. Hynes, unpub-lished). In contrast to its apparently limited distribution in vivo,α3β1 is present in the majority of adherent cell cultures (Fradetet al., 1984; Rettig et al., 1986; Hemler et al., 1987), suggest-ing that either it has a broader in vivo distribution than has beendetected by immunofluorescence, or that its expression isinduced upon adherent cell culture (Fradet et al., 1984; Rettiget al., 1986). The latter possibility could reflect an activationof α3 during certain developmental or physiological processesthat involve changes in the adhesive or proliferative propertiesof a cell. Indeed, upon oncogenic transformation, cell surfaceexpression of α3β1 often increases (Tsuji et al., 1990) orpersists while levels of other integrins decrease (Plantefaberand Hynes, 1989; Dedhar and Saulnier, 1990). Furthermore,recent immunohistochemical studies showed that α3β1

expression was conspicuously maintained in most primary andmetastatic tumors, and in some cases increased upon metasta-sis (Bartolazzi et al., 1994).

It is not yet known whether most of the extracellular matrixmolecules tested in this study colocalize with α3Aβ1 in vivo,and the physiological relevance of the broad responsivenessof α3Aβ1 is not yet clear. However, whether α3Aβ1 iscommon to many cell types or restricted to a few cell types invivo, an integrin with such a broad matrix-responsivenessmight be useful in a number of ways. For example, α3Aβ1may provide certain cell types with an inherent flexibility torespond to changes in matrix composition that they mightencounter during transient processes such as tissue develop-ment or wound healing. Such broad matrix-responsivenessmay also be important in tumorigenesis and metastasis.Indeed, antibody-blocking experiments suggest that metasta-sis of breast carcinoma cells is correlated with increasedadhesion to fibronectin by α3β1, and α3A is detected infibronectin-induced focal contacts of these cells with the BS3

cross-linking method (N. Tawil and P. Brodt, personal com-munication).

As discussed above, α3Aβ1 may also mediate post-adhesion, secondary responses to various extracellularmatrices. In this way, α3Aβ1 and other integrins/receptors maycooperate to induce an appropriate cellular response to a givenextracellular matrix. For example, following initial adhesionby a primary receptor, α3Aβ1 may modulate cell migration ortransduce a transmembrane signal that the primary receptor isunable to transduce. Indeed, clustering of α3β1 in epidermalcarcinoma cells stimulates tyrosine phosphorylation of cyto-plasmic proteins (Kornberg et al., 1991). The α3A cytoplas-mic domain may also be required for recruitment of cytoskel-etal or cytoplasmic proteins that are common to focal contactson different matrices. Careful comparative studies of α3Aβ1in cells containing different primary adhesion receptors, aswell as transfection of α3 isoforms into cells lacking endogenous α3, should allow identification of signals andadhesion functions mediated by α3β1 integrins in response todiverse extracellular matrices.

We are grateful to Robert Burgeson for providing us with kalinin,and to Martin Hemler and Louis Reichardt for antibodies. We thankNabil Tawil and Grant Wheeler for critical reading of the manuscript.C.M.D. was supported by a fellowship from the Muscular DystrophyAssociation. This work was supported by grant CA17007 from theNational Institutes of Health to R.O.H.; R.O.H. is an investigator ofthe Howard Hughes Medical Institute.

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(Received 12 December 1994 - Accepted 9 March 1995)

3Aβ1 integrin localizes to focal contacts in response to ... · INTRODUCTION The integrin family...

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