The vitronectin receptor associates with clathrin-coated ... · Peptides were synthesized and...

2729Journal of Cell Science 111, 2729-2740 (1998)Printed in Great Britain © The Company of Biologists Limited 1998JCS4539

The vitronectin receptor associates with clathrin-coated membrane domains

via the cytoplasmic domain of its β5 subunit

Patrick G. De Deyne1,2,*, Andrea O’Neill1, Wendy G. Resneck1, George M. Dmytrenko3,‡, David W. Pumplin4

and Robert J. Bloch1

Departments of 1Physiology, 2Surgery, 3Neurology and 4Anatomy & Neurobiology, School of Medicine, University of Maryland atBaltimore, Baltimore, MD 21201, USA*Author for correspondence (e-mail: [email protected])‡Present address: The Neurosurgical Group, Pensacola, FL 32501, USA

Accepted 3 July; published on WWW 27 August 1998

Rat myotubes cultured in fetal calf serum adhere tovitronectin-coated substrates through two distinctstructures, focal contacts and clathrin-coated membranedomains. We studied the integrins in myotubes to learn howthey associate with these two domains. Double labelimmunofluorescence studies with antibodies specific forclathrin, vinculin and several forms of integrin showed thatfocal contacts and clathrin-coated membrane domainscontain both vitronectin receptors (VnR, containing β3-and β5-integrins) and fibronectin receptors (FnR,containing β1-integrin). VnR but not FnR associates tightlywith the substrate in both domains, as the VnR aloneremains attached to the coverslip when the lipid bilayer andother membrane proteins are removed by detergent.Ultrastructural studies confirmed the localization of the β5subunit of the VnR at both domains.

We used intracellular injection and affinitychromatography to test the possibility that clathrin atcoated membrane domains associates with thecytoplasmic sequence of the β5 subunit of the VnR.

Injection of a synthetic peptide containing the NPXYmotif from the cytoplasmic domain of the human β5subunit, SRARYEMASNPLYRKPIST, depleted clathrinfrom coated membrane domains without affectingclathrin in perinuclear structures or vinculin at focalcontacts. Injection of the homologous β1 peptide,MNAKWDTGENPIYKSAVITT, also containing an NPXYmotif, had no significant effect on any of these structures.Affinity matrices containing the β5 but not the β1 peptideselectively retained clathrin from myotube extract, andbound clathrin could be selectively eluted by soluble formsof the β5 but not the β1 peptide. Thus, a sequence includingthe NPXY motif in the integrin β5 subunit is involved in thespecific anchoring of the VnR, but not the FnR, to clathrin-coated membrane.

Key words: Integrin, Vitronectin receptor, Fibronectin receptor,Vitronectin, Clathrin, Vinculin, Acetylcholine receptor, Muscle,Neuromuscular junction

SUMMARY

INTRODUCTION

Rat myotubes grown in tissue culture adhere to a vitronectin-coated glass substrate through two distinct structures, focalcontact domains and clathrin-coated membrane domains.Intracellularly, the focal adhesions contain vinculin, talin,bundles of actin microfilaments, and associated proteins (e.g.Bloch and Geiger, 1980; Bloch et al., 1989; Samuelsson et al.,1993; Plopper and Ingber, 1993), and so are typical of focaladhesions in mononucleated cells. Clathrin-coated membranedomains are characterized by extensive arrays of clathrin thatare too large to form as a result of individual coated vesiclesfusing with the membrane (Pumplin and Bloch, 1990).Glycoproteins in these domains are so closely bound to thesubstrate that they block access of antibodies to the vitronectincoating the glass (Baetscher et al., 1986). The identity of theseglycoproteins and their mode of adhesion to the tissue culturesubstrate have remained unknown.

Here we report that integrins, particularly vitronectinreceptors (VnR), are present at the two types of contactdomains, and so are likely to account for the close interactionswith the vitronectin substrate. We focused our attention on theVnR at the clathrin-coated membrane domains, as anassociation between clathrin and this family of integrins hasnot been widely reported. We used intracellular injection andaffinity chromatography to show that the association of clathrinwith the VnR is mediated by a limited region of the shortcytoplasmic sequence in the β5 subunit of the VnR. This regioncontains an NPXY motif that has been suggested to be a signalfor assembly of clathrin complexes at the plasma membrane(Chen et al., 1990; Vaux, 1992).

MATERIALS AND METHODS

Culture of primary muscle cellsPrimary cultures of rat myotubes were prepared as described (Bloch

2730 P. G. De Deyne and others

and Geiger, 1980; Bloch, 1979). Briefly, muscles from neonatal rathindlimb were dissociated enzymatically and suspended at 106

cells/ml in Dulbecco-Vogt modified Eagle’s medium containing 10%fetal calf serum (medium). Cultures were initiated by applyingaliquots (0.4 ml) to glass coverslips (Van Labs, Oxnard, CA) and weresupplemented the next day with 1.5 ml medium. Medium wasremoved 3 days later and replaced with medium containing 2×10−5 Mcytosine arabinoside to kill dividing cells. Cultures were used 6 daysafter initial plating for most immunolocalization experiments and 4-5 days after plating for intracellular injections.

Isolation of substrate-attached materialTo isolate substrate-attached material (SAM), cultures were eitherextracted with saponin (Bloch, 1984) or treated briefly with ZnCl2 andsheared with a stream of ice cold buffer (Pumplin and Bloch, 1990).Saponin removes >99% of total cellular protein from cultures, leavingonly SAM remaining on the coverslip (Bloch, 1984). Shearing aftertreatment with ZnCl2 removes most of the myotubes andcontaminating fibroblasts, but leaves membrane and associatedperipheral membrane proteins on the coverslips. Some cultures werefirst incubated with monotetramethylrhodamine-α-bungarotoxin (R-BT; 5 µg/ml in Hepes-buffered DMEM containing 5% fetal calfserum) to label acetylcholine receptors (AChR). Large clusters ofAChR form preferentially at the myotube-substrate interface (e.g.Bloch and Geiger, 1980) and serve as a useful marker for SAM inthese cultures.

Preparations of SAM were usually fixed with 2% paraformaldehydein phosphate-buffered saline (PBS) immediately after saponinextraction or shearing. Samples were often treated briefly either beforeor after fixation with 0.05% or 0.5% Triton X-100 to exposeextracellular determinants (Baetscher et al., 1986). Shearing andextraction with saponin yielded similar distributions of integrins, butFnR was only visualized in samples that were fixed before extractionwith Triton X-100 (see Results).

ImmunolabelingSome samples, labeled with R-BT to visualize AChR clusters in SAM,were prepared as above and incubated with rabbit antibodies tointegrins or with control antibodies, followed by fluoresceinated goatantibodies to rabbit IgG (FGAR). Samples that had not beenprelabeled with R-BT were double-labeled with mouse monoclonalantibodies to vinculin or to the heavy chain of clathrin and with rabbitantibodies to the integrins. Bound antibodies were visualized withrhodaminylated goat antibodies to rabbit IgG (RGAR) andfluoresceinated antibodies to mouse IgG (FGAM). Appropriatecontrols demonstrated the species specificity of the secondaryantibodies.

Labeled coverslips were mounted in glycerol, 1 M Tris-HCl, pH8.0 (9:1, v:v), supplemented with 1 mg/ml p-phenylenediamine toreduce photobleaching (Longin et al., 1993). Samples were observedwith a Zeiss IM-35 microscope equipped for epifluorescence and werephotographed using Kodak TMAX 3200 film that was exposed for 5-20 seconds and developed to an ASA of 1600 using Kodak TMAXdeveloper (Eastman-Kodak, Rochester, NY). Alternatively, sampleswere observed with a Zeiss 410 confocal laser scanning microscope,with pinhole settings to yield the highest possible resolution. ‘Bleed-through’ of fluorescence from one channel to the other was minimalin both epifluorescence and confocal images.

AntibodiesAntibodies against vitronectin and the vitronectin and fibronectinreceptors were obtained from Life Technologies (Gaithersburg, MD).Antibodies specific for the β1, β3 and β5 subunits of integrin werepurchased from Chemicon (Temecula, CA). Monoclonal antibodies tovinculin (11.5) and to the heavy chain of clathrin (X-22) were kindlyprovided by Dr B. Geiger (Weizmann Institute, Rehovot, Israel), andDr F. Brodsky (University of California, San Francisco, CA),

respectively. An affinity-purified rabbit antibody to the COOH-terminal sequence of the β5 subunit of integrin (Ramaswamy andHemler, 1990) was also generously provided by Dr M. Hemler (DanaFarber Cancer Research Institute, Boston, MA) and affinity-purifiedrabbit antibodies to the COOH-terminal sequences of the β1- and β3-integrin subunits were generously provided by Dr E. Ruoslahti (TheBurnham Institute, La Jolla, CA). A non-immune mouse monoclonalIgG (MOPC-21) and normal rabbit serum were routinely used ascontrols in immunolabeling experiments. The specificity of the anti-integrin antibodies were tested by immunoblotting of proteins frommyotube cultures, separated by SDS-PAGE and transferredelectrophoretically to nitrocellulose (see below). The specificities ofthese and the other antibodies we have used have been established(Mahaffey et al., 1990; Pasqualini et al., 1993; Adams and Watt, 1991;Leavesley et al., 1992; Ruoslahti and Pierschbacher, 1987). Secondaryantibodies conjugated to fluorescein or rhodamine were obtained fromJackson Laboratories (West Chester, PA).

Ultrastructural studiesSAM was prelabeled with R-BT, isolated by shearing, fixed inparaformaldehyde and labeled for 1 hour at RT with affinity-purifiedrabbit antibody to the COOH-terminal region of β5-integrin (diluted1:100 in 1% bovine serum albumin in PBS), followed byfluoresceinated mouse anti-rabbit IgG (diluted similarly) for 1 hour atRT. After brief washing, samples were incubated overnight at 4°Cwith goat anti-mouse IgG adsorbed to 10 nm colloidal gold (JanssenPharmaceuticals, Beerse, Belgium), diluted 1:10 in BANT (0.1%BSA, 0.5 M NaCl, 10 mM MgCl2, 20 mM Tris-HCl, 20 mM NaN3,pH 7.4; Luther and Bloch, 1989). AChR clusters labeled with the anti-integrin were chosen for the clarity of the fluorescence label and aminimum of material visible by phase contrast microscopy. Circlesaround these structures were scribed with a diamond markingobjective and broken out of the coverslips. The SAM was subjectedto quick-freezing and deep-etch rotary replication as describedpreviously (Pumplin and Bloch, 1990). Electron micrographs ofreplicas were taken at low magnification (×1,400) to compare withcorresponding fluorescence images and at high magnification(×30,000 or ×45,000) in stereo pairs with 6° of tilt.

Intracellular injectionsPeptides were synthesized and purified by Dr N. Ambulos(Biopolymer Core Facility, University of Maryland at Baltimore). Thelyophilized peptides were reconstituted in microinjection buffer (seebelow) and stored as a 10 mM solution at 4°C. One 19mer peptide,SRARYEMASNPLYRKPIST, contained an ‘NPXY’ motif andflanking sequence of the cytoplasmic domain of the β5 subunit ofhuman integrin (Williams et al., 1994). The other 19mer,MNAKWDTGENPIYKSAVTT, contained the ‘NPXY’ motif and theflanking sequence from the homologous region of the β1 subunit(Williams et al., 1994). Peptides (5 µM) and fixable dextran-rhodamine (0.75 mg/ml; Molecular Probes, Oregon) were dissolvedin microinjection buffer (40.5 mM K2HPO4, 39 mM KH2PO4, 12 mMNaH2PO4, pH 7.25) for injection into cells.

Prior to injection, myotubes were incubated with R-BT to assist inidentifying substrate-apposed membrane. Only unbranched myotubesof similar size (~400 µm in length, 15-20 µm in width) and showinglarge, well-defined clusters of AChR were selected for injection.Micropipettes with an inner diameter of 0.5 µm were made fromborosilicate glass capillaries containing an internal filament (WorldPrecision Instruments, Sarasota, FL) on a model P-87 Flaming/Brownmicroelectrode puller (Sutter Instrument Co., Novato, CA).Micropipettes were back-filled with the peptide-dextran solutionsdescribed above. For injection, the tip of the micropipette was pushedinto the myotube by a smooth movement of the micromanipulator(MMO-203 Narashige, Tokyo, Japan). Approximately 3 nl of solution(an estimated 10% of the intra-cellular volume) was injected into thecell at a constant flow rate, and typically the microinjection took 1-2

2731The association of vitronectin receptors with clathrin in cultured myotubes

kDa

Fig. 1. Specificity of antibodies to the β subunits of integrin incultures of rat myotubes. Proteins extracted from cultures of ratmyotubes were separated by SDS-PAGE under reducing conditions,transferred electrophoretically to nitrocellulose paper, and incubatedwith: (A) subunit specific rabbit antibodies to the β1, β3, and β5subunits of integrin or with non-immune rabbit serum (NRS); and(B) rabbit antibodies to the heterodimeric integrins, FnR and VnR.Bound antibody was visualized chromogenically after labeling with asecondary antibody conjugated to alkaline phosphatase. (A) Each ofthe anti-β-subunit antibodies specifically labeled 1-3 bands withapparent molecular masses appropriate for their respective antigens.The antibodies to the β1 subunit labeled a doublet at 150 and 130kDa, antibodies to the β3 subunit reacted with a single band at 135kDa, and antibodies to the β5 subunit recognized three bands at 140,120 and 65 kDa. The band visualized at lower molecular masses(around 40 kDa) associated with antibodies non-specifically, asindicated by the reaction with non-immune serum. The positions ofthe molecular mass standards are shown to the left. (B) Antibodies tothe FnR specifically reacted with 4 bands, at 150, 130, 95 and 90kDa. The former 2 bands (arrows) were also recognized by anti-β1-integrin (See A). The latter pair of bands (arrowheads) are likely torepresent the extracellular domains of α-integrins. Antibodies to theVnR labeled bands at 140, 120 and 65 kDa (arrows), as also seenwith the anti-β5-integrin (see A).

seconds per myotube. The micropipette was then withdrawn andmoved to the next cell. Microinjection was followed under fluorescentillumination to ensure that the injected fluorescent dextran marker wasretained by the cell, consistent with the cell remaining intact andadhering to the substrate.

Following injection, cultures were incubated at 37°C for 1.5 hour,then fixed, permeabilized with 0.5% Triton X-100 for 5 minutes, andlabeled with antibodies to clathrin or vinculin, followed by FGAM.Due to the presence of high concentrations of rhodamine-dextran inthe myoplasm of injected cells, we usually used confocal laserscanning microscopy to examine these samples.

Affinity chromatographyMyotubes were collected at 4°C by scraping the cultures into 0.5%Triton X-100 in phosphate-buffered saline (PBS; pH 7.4) containinga cocktail of protease inhibitors (220 units/µl aprotinin, 1 mMbenzamidine, 10 µg/ml leupeptin, 10 µg/ml antipain, 200 µg/mlsoybean trypsin inhibitor, 100 µM phenylmethylsulfonyl fluoride).The cell extract was centrifuged at 3,000 rpm for 3 minutes at 4°C ina clinical centrifuge and the supernatant was subjected to affinitychromatography on matrices containing peptides from thecytoplasmic domains of the β5 or β1 subunits of integrin (see above),linked to a Multiple Antigenic Peptide (MAP) system (Tam, 1988).

The MAP-peptides were covalently bound to CNBr-activatedSepharose 4B (Pharmacia Biotech, Sweden), following therecommendations of the manufacturer. In addition to the β1- and β5-MAP conjugates, we also used an affinity matrix with an unrelatedcontrol peptide, SK-1, having the sequence, SEGLSDDEET. Thecolumns were washed with PBS to elute unbound proteins; boundprotein was then eluted in 0.5% SDS in PBS. Alternatively, materialbound to the column was eluted with solutions containing 1 µg/ml ofthe β1 and β5 peptides dissolved in PBS. Fractions (1 ml) werecollected, analyzed by SDS-PAGE in 7% acrylamide gels, andimmunoblotted with antibodies to clathrin (X-22). Bound antibodieswere visualized colorimetrically with alkaline phosphatase-conjugated secondary antibodies and a kit obtained from Kierkegaardand Perry (Gaithersburg, MD).

RESULTS

We isolated SAM from myotubes by extraction with saponin,to study AChR, or by shearing, to study structural proteins, andexamined the distribution of the integrins in these preparations.We then focused on the interactions of integrin with clathrin-coated membrane, using intracellular injection techniques andaffinity chromatography to identify a sequence on thecytoplasmic ‘tail’ of the integrin responsible for anchoringclathrin to coated membrane domains.

Antibodies to the integrins in muscle culturesRabbit antibodies to the β1, β3 and β5 subunits of integrin wereobtained from two independent sources. We tested theseantibodies by immunoblotting proteins isolated from culturesof rat myotubes that had been subjected to SDS-PAGE underreducing conditions (Fig. 1A). Antibodies specific for the β1and β5 subunits both reacted with doublets, of 150 and 130kDa, and 140 and 120 kDa, respectively, consistent with otherreports (Damsky et al., 1985; Bozyczko et al., 1989).Antibodies to the β5 subunit also recognized a smaller band, at~65 kDa (Fig. 1A). Antibodies to VnR and FnR labeled thesame bands recognized by anti-β1 and anti-β5-integrinantibodies (Fig. 1B, arrows). In addition, anti-FnR also reactedwith a pair of bands at 95 and 90 kDa (Fig. 1B, arrowheads)

that are likely to represent the extracellular domains of α-integrins, expected under the reducing conditions we used.Only one band in the immunoblots was not accounted for byintegrin subunits, but this was also present in the sample blottedwith non-immune rabbit serum (Fig. 1, lane 4), indicating thatit was due to non-specific binding of antibodies. Our resultsindicate that the subunit-specific antibodies and the antibodiesto the heterodimeric integrins specifically recognize bands ofthe appropriate molecular masses in SDS-PAGE. Onlyantibodies that reacted with the integrins, and not non-immune


serum or control antibodies, labeled SAM specifically (seebelow).

Distribution of the integrinsWe used all the antibodies described above to localizeintegrins within AChR clusters. Neither the antibodies to theFnR and VnR, nor the subunit-specific antibodies to β1 or β5integrin labeled the AChR-rich domains of clusters, butinstead they all labeled the receptor-poor regions (e.g. Fig. 2).These regions have been previously shown to contain focalcontacts and clathrin-coated domains (Bloch and Geiger,1980; Pumplin and Bloch, 1990; see below). In general,antibodies to the FnR (not shown) or the β1-integrin (Fig.2A,B,C) labeled these domains of clusters less intensely thanantibodies to the VnR (not shown) or to β5-integrin (Fig.2D,E,F). The clusters were not labeled by non-immune rabbitantibodies (not shown), and labeling by anti-β5-integrin wasinhibited by its peptide antigen (Fig. 2G,H,I). These resultsstrongly suggest that labeling of AChR-poor domains by theanti-integrin antibodies was specific. Antibodies to the β3subunit of integrin did not label clusters to any significantextent, although they labeled focal contacts in fibroblastsbrightly (not shown). These results suggest that the FnR and

Fig. 2. Integrins in regions ofAChR clusters poor inAChR. Cultures of myotubeswere labeled with R-BT(A,D,G), and clusters wereisolated by extraction withsaponin (see Materials andMethods). Samples werefixed, permeabilized brieflywith Triton X-100, andlabeled with antibodies to theβ1 (B) or β5 (E) subunits ofintegrins, followed byFGAR. The results show thatboth the β1- and β5-integrinsubunits are present in dart-like structures resemblingfocal contacts (arrows in B,Cand E,F), and in small spotsresembling clathrin-coateddomains (arrowheads in B,Cand E,F). Double coloroverlay (C and F) shows thatneither protein codistributeswith AChR (AChR is shownin red and the integrinsubunits are shown in greenin C,F). The binding ofantibodies to the β5-integrinsubunit was blocked in a 10µM solution of theimmunogenic peptide (G,H,and overlay in I), suggestingthat the labeling shown in Eis specific. Bar, 10 µm.

its β1 subunit, and the β5 but not the β3 form of the VnR, arepresent in AChR clusters.

We compared the distribution of the FnR and VnR tovinculin and clathrin, which are markers for focal contacts andcoated membrane domains in SAM derived from myotubes(Bloch and Geiger, 1980; Pumplin and Bloch, 1990). Anti-VnRand anti-FnR co-localized with anti-vinculin in focal contactsand with anti-clathrin in coated membrane domains (Fig. 3A-I). Similarly, antibodies to β1-integrin (not shown) and β5-integrin (e.g. Fig. 3J,K,L) colocalized with these markers.These results indicate that the FnR, VnR, and their constituentβ subunits are present in both focal contacts and clathrin-coated domains. In the following, we focus on the relationshipbetween the clathrin-coated domains and the β1- and the β5-integrins.

As the antibody to the COOH-terminal sequence of β5-integrin gave strong and specific labeling, we used it to confirmthe localization of the VnR to focal contacts and coatedmembrane at the ultrastructural level. Colloidal gold particles,representing sites of antibody binding, appeared at both focalcontact domains and at clathrin-coated membrane domains.Stereo views (Fig. 4) showed that gold particles at focalcontacts were concentrated where actin filaments closely


approached the inner membrane surface, consistent with thelocation of the cytoplasmic tail of β5-integrin. Clathrin-coateddomains had more label around their edges, suggesting that theclathrin coat limits access of gold-adsorbed antibodies to thecytoplasmic tails of β5-integrin molecules. Alternatively, the β5subunit of integrin may be concentrated in these regions, whilemore central areas may be enriched in other integrins.

VnR, but not FnR, blocks access to the substrateWe showed previously that vitronectin coats the coverslip glasson which myotubes are grown, and that extraction of AChR

Fig. 3. VnR and FnRcodistributes with vinculinand clathrin. Substrate-attached membrane (SAM)from cultures of rat myotubeswas isolated by treatmentwith ZnCl2 followed byshearing, fixed, permeabilizedwith Triton X-100, andlabeled with rabbit antibodiesto the FnR (B,E), VnR (H), orβ5-integrin subunit (K),together with mousemonoclonal antibodies toeither vinculin (D,J) or theheavy chain of clathrin (A,G).The bound antibodies werevisualized with RGAM andFGAR. Double color overlayimages (C,F,I,L) show thatlabeling of the VnR and theFnR overlap (yellow) withlabeling of both vinculin (atfocal contacts, arrows) andclathrin (at coated membranedomains, arrowheads).Similar results to those shownin B and E were obtainedwith the antibodies specificfor the β1 subunit of integrin(Fig. 2). Bar, 20 µm.

clusters with Triton X-100 leaves material remaining on thecover glass that binds concanavalin A (Dmytrenko et al., 1990).This material is so tightly attached to the substrate that it blocksaccess of antibodies to the vitronectin beneath it (Baetscher etal., 1986). We examined SAM further to learn if this materialcontains integrins.

Even after extraction of unfixed, isolated membranefragments with Triton X-100, we detected significant amountsof VnR attached to the substrate (Fig. 5A,B). The labelingpattern was consistent with the presence of the VnR in bothfocal contact and clathrin-coated membrane domains.


Fig. 4. Ultrastructurallocalization of β5-integrin infocal contact and clathrin-coated membrane domains.Substrate-associatedmembrane fragments wereisolated by treatment withZnCl2 followed by shearing,and labeled with antibodiesdirected to the C-terminal,cytoplasmic sequence of β5-integrin. Samples werelabeled first with FGAR andthen with rabbit anti-goat IgGcoupled to 10 nm particles ofcolloidal gold, and processedby quick-freeze, deep-etch,rotary-replication electronmicroscopy. Samples showimmunogold labeling at thecytoplasmic surface of thecell membrane at sites whereactin filaments associate(double arrowheads), and atlarge clathrin-coatedmembrane domains(C, arrowheads). Bar, 0.1 µm.

Similarly treated samples were not labeled by a fluorescentlipid probe, C18-diI (not shown), indicating that the lipidbilayer had indeed been removed by the detergents. AChR hadalso been extracted from these samples, as R-BT labeling wasabsent. Despite strong labeling by antibodies to the VnR, littleFnR labeling remained in these samples (Fig. 5C,D),suggesting that most of the FnR had also been extracted bydetergent. Thus, the binding of VnR to the substrate is notdependent on FnR. Furthermore, the VnR probably associatesmore stably with the substrate than the FnR and is thereforemore likely to block access of anti-vitronectin antibodies to thevitronectin coating the coverslip.

To test this idea we compared labeling by anti-VnR and anti-vitronectin antibodies. We observed a complementary stainingpattern (Fig. 5E,F): where VnR was present, vitronectin couldnot be immunolabeled, and where vitronectin was brightlyimmunolabeled, VnR was not detectable. The VnR-richdomains were also stained with fluoresceinated concanavalinA (e.g. Fig. 5A,B), which we had previously used to identifyglycoproteins that were closely apposed to vitronectin on theglass coverslip (Baetscher et al., 1986). These results suggestthat the tight adhesion of myotubes to the substrate is mediatedby VnR rather than by FnR.

Perturbing clathrin-coated domains bymicroinjectionClathrin is bound to integral membrane proteins via the adaptincomplex (reviewed by Robinson, 1994; Schmid, 1997). Duringendocytosis from the surface, AP-2 binds to integral membraneproteins at or near sequences of amino acids, such as FRXYand NPXY, that are thought to form tight turns that serve as

signals for assembly of coated membrane (Chen et al., 1990;Collawn et al., 1990; Vaux, 1992). We therefore studied therole of the NPXY motif common to both integrin subunits inanchoring clathrin complexes at coated membrane domains.

We synthesized oligopeptides containing these NPXYmotifs (see Materials and Methods) and injected them intomyotubes. After injection, cells were incubated briefly, thenfixed, permeabilized and immunolabeled. Given that the VnRis more stably associated with clathrin-coated membranedomains than the FnR, we anticipated that the oligopeptidecontaining the NPXY motif from the β5 subunit would have amore potent effect than the homologous β1 oligopeptide.

Shortly after injection of the β5 peptide, the labeling ofclathrin-coated membrane domains by anti-clathrin wassignificantly reduced (Fig. 6B). This was not due to directeffects of the β5 peptide on the anti-clathrin antibody, asimmunoblotting by the antibody was unaffected in the presenceof the peptide (Fig. 8, lanes 1,2), even at concentrations muchhigher (µM) than those achieved intracellularly followinginjection. Injection of the β1 peptide had no detectable effectson clathrin labeling and, indeed, cells injected with the β1peptide could usually not be distinguished from uninjectedcontrols (Fig. 6D,F). Double-blind evaluation revealed that asignificant reduction of labeling by anti-clathrin occurred in 21of the 27 cells (77%) injected with the β5 peptide, but only in9 of the 33 cells (27%) injected with the β1 peptide (n=33).This difference was highly significant by Chi2 analysis(P<0.0001).

We also examined cells injected with the β5 peptide to learnif clathrin was altered elsewhere in the cell. Clathrin labeling atperinuclear structures seemed unaffected by the β5 peptide,


Fig. 5. VnR, but not FnR, is tightly bound to thesubstrate. SAM was isolated by extraction withsaponin, treated with 0.5% Triton X-100, fixed, anddouble labeled with fluoresceinated concanavalin A(A,C) or with mouse antibodies to vitronectin (E)together with rabbit antibodies to the FnR (D) or theVnR (B,F) and followed by FGAM (E) and RGAR(B,D,F). The results show significant amounts ofVnR (B) but not FnR (D) remaining associated withthe glass coverslip. Fluorescently labeled vitronectinis present over most of the substratum, but is notlabeled by anti-vitronectin antibodies where theVnR is also present (e.g. arrows in E,F). Bar, 20 µm.

suggesting that this sequence was not involved in the bindingof clathrin to Golgi membranes (Fig. 7D). Vinculin at focalcontacts was also not affected by the injected β5 peptide (Fig.7F), consistent with evidence that other integrin sequences helpto mediate the binding of cytoskeletal proteins at focal contacts(Reszka et al., 1992; Otey et al., 1993; Lewis and Schwartz,1995). These results, together with the lack of effect of the β1peptide, show that when myotubes are grown on a vitronectinsubstrate, the clathrin-AP-2 complex associates with membranedomains that are enriched in the β5 subunit of integrin by virtueof its ability to recognize the cytoplasmic sequence containingthe NPXY motif and adjacent amino acids.

Selective association with the clathrin complex withβ5-integrin demonstrated by affinity chromatography The effect of the injected β5 peptide could be due to interaction

of this peptide with the clathrin-AP-2 complex, or it could beindirect. For example, proteins with NPXY motifs are knownto be involved in downstream signaling initiated by theactivation of receptor tyrosine kinases (reviewed by Fantl et al.,1993). Injection of the β5 oligopeptide might therefore havepleiotropic effects on cells that result in the selectivedisaggregation of clathrin-coated membrane domains. In thiscase, we would not expect to see binding of the clathrincomplex to the β5 peptide in biochemical assays. If, however,the injected oligopeptide directly inhibits the ability of theclathrin complex to associate with integrins at coatedmembrane domains, the oligopeptide should associate withclathrin in vitro.

We performed affinity chromatography on resins containingthe β5 and β1 peptides to try to distinguish between thesepossibilities. We prepared the oligopeptides as MAP


Fig. 6. Injection of a peptidecontaining the NPXY cytoplasmicsequence of the β5 subunit perturbsclathrin distribution. Myotubes werelabeled with R-BT and injected with‘injection buffer’ containingrhodamine-dextran (0.75 mg/ml) andan oligopeptide (0.5 µM) thatincludes the cytoplasmic NPXYsequence of either β5-integrin (A,B)or β1-integrin (C,D). Samples werethen permeabilized andimmunolabeled for clathrin.Rhodamine-dextran was used toidentify injected cells. The AChRclusters present in substrate-attachedmembrane were visualized by R-BT(A,C,E). Confocal microscopy wasused to observe the immunolabeledclathrin at the same focal plane(B,D,F). The uninjected control isshown in E and F. The β5 but not theβ1 peptide perturbed clathrinorganization at the substrate-attachedmembrane. Bar, 20 µm.

conjugates, to facilitate coupling to CNBr-activated Sepharoseand to increase the binding capacities of the affinity resins.Myotube extracts were prepared in PBS containing 0.5%Triton X-100 and passed over the affinity columns; boundprotein was eluted with SDS and analyzed by SDS-PAGE andimmunoblotting. The affinity column carrying the β5 peptidewas considerably more effective in retaining clathrin than thecolumn carrying the β1 peptide (Fig. 8A, lanes 4, 6). Anunrelated sequence, SK-1, prepared as a MAP peptide andconjugated to Sepharose identically, was unable to retain anydetectable clathrin (Fig. 8A, lane 8), confirming the specificityof the interaction between clathrin and the β5 oligopeptide.Columns carrying the β1 or β5 peptides did not bind myosin,talin, or vinculin (not shown), all of which were present in themyotube extract. Although previous studies suggested that thisregion of the β1 sequence could interact with talin (Tapley etal., 1989; see also Lewis and Schwartz, 1995), the binding isweak (Horwitz et al., 1986) and would probably not be detectedunder our experimental conditions.

We further examined the specificity of binding by usingsoluble peptides to compete for clathrin binding with theimmobilized β5 peptide. Clathrin that had been prebound to theaffinity column carrying the MAP-β5 peptide was eluted by the

β5 peptide but not by the β1 peptide (Fig. 8B). Similarly, theβ5 peptide, but not the β1 peptide, prevented clathrin frombinding the immobilized ligand (not shown). These resultsindicate that the clathrin complex associates with a specificcytoplasmic sequence of β5-integrin, consistent with the ideathat this sequence selectively anchors clathrin at coatedmembrane domains.

DISCUSSION

Rat myotubes in tissue culture attach to the substrate throughtwo distinct membrane domains. One domain resembles focalcontacts in mononucleate cells (for a review see Kinch andBurridge, 1995). The other is composed of clathrin-coatedmembrane plaques that are flat and several times larger thanindividual clathrin-coated vesicles (Pumplin and Bloch, 1990).Our results suggest that myotube-substrate adhesion ismediated primarily by integrins of the VnR family located inboth focal contacts and clathrin-coated membrane domains.We show further that a small region of the β5 subunit of theVnR mediates the binding of the clathrin complex to thecytoplasmic surface of coated membrane domains.


Fig. 7. Microinjection of the β5peptide selectively displaces clathrinfrom coated membrane domains at theplasma membrane. Myotubes werelabeled with R-BT and then injectedwith a solution containing the β5 or β1oligopeptides (0.5 µM) together withrhodamine-dextran (0.75 mg/ml), asoutlined in the legend to Fig. 6.Samples were then fixed,permeabilized, and labeled with anti-clathrin (B,D) or anti-vinculinantibodies (F), followed by secondaryantibodies (FGAM). Injected cells,identified from their intracellularcontent of rhodamine-dextran, wereobserved and photographed underconfocal laser optics. Fluorescentlylabeled clusters of AChR marked thesubstrate-attached membrane in A andE; the AChR cluster shown in A is notin the focal plane in C. Microinjectionof the β5 peptide perturbed theclathrin-coated domain at thesubstratum (B). The perinuclearstaining of clathrin in the same cellwas unaffected (D, arrow). Injectionof the β5 peptide did not affectvinculin at focal contacts (E,F). Bar,20 µm.

The identity of VnR as the molecule that mediates substrateadhesion at the clathrin-coated membrane domains could nothave been predicted easily from earlier studies. Smallerclathrin-coated structures have been reported to mediate cell-substrate adhesion (Maupin and Pollard, 1983; Nicol andNermut, 1987; Nermut et al., 1991; Wayner et al., 1991; Tawilet al., 1993) but, with the exception of podosomes or ‘pointcontacts’, the adhesive molecules that anchor them to thesubstrate have not been identified. Podosomes and pointcontacts are small, punctate structures that mediate adhesionthrough integrins and that associate with vinculin and actin(Marchisio et al., 1988; Nermut et al., 1991; Tawil et al., 1993;Arregui et al., 1994). Although the much larger clathrin-coatedmembrane domains of rat myotubes lack vinculin and actin(Figs 3 and 4; see also Pumplin and Bloch, 1990), they are alsoanchored to the substrate by integrins and, in particular, by theVnR.

The presence of extracellular ligands is known to influencethe accumulation and organization of integrins in the plasmamembrane (e.g. Singer et al., 1988; Miyamoto et al., 1995).Consistent with this, the presence of vitronectin on the glasssubstrate (Baetscher et al., 1986) is probably the single mostimportant determinant of integrin localization in cultured

myotubes. The vitronectin-coated glass coverslip binds cellularmaterial tightly enough to resist extraction by neutral detergentand to inhibit access of antibodies to the vitronectin on thesubstratum (Baetscher et al., 1986). Although myotubesexpress both VnR and FnR, and both integrins associate withsubstrate-attached membrane (SAM), the FnR binds to thesubstrate only weakly (see below). By contrast the VnR bindsvery tightly indeed, so that it remains attached to the substrateeven after many other integral and peripheral membraneproteins have been extracted by detergent (Fig. 5B). This tightbinding is almost certainly the result of the specificity of VnRfor vitronectin and the high local concentrations of both ligandand receptor achieved at the myotube-substrate interface.

A vitronectin substrate cannot explain the presence of VnRat two different membrane-cytoskeletal domains, however.Why should VnR in one domain associate with vinculin andproteins of focal contacts, but at the other associate only withclathrin, adaptin, and other proteins of coated membrane?One possible explanation is that different forms of the VnR(e.g. containing β3 or β5 subunits) tend to associatepreferentially with different structural proteins. Although theinconsistent labeling by anti-β3-integrin antibodies has madethis difficult to confirm in myotube membranes, this


Fig. 8. Affinity chromatographyusing the β1- and β5-integrinoligopeptides. (A) Lanes 1 and 2show rat hippocampalhomogenates that were subjectedto SDS-PAGE, transferredelectrophoretically tonitrocellulose paper, andimmunoblotted with X-22 anti-clathrin in the absence (lane 1)or the presence (lane 2) of thesoluble β5 peptide (1 µM). Thepeptide did not inhibit thereaction of anti-clathrin with itsantigen. Lane 3 shows animmunoblot of myotube extracts

probed with a non-specific mouse IgG, MOPC-21. No clathrin wasdetected. Lanes 4-6 show the results of immunoblotting the fractionsfrom myotube extracts that were eluted from affinity columns linkedto MAP-peptides. Synthetic MAP-peptides, with sequences identicalto the sequences containing and flanking the NPXY motifs of the β5and β1 subunits of human integrins, or a control MAP-peptide, werecovalently coupled to Sepharose beads. Extracts of myotubesprepared with non-denaturing detergent were applied to the columns.After unbound material was washed free of the columns, boundmaterial was eluted with a solution containing SDS (lanes 4-6). Lane4: MAP-β5-Sepharose; lane 5: MAP-β1-Sepharose; lane 6: MAP-SK1-Sepharose, which serves as an additional control. Clathrin,detected in immunoblots using monoclonal antibody X-22, wasretained specifically by the MAP-β5-Sepharose column. (B) Myotubeextracts were applied to MAP-β5-Sepharose and unbound materialwas removed by washing. The column was then eluted with solutionscontaining either the β5 (lane 2) or β1 (lane 1) oligopeptides (1 µg/mlbuffered saline). Clathrin, detected in immunoblots using monoclonalantibody X-22, was selectively eluted by the β5 oligopeptide.

kDa

kDa

explanation would be consistent with our results withfibroblasts (P. De Deyne and R. J. Bloch, unpublished), andwith the results reported by others in carcinoma cells andastrocytes (Wayner et al., 1991; Tawil et al., 1993). In theformer, αvβ3 partitioned preferentially into focal adhesionsand αvβ5 partitioned into distinctive structures at substrate-associated membrane that appeared as small spots or irregularlines (see also Nicol and Nermut, 1987). From our results, itis likely that the regions enriched in αvβ5 in carcinoma cellsand astrocytes are clathrin-coated membrane domains. Thus,these structures are probably more widespread than

previously recognized and may play an important role inadhesion in many cells other than myotubes.

Although clathrin associates with VnR at coated membranedomains, it is still not clear if this interaction is direct orindirect. The oligopeptides we used for microinjection andaffinity chromatography contain an NPXY motif, a conservedsequence present in the cytoplasmic tails of the β1, β3, and β5subunits. In other molecules this sequence is believed to forma tight turn that interacts with the adaptin complex at theplasma membrane (Chen et al., 1990; Collawn et al., 1990;Chang et al., 1993; for reviews, see Robinson, 1994; Schmid,1997). It therefore seems likely that the β5 oligopeptideinteracts directly with the adaptin complex and only indirectlywith clathrin. Preferential binding of the β5 oligopeptide toadaptins at the plasmalemma would also explain its inabilityto disturb clathrin binding to Golgi membrane, which ismediated by a different set of adaptins (reviewed by Sandovaland Bakke, 1994; Robinson, 1994; Schmid, 1997). We havenot been able to determine if the β5 peptide does indeed bindto the adaptins, because these proteins have not yet beencharacterized in developing skeletal muscle. In preliminaryexperiments, antibodies to α1-adaptin (kindly provided by DrF. Brodsky) labeled the clathrin-coated membrane domains ofmyotubes and also recognized several proteins that boundpreferentially to the affinity matrix coupled to the β5oligopeptide (not shown). The apparent molecular masses ofthese proteins were significantly greater than 100,000, themolecular mass of α1-adaptin, suggesting that muscle, likenerve (Zhou et al., 1993; Newman et al., 1995) may havepredominantly large isoforms of this molecule.

Cytoplasmic tails of β subunits of integrins have manybinding activities that have been ascribed to particularsequences (Reszka et al., 1992; Otey et al., 1993; Cone et al.,1994; Filardo et al., 1995; Lewis and Schwartz, 1995;reviewed by Williams et al., 1994). Our results stronglysuggest that the ability of VnR to concentrate clathrin atcoated membrane domains depends largely on the NPXYsequence and surrounding amino acids in the cytoplasmic tailof the β5 subunit. The homologous peptide of the β1-integrin,which helps to mediate the association of FnR withcytoskeletal proteins at focal contacts (Tapley et al., 1989;Reszka et al., 1992; Lewis and Schwartz, 1995), contains anNPXY sequence that is almost identical to that of the β5peptide, differing only in the substitution of isoleucine forleucine at the X position. Nevertheless, it has no noticeableeffect on the organization of clathrin at coated membranedomains. This suggests that an NPXY motif in this region ofthe cytoplasmic sequence of β5-integrin is not sufficient toaccount for its association with clathrin. Flanking the NPXYmotif of the β1 sequence, however, only 9 of 19 amino acids(47%) are conserved or identical to those of the β5oligopeptide, with the largest changes immediately N- and C-terminal to the NPXY motif. These sequences also differgreatly between the β5 and the β3 subunits (Williams et al.,1994). It therefore seems likely to us that the ability of the β5subunit to interact preferentially with clathrin relies in largepart on amino acids that surround the NPXY motif, ratherthan on the NPXY signal itself. This is consistent with thefact that mutation of the NPXY sequence of β1-integrin toNPXS did not affect the clathrin-mediated internalization ofthe FnR in Chinese hamster ovary cells (Vignoud et al., 1994;


but see Vaux, 1992). Our results suggest that the specificityof association of clathrin with β5-integrin can be studiedeffectively using homologous oligopeptides in which selectedamino acids have been altered.

Although association of the β5 oligopeptide with theclathrin complex is sequence-specific, it is probably notmodulated by phosphorylation. Our affinity chromatographyprotocols used synthetic peptides that were notphosphorylated and that were unlikely to be modified to anysignificant extent during the course of the experiment. Thissuggests that, unlike the interaction of β1-integrin with talin(Tapley et al., 1989), phosphorylation of the tyrosine in theNPXY sequence does not significantly alter the ability of β5-integrin to interact with the clathrin-adaptin complex.Phosphorylation of the NPXY motif of β5-integrin probablyhas other important effects, however. Following ligand-integrin binding and activation of soluble tyrosine kinases(Blystone et al., 1996; Miyamoto et al., 1995; Law et al.,1996), phosphorylation of the NPXY sequence of integrinmight lead to the binding of proteins containing SH2 or PTBdomains, with subsequent activation of several signalingcascades (Kuriyan and Cowburn, 1997; Law et al., 1996).This raises the interesting possibility that the NPXY motif ofβ5-integrin serves as an intermediate in intracellular signalingwhen phosphorylated, but as a structural element whenunphosphorylated.

A reciprocal relationship between the activation of a tyrosinekinase cascade and the association of clathrin with largeplaques of VnR, while of potential interest in a wide variety ofcells, may be especially relevant in embryonic muscle cells atthe time of innervation. Recent evidence indicates that agrin, amacromolecule deposited on muscle cells by motor neurons,can both bind to integrins (Martin and Sanes, 1997) andactivate a muscle-specific receptor tyrosine kinase (MuSK: DeChiara et al., 1996) to initiate the process of AChR clustering(for reviews, see Glass and Yancopoulos, 1997; Slater, 1997;Wells and Fallon, 1997). Simultaneously, neuregulin releasedby the motor neuron activates erbB receptors in the musclemembrane that in turn modulate the synthesis of new AChR(Francoeur et al., 1995). These new AChR are processedthrough clathrin-coated vesicles (Bursztajn and Fischbach,1984; Porter-Jordan et al., 1986; see also Matthews-Bellingerand Salpeter, 1983) and may be inserted preferentially into thepostsynaptic membrane as it is assembled (Campanelli et al.,1992). The association of integrins with both agrin in thesynaptic cleft and large arrays of clathrin on the cytoplasmicsurface of the sarcolemma suggests that clathrin-coatedmembrane domains may help both to direct the local insertionof AChR and to organize the signal transducing mechanismsresponsible for postsynaptic differentiation of theneuromuscular junction.

We are grateful to R. Meyer, J. Strong and D. Yarnell for theirtechnical assistance, to Drs M. Hemler, F. Brodsky, B. Geiger, and E.Ruoslahti for their generous donations of antibodies, and to Dr J.Barker for her gift of the MAP conjugate of the SK-1 peptide. Ourresearch has been supported by grants from the National Institutes ofHealth (NS 15513 to D.W.P.; NS17282 to R.J.B.) and the MuscularDystrophy Association (to R.J.B.), by a Clinical InvestigatorDevelopment Award (to G.M.D.), and by funds made available toP.G.D.D. by the School of Medicine, University of Maryland atBaltimore.

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