©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Specific Association Of CD63 with the VLA-3 and VLA-6 Integrins (*)

(Received for publication, March 20, 1995; and in revised form, April 26, 1995)

Fedor Berditchevski Gianfranco Bazzoni Martin E. Hemler (§)

From the Dana-Farber Cancer Institute, Harvard Medical School, Boston Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We screened monoclonal antibodies to cell-surface proteins and selected an antibody, called 6H1, that recognizes a putative integrin-associated protein. The 6H1 monoclonal antibody (mAb) indirectly coprecipitated and/or , but not , or from Brij 96 detergent lysates of multiple cell lines. Large scale purification using the 6H1 mAb yielded a single protein of 45-60 kDa with an amino-terminal sequence that exactly matched CD63. Confirming that the 6H1 mAb recognized the CD63 protein, 6H1 and a known anti-CD63 mAb yielded identical coprecipitation results and identical colocalization into lysosomal granules containing cathepsin D. Furthermore, we used an established anti-CD63 mAb to detect this protein in an immunoprecipitate, and also we observed VLA-3 and CD63 colocalization in cellular ``footprints.'' Notably, the cytoplasmic domain of was neither required nor sufficient for CD63 association, suggesting that it occurred elsewhere within the complex. Knowledge of these specific CD63- and CD63- biochemical associations should lead to critical insights into the specialized functions of , , and CD63.


INTRODUCTION

The adhesion of cells to the extracellular matrix is dependent on the function of integrins, a large family of ubiquitous heterodimeric cell-surface proteins(1) . Cell attachment to extracellular matrix through integrins results in generation of biochemical signals (e.g. changes in protein phosphorylation, intracellular pH, and Ca levels) and can regulate a variety of biological responses including cell migration, proliferation, apoptosis, and differentiation(1, 2, 3, 4) .

Notably, different integrins binding to the same ligands may induce distinct post-ligand binding events(5, 6, 7) , consistent with specific signaling pathways. Although the mechanisms for specific integrin-mediated signal transduction remain largely unexplored, different integrin and chain cytoplasmic domains have been associated with particular post-ligand binding events(5, 8, 9, 10, 11) . Because integrins themselves lack obvious enzymatic activity, we hypothesize that their differential associations with other proteins might be important for specific integrin signaling.

Only a few integrin-associated proteins have been identified that could potentially facilitate specific signaling. A 50-kDa protein (IAP-50/CD47), associated with a -like integrin, is essential for neutrophil phagocytosis enhanced by fibronectin but not by laminin(12) . The phosphorylation of other -associated proteins occurs in response to platelet-derived growth factor (13) or insulin (14) stimulation. However, no proteins have yet been identified that specifically associate with particular integrins. Cytoskeletal proteins such as -actinin and talin seem to associate with many different integrins, including those containing , , and subunits (15, 16) .

This study is aimed at identifying proteins that associate selectively with particular integrins. We used mild immunoprecipitation conditions to select for a new mAb()against a protein specifically associated with the and integrins. This protein was subsequently identified as the CD63 antigen, a member of the TM4 (or tetraspans) family of proteins(17) . Relatively little is known regarding CD63 other than that it is widely expressed on the surface of many cell types (18) and is present in the membranes of many types of intracellular granules (see ``Discussion'').


MATERIALS AND METHODS

Cell Lines

The human fibrosarcoma cell line HT1080, the human erythroleukemia cell line K562, and the various K562 transfectants were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS). The K562 transfectants K562-A2 and K562-A3 were transfected with (19) or (20) cDNA as previously described. To make K562-A6 cells, cDNA cloned into the pRc/CMV expression vector (21) was transfected into K562 cells by electroporation, and then 18 days after transfection, G418-resistant colonies were selected, and expression of was verified by flow cytometry. The K562-A3C0 transfectant contained cDNA with the cytoplasmic tail region terminated after GFFKR, and K562-A2C3 cells contained cDNA in which the tail of replaced the tail.()The P388D1 mouse macrophage cell line and the transfected variant P388D1-(22) were maintained in RPMI 1640 media with 15% FBS. Chinese hamster ovary cells (CHO) and variants CHO-A2(23) , CHO-A3(20) , and CHO-B1 (24) transfected with human , , and cDNAs, respectively, were grown in -minus minimum essential media (Life Technologies, Inc.) supplemented with 10% FBS. The CHO-A3B1 cell line (provided by Dr. J. Weitzman) expressing both human and subunits, was similarly maintained, except that 2 mg/ml G418 was added to the growth medium. A mutant strain of CHO cells transfected with human cDNA (25) (CHOB2a27 is called CHOB2-A5 in this paper) was obtained from Dr. R. Juliano. The human breast carcinoma MDA-MB-231 and mammary epithelial MTSV 1-7 cell lines were cultured in DMEM supplemented with 10% FBS, with the latter additionally supplemented with 5 µg/ml hydrocortisone (Sigma) and 10 µg/ml insulin (Sigma) as previously described(26) .

Antibodies

Anti-integrin mAbs used were anti-, A2-2E10, and A2-3E6(27) ; anti-, IA3(28) ; P1B5(29) ; A3-IIF5(20) ; anti-, A5-PUJ2(6) ; anti-, A6-ELE(6) ; and anti-, A-1A5(30) . Also, we used anti-CD63 mAbs RUU-SP 2.28(31) , AHN-16.1 (From M. Skubitz, University of Minnesota), and the negative control mAb P3(32) . The anti-CD109 mAb 8E11 was generated as previously described(33) . Rabbit polyclonal serum against the cytoplasmic domains of the integrin (34) , (35) , (36) (serum 161-C2), and (36) (serum 363) subunits were previously described. Rabbit sera against alternatively spliced variants of the cytoplasmic domain (6845, anti-; 382, anti-) were generously provided by Dr. V. Quaranta. Rabbit polyclonal serum against human cathepsin D was purchased from Sigma.

Antibody Production

RBF/DnJ mice were immunized four times with MTSV 1-7 mammary epithelial cells collected in PBS with 5 mM MgCl, and hybridoma clones were then produced essentially as previously described(33) , except that the fusion of splenocytes with the P3X myeloma cell line was done at a 2:1 ratio. After 12 days of selection in hypoxantihine/aminopterin/thymidine-containing medium, hybrid clones were analyzed by immunoprecipitation as described below.

Immunofluorescence

For immunofluorescence studies, HT1080 cells were grown on coverslips for 48 h, fixed for 20 min with 2% formaldehyde in PBS containing 5 mM MgCl and 5% sucrose, permeabilized for 5 min with 0.5% Triton X-100 in PBS, and then blocked with 10% goat serum in PBS. Cells were stained with 6H1 mAb (20 µg/ml in culture supernatant) or with RUU-SP 2.28 mAb (1:200 dilution of ascites in PBS with 10% goat serum) and subsequently with FITC-conjugated goat anti-mouse serum (Cappel, Durham, NC). In double-staining experiments, fixed and permeabilized HT1080 cells were incubated with both anti-CD63 mAb (6H1 or RUU-SP 2.28) and rabbit polyclonal anti-cathepsin D serum (1:500 dilution) and then developed with both FITC-conjugated goat anti-mouse and rhodamine-conjugated goat anti-rabbit sera (Cappel). For CD63/ colocalization experiments, the 6H1 and A3-IIF5 mAbs were directly conjugated with rhodamine and fluorescein (Pierce), respectively, using the manufacturer's protocol. HT1080 cells grown on coverslips (for 48 h) were preincubated in PBS containing 5 mM MgCl for 15 min, fixed with 2% paraformaldehyde, and then blocked with 10% goat serum/PBS. ``Footprints'' left behind by cells were then stained with the fluorescent 6H1 and A3-IIF5 mAbs. The coverslips were mounted with FluorSave Reagent (Calbiochem), and immunofluorescence was examined using a Zeiss Axioscop equipped with optics for epifluorescence.

Immunoprecipitation

Cells were labeled with ImmunoPure NHS-LC-Biotin (Pierce) according to the manufacturer's protocol, and cellular proteins were extracted for 1 h at 4 °C using either mild conditions (25 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM MgCl, 1% Brij 96, 2 mM phenylmethylsulfonyl fluoride (Sigma), 10 µg/ml aprotinin (Sigma), 10 µg/ml leupeptin (Sigma), 2 mM NaF) or more stringent conditions (lysis buffer supplemented with 0.2% SDS). Insoluble material was pelleted at 12,000 rpm for 10 min, and the cell lysate was incubated with protein A-Sepharose 4CL (Pharmacia Biotech Inc.) for 1 h to remove background-binding material. The cell extract was then incubated with specific mAb for 1-2 h at 4 °C, and the resulting immune complexes were isolated using rabbit anti-mouse polyclonal antibody (Sigma) preadsorbed onto protein A-Sepharose and then washed four times with the appropriate immunoprecipitation buffer. Proteins were eluted from protein A beads using Laemmli sample buffer, resolved on SDS-PAGE gels, and transferred to nitrocellulose. Biotinylated proteins were detected by incubation with Extravidin horseradish peroxidase (Sigma) followed by the Renaissance chemiluminescent developing reagent (DuPont NEN). For some experiments, cells were surface labeled with NaI using lactoperoxidase/glucose oxidase according a published protocol(37) , and immunoprecipitation proceeded as described above. For initial experiments, several other detergents (Nonidet P-40, SDS, Triton X-100, Digitonin) were substituted for Brij 96 in the extraction buffer. For metabolic labeling experiments, HT1080 cells (2 10) were starved for 1 h in methionine-free DMEM and then labeled with 3 mCi of [S]methionine for 1.5-2 h. After a chase with complete DMEM, 10% FBS for 3.5-5 h, cells were lysed and immunoprecipitation was carried out as above.

Reimmunoprecipitation

For reprecipitation of integrin subunits, HT1080 cells (5 10-10) were surface labeled with ImmunoPure NHS-LC-Biotin, lysed in extraction buffer, and immunoprecipitated either with 6H1 or RUU-SP 2.28 mAb (anti-CD63). Immune complexes were collected on protein A-Sepharose beads, washed four times with extraction buffer, and subsequently eluted from the beads at 70 °C for 10 min in 0.5 ml of extraction buffer supplemented with 0.5% SDS. The eluate was diluted 3-fold with extraction buffer and reprecipitated using specific anti-integrin cytoplasmic domain polyclonal antibodies that were preadsorbed onto protein A beads. In positive control experiments, protein lysate prepared in extraction buffer containing SDS was heat treated in identical fashion but then directly immunoprecipitated with anti-integrin polyclonal sera.

For reprecipitation of CD63 protein, HT1080 cells (7 10) were metabolically labeled with 5 mCi [S]methionine in a methionine-free DMEM for 1.5 h and chased in a complete DMEM, 10% FBS for 4-5 h. After lysis in 1% Brij 96-containing buffer, the and immune complexes were recovered on Sepharose 4B beads directly conjugated to anti- (A2-IIE10) and anti- (A3-IVA5) mAbs, respectively. The integrin-associated proteins were eluted from the immune complexes in lysis buffer additionally supplemented with 0.2% SDS at 4 °C for 30 min and subsequently reprecipitated with 6H1 mAb. The reprecipitated material was resolved on SDS-PAGE and analyzed as above.

Purification and Sequencing of 6H1 Antigen

From K562 cells (30 g), lysate was prepared in 100 ml of buffer containing 1% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml aprotinin. To remove background bead-binding material, the lysate was sequentially preincubated with protein A-Sepharose (for 5 h at 4 °C) and Sepharose beads conjugated with irrelevant mAb (for an additional 4 h). To isolate the 6H1 antigen, the lysate was incubated for 4 h in batch with immobilized 6H1 mAb, coupled at 3 mg/ml packed Sepharose 4B beads (Pharmacia Biotech Inc.). After washing the beads with 50 column volumes of lysis buffer, the 6H1 antigen was eluted from the beads using 0.1 M glycine (pH 2.7), and the fractions were immediately neutralized with 0.1 volume of 1 M Tris-HCl (pH 8.8). Eluted fractions were analyzed by SDS-PAGE (8% acrylamide), followed by silver staining. Larger quantities of the fractions containing the 6H1 antigen were then subjected to SDS-PAGE, and proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad). The major protein band was visualized by Ponceau S staining, excised from the membrane, and then amino-terminal sequencing was carried out using an Applied Biosystems 470A gas-phase sequenator equipped with a 120A phenylhydantoin amino acid analyzer (Harvard microsequencing facility, Cambridge, MA). The resulting amino-terminal sequence was compared to published sequences available in the GenBank data base using the FASTA program.

Flow Cytometry

Cells were harvested, washed once with PBS containing 5 mM MgCl, and preincubated in WB5 buffer (PBS containing 5% bovine serum albumin, 0.1% fetal calf serum, and 0.02% sodium azide) at 4 °C for 10-15 min. Typically, 5 10 cells in 0.1 ml were incubated either with an equal volume of culture supernatant or with 1:100 dilution of ascites or with 20 µg/ml of purified primary mAb at 4 °C for 45 min followed by staining with FITC-conjugated goat anti-mouse secondary Ab (1:100 dilution in WB5 buffer). In experiments testing for possible cross-reactivity of the 6H1 mAb with integrins, cells were incubated with 100 µg of the purified primary antibody (well above the saturation level). Fluorescent staining was analyzed using a Becton Dickinson FACScan device.


RESULTS

Identification of Integrin-associated Proteins

To search for integrin-associated molecules, we chose immunoprecipitation as our major approach. After testing many different combinations of various detergents, we found that buffer containing 1% Brij 96 most consistently immunoprecipitated integrins together with putative associated molecules and with relatively low background. Also, we found that cell-surface labeling with biotin was more informative than either labeling with I-labeled iodine or metabolic labeling with [S]methionine. With this background information, we then immunized mice with intact MTSV 1-7 mammary epithelial cells and attempted to select mAbs against putative integrin-associated proteins.

Of more than 600 hybridoma supernatants screened by immunoprecipitation, 131 precipitated various cell-surface proteins but failed to precipitate or coprecipitate any molecules resembling integrins. Another 28 antibodies precipitated biotin-labeled material that appeared on SDS-PAGE as two protein bands of sizes characteristic for integrin and subunits. To select against clones secreting antibodies that directly recognize integrins, flow cytometry was carried out using transfected cells expressing various integrin and subunits. Two new mAbs bound to K562-A2 but not K562 cells, six recognized K562-A3 but not K562, one bound to CHOB2-A5 but not CHO, two bound to K562-A6 but not K562, and one recognized CHO-B1 but not CHO. Because these were likely to be anti-integrin antibodies, they were not considered further. From the remaining antibodies, the mAb secreted by the hybridoma clone 6H1 was analyzed in more detail.

From a biotinylated extract of HT1080 cells, the 6H1 antibody immunoprecipitated two integrin-like proteins of 150 and 130 kDa (Fig. 1, lanea). In addition, this antibody coprecipitated a 67-kDa protein, similar in size to one of the bands coprecipitated with (lanec) but distinct from bands coprecipitated along with other integrins (lanesb, d, and e). Other protein bands in lanea are probably background bands since they also appeared in the negative control lane (lanef). However, as discussed below, the antigen directly recognized by 6H1 is not shown in this figure since it is not surface labeled by biotin.


Figure 1: Coimmunoprecipitation of an integrin-like heterodimer by mAb 6H1. HT1080 cells were surface labeled with biotin, lysed in 1% Brij 96 buffer, and immunoprecipitated with mAb 6H1 (lanea), mAb to the indicated integrins (lanesb-e), or with the P3 negative control (lanef). Precipitates were resolved in 9% SDS-PAGE under non-reduced conditions.



When analyzed by flow cytometry with a saturating amount of mAb (100 µg/ml), the 6H1 antigen was detected on the surface of K562, K562-A2, K562-A3, and K562-A6 cells at approximately similar levels, indicating that expression was not specifically dependent on the presence of the , , or integrin subunits (not shown). Accordingly, the 6H1 mAb at a concentration of 100 µg/ml did not recognize CHO-A2, CHO-A3, CHOB2-A5, P388-A6, or CHO-B1 transfectants (Fig. 2, middlecolumn), indicating that the antibody did not directly recognize the human integrin , , , , or subunits expressed on either mouse (P388D1) or hamster (CHO) cells. The rightcolumn in Fig. 2confirms that each transfected integrin subunit is expressed at levels substantially higher than the negative controls (leftcolumn). To exclude the possibility that the 6H1 mAb may recognize a combinatorial epitope formed by the coexpression of both human and integrin subunits, an additional flow cytometry experiment was carried out. CHO cells transfected with both and subunits could not be stained with the 6H1 mAb even at a concentration as high as 150 µg/ml (not shown), confirming that this mAb was not cross-reacting with integrin.


Figure 2: The 6H1 mAb fails to recognize human integrin subunits on hamster or mouse cells. CHO-A2, CHO-A3, CHO/B2-A5, CHO-B1, and P388-A6 cells were stained with control mAb P3 (firstcolumn), mAb 6H1 (secondcolumn), or anti-integrin mAbs (thirdcolumn), and expression was analyzed by flow cytometry. Anti-integrin mAbs were: anti-, A2-2E10; anti-, A3-IVA5; anti-, A5-PUJ2; anti-, A6-ELE; anti-, A-1A5. All antibodies were used at 20 µg/ml, except for 6H1, which was used at 100 µg/ml.



The 6H1 antigen was expressed on the surface of all human cell types analyzed as well as platelets (not shown). Notably, the 6H1 antibody stained a variant of JY cells that lacks all integrins (38) , again indicating that integrins are not required for the cellular reactivity of this antibody.

To determine whether any proteins immunoprecipitated by the 6H1 mAb indeed represent integrin and subunits, a reprecipitation experiment was carried out (Fig. 3). Following SDS disruption of a 6H1 immune complex isolated from biotin-labeled HT1080 cells, proteins of 125-130 kDa could be reprecipitated using polyclonal antibodies against the cytoplasmic domains of , , and (lanesj, l, and n). In contrast, neither nor integrin subunits were reprecipitated from the 6H1 mAb immune complex (lanesi and k). Positive control experiments showed that anti-, -, -, -, -, and - sera were each fully capable of recognizing the appropriate integrins from HT1080 cell lysates (Fig. 3, lanesa-f). The subunit, expressed at only a low level on HT1080 cells, also could be reprecipitated from the 6H1 immunoprecipitate (data not shown). In two additional cell lines (MTSV 1-7, MDA-MB-231), the 6H1 mAb again coprecipitated the 1 integrin as determined by reprecipitation experiments (not shown). The 6H1 protein itself was again not visible because it is not biotin labeled (see below).


Figure 3: Reprecipitation of and integrins from a 6H1 immunoprecipitate. HT1080 cells were surface labeled with biotin, lysed in 1% Brij 96 buffer, and then immunoprecipitated with the 6H1 mAb (laneh). Material precipitated by mAb 6H1 was eluted from protein A beads in 1% Brij 96 buffer supplemented with 0.2% SDS at 70 °C and then reprecipitated with specific polyclonal sera against the indicated cytoplasmic domains (lanesi-m) or rabbit preimmune sera (laneo). In a control experiment (to ensure that all anti-cytoplasmic domain sera were active), surface-labeled HT1080 cells were lysed in buffer containing 1% Brij 96 and 0.2% SDS, preheated for 10 min at 70 °C, and directly precipitated with specific polyclonal sera against the indicated integrin cytoplasmic domains (lanesa-f) or with preimmune sera (laneg). Precipitates resolved in 10% SDS-PAGE under reduced conditions.



The apparent specific association of the 6H1 antigen with the and integrins was additionally confirmed in direct immunoprecipitation experiments, using extracts prepared from biotin-labeled K562, K562-A2, K562-A3, and K562-A6 cells (Fig. 4). From all four cell lines, the 6H1 mAb consistently precipitated several proteins of 67, 95, and 110-115 kDa. However, bands in the region of integrin subunits (140 and 150 kDa) were detected in immunoprecipitates from K562-A3 and K562-A6 cells (Fig. 4, lanesf and h, respectively) but not from K562 or K562-A2 cells (lanesd and e). Taken together, these data suggest that the antigen recognized by the 6H1 mAb could selectively associate with the and integrins but not the or integrins.


Figure 4: Selective coprecipitation by the 6H1 mAb of integrins from K562 cell transfectants. Equal numbers of K562, K562-A2, K562-A3, and K562-A6 cells were surface labeled with biotin, lysed in 1% Brij 96 buffer, and immunoprecipitated with either 6H1 mAbs (lanesd-f, h) or with anti-a mAb A5-PUJ2 (lanea), anti-a mAb A2-2E10 (laneb), anti-a mAb A3-IVA5 (lanec), or anti-a mAb A6-ELE (laneg). Precipitates were eluted into Laemmli loading buffer and resolved in 10% SDS-PAGE under non-reduced conditions. The negative control mAb P3 did not precipitate labeled material from any of the cell extracts used (not shown).



The 6H1 mAb Recognizes CD63

To characterize the 6H1 antigen further, HT1080 cells were metabolically labeled with [S]methionine, and then immunoprecipitation was performed under stringent conditions (with 0.2% SDS included in extraction and washing buffers). For the first time, we could see that material precipitated by mAb 6H1 appeared as a single broad band ranging from 45 to 60 kDa (Fig. 5, laned). This band was not precipitated by a control antibody (lanef). Notably, when mild conditions were utilized, 6H1 immunoprecipitation yielded other S-labeled proteins, including proteins of 150 and 130 kDa that are likely to be integrin , , and subunits (Fig. 5, lanec). The prominent protein of 45-60 kDa seen with S labeling (lanesc and d) was not obvious upon I-labeled iodine (not shown) or biotin labeling (lanesa and b). Nonetheless, 130-150-kDa integrin proteins were coprecipitated under mild conditions (lanea), consistent with these being indirectly associated with the 6H1 antigen.


Figure 5: Biochemical characterization of the 6H1 antigen. HT1080 cells were surface labeled with biotin (lanesa and b) or metabolically labeled with [S]methionine (lanesc-h). Lysis buffer contained 1% Brij 96 (lanesa, c, e, and g) or 1% Brij 96 and 0.2% SDS (lanesb, d, f, and h). Lysates were immunoprecipitated with mAb 6H1, the established anti-CD63 mAb RUU-SP 2.28, or the negative control mAb P3 as indicated. Precipitates were resolved in 9% SDS-PAGE under non-reduced conditions.



To further characterize the 45-60 kDa antigen, 5-10 pmol of this protein was purified from K562 cells using a 6H1 immunoaffinity column, and the sequence of the first 30 amino-terminal amino acids was determined (Fig. 6). A search of protein sequences available from the GenBank data base revealed a perfect match between the 6H1 antigen and residues 2-31 from the amino terminus of the CD63 protein, except for three missing cysteines. Consistent with this result, both 6H1 and a previously characterized anti-CD63 mAb called RUU-SP 2.28 precipitated a very similar pattern of major and minor S-labeled proteins (Fig. 5, lanesc and g) that were not seen in control lanes (lanese and h). Also, even though the 45-60-kDa CD63 protein itself was not labeled with biotin, nearly identical patterns of biotin-labeled proteins were coprecipitated by both the 6H1 and RUU-SP 2.28 mAbs (Fig. 7, lanesb and c). Additional reprecipitation experiments confirmed that the RUU-SP 2.28 mAb, like 6H1, could coprecipitate and integrins from a Brij-96 HT1080 cell lysate (data not shown). Notably, the proteins coprecipitated by these two antibodies closely resemble a subset of those proteins coprecipitated by the anti- antibody (Fig. 7, lanea). Furthermore, immunofluorescent staining of HT1080 cells showed that the antigenic determinants recognized by both the 6H1 and RUU-SP 2.28 mAbs were not only present on the outer cell membrane of HT1080 cells but also were highly abundant in perinuclear granules where they colocalized with cathepsin D (not shown).


Figure 6: Amino-terminal sequence of the 6H1 antigen. The 6H1 antigen was immunopurified from K562 cells, the amino-terminal sequence was determined as described under ``Materials and Methods,'' and this sequence was aligned with the sequence from the CD63 antigen obtained from GenBank. No determination could be made at three positions (indicated by ``-''), corresponding to cysteine residues in CD63.




Figure 7: The 6H1 mAb and a known anti-CD63 mAb yield similar patterns of coprecipitating proteins. HT1080 cells were surface labeled with biotin, lysed in 1% Brij 96 buffer, and immunoprecipitated with anti- mAb P1B5 (lanea), 6H1 mAb (laneb), or RUU-SP 2.28 mAb (lanec). Precipitates were resolved in 9% SDS-PAGE under non-reduced conditions.



To further demonstrate specific CD63 association with integrins, we carried out immunoprecipitations of metabolically labeled and from HT1080 cells and then looked for the presence of CD63. The reprecipitation experiment showed that a protein band corresponding to CD63 was detected in the immunoprecipitate (Fig. 8, laneb) that represents 2-5% of the total CD63 directly immunoprecipitated in lanea. No CD63 was detected in an immunoprecipitate (lanec), even though is highly expressed on HT1080 cells (e.g. see Fig. 3). In addition, we looked for possible cellular colocalization of CD63 with the integrin (Fig. 9). Within HT1080 cells that were fixed but not permeabilized, staining for integrin showed abundant distribution throughout the membrane, with some punctate staining (panelA). The CD63 molecule was also present through the cell membrane, but the punctate expression pattern was more obvious (panelB). Colocalization of integrin (panelC) and CD63 (panelD) was most obvious in cellular footprints left following the retraction of spread cells on a coverslip. In a control experiment, another abundant protein (CD109) was not localized into the same footprints, and neither was the VLA-2 integrin (not shown). From the above results, we conclude that the 6H1 mAb recognizes the CD63 protein (39) and that this protein may associate with integrin within intact cells as well as in cell lysates.


Figure 8: Detection of CD63 in an immunoprecipitate. Integrin immune complexes were prepared from S-labeled HT1080 cells using A2-2E10 (anti-) and A3-IVA5 (anti-) mAbs directly coupled to Sepharose 4B beads. Integrin-associated molecules were eluted from the beads with 0.2% SDS, reprecipitated with an anti-CD63 mAb (6H1), and resolved on 11% SDS-PAGE (lanesb and c). A positive control experiment shows the broad CD63 protein (45-60 kDa) specifically precipitated by mAb 6H1 (lanea).




Figure 9: Colocalization of CD63 and integrin in cell footprints. HT1080 cells were grown overnight on coverslips, fixed with 2% paraformaldehyde, stained with anti- mAb A3-IVA5 (panelA) and 6H1 anti-CD63 (panelB), and visualized using FITC-conjugated goat anti-mouse Ig. Also, cell footprints were prepared and stained as described under ``Materials and Methods,'' and double staining was carried out using FITC-conjugated anti- A3-IVA5 (panelC) and rhodamine-conjugated 6H1 mAb (panelD). The arrows indicate colocalization of CD63 and integrin.



The Cytoplasmic Domain Is Not Required for CD63 Association

Because integrin subunit cytoplasmic domains have diverse functional roles(5, 40) , we hypothesized that they might also mediate selective biochemical associations with proteins such as CD63. To ascertain whether the cytoplasmic tail of is required for the association of the integrin with CD63, we analyzed K562-A3C0 cells (K562 cells expressing the subunit with no cytoplasmic domain). As shown in Fig. 10, both wild type (laneb) and the deletion mutant (laned) could be coprecipitated by the 6H1 mAb from biotinylated cell extracts. In each case, similar patterns of additional coprecipitated bands of 67 and 97 kDa were present, and these closely resembled proteins coprecipitated by the anti- mAb (lanesa and c). In a separate experiment, we analyzed K562-A2C3 cells (K562 cells expressing the chimera, which contains the transmembrane and extracellular domains of and the cytoplasmic domain of ). As shown (Fig. 10, lanef), the 6H1 mAb failed to coprecipitate the chimeric A2C3 or integrin subunits. Together, these results suggest that the cytoplasmic domain of the subunit is neither required nor sufficient for the formation of the -CD63 complex.


Figure 10: Role of the cytoplasmic domain in CD63 association. K562-A3, K562-A3C0, and K562-A2C3 cells were surface labeled with biotin, lysed in 1% Brij 96 buffer, and immunoprecipitated using mAb 6H1 (lanesb, d, and f), anti- mAb P1B5 (lanesa and c), or anti- mAb A2-2E10 (lanee). Precipitates were resolved in 10% SDS-PAGE under non-reduced conditions. The negative control mAb P3 did not yield labeled material from the cell lines used (not shown).




DISCUSSION

Specific interactions of particular integrins with other proteins have not previously been described. Now, our reciprocal immunoprecipitation data and cellular colocalization data provide strong evidence for specific CD63- and CD63- associations.

Our detection of CD63-integrin associations is unlikely to result simply from spurious cross-reactivity of anti-CD63 antibodies with integrins. First, the 6H1 mAb to CD63 failed to coprecipitate and in stronger detergent conditions, consistent with an indirect reactivity. Second, we have utilized three different anti-CD63 monoclonal antibodies to detect CD63 association with integrins. Furthermore, high levels of the anti-CD63 mAb 6H1 (well above saturating) failed to recognize CHO cells transfected with human , , or integrin subunits, and conversely, no integrins could be detected on a cell line (JY) that expresses CD63.

The specificity and relevance of the CD63-integrin interactions are apparent from several different results. First, in multiple cell lines, CD63 associated with and but not or , even though the latter were present at similar levels. Second, reciprocal experiments showed not only that VLA-3 (and VLA-6) were present in CD63 immunoprecipitates but also that CD63 was present in VLA-3 (but not VLA-2) immunoprecipitates. Third, both the CD63 and VLA-3 proteins are colocalized in cellular footprints, as seen by immunofluorescent staining.

The 6H1 antigen is identical to the CD63 protein, based on amino-terminal sequence identity. Consistent with this, 6H1 and an established anti-CD63 mAb (RUU-SP 2.28) both 1) specifically coprecipitated and integrins and showed a similar pattern of other coprecipitated proteins on SDS-PAGE and 2) showed similar profiles of cell-surface staining and localization to cathepsin D-containing intracellular granules. The latter data are in agreement with previous reports describing a dual subcellular localization of CD63(39, 41) .

The ability of CD63 to complex specifically with and integrins is of interest because these integrins have some degree of functional overlap(42, 43, 44, 45) , and their primary structures are more similar to each other (37%) than to most other integrin subunits (46, 47, 48) . Thus, regions specifically shared between and are likely to be critical for their association with CD63. Because distinct integrin subunit cytoplasmic domains sometimes exhibit diverse functions during both inside-out (40) and outside-in (5, 8) signaling events, we thought that the and cytoplasmic domains might play key roles in CD63 association. However, the integrin cytoplasmic domain was neither necessary nor sufficient for CD63 association, and -CD63 association occurred regardless of whether the or cytoplasmic domain was present. Thus, it appears that extracellular (or transmembrane) regions, rather than cytoplasmic domains, must be critical for CD63 association. Also, the integrin subunit might influence CD63 interactions. In preliminary experiments, anti-CD63 mAb failed to coprecipitate the subunit from cells where it was predominantly paired with the subunit, even though an -CD63 complex was readily detectable in the same cells.

The CD63 protein has been separately described as a platelet activation antigen and as a stage-specific melanoma membrane antigen, ME-491(41, 49) . It belongs to the TM4 (or tetraspans) family of proteins, which includes CD9, CD37, CD53, CD81 (TAPA-1), CD82 (C33/IA4 antigen), and some other proteins(17) . These all contain four putative transmembrane domains and show some similarity in their primary structures(50) . As we have confirmed, CD63 appears on the surface of most cultured cell lines at a moderate level(18) . In addition, it is incorporated into the membranes of different types of intracellular granules, including lysosomes(18, 39, 41) , endothelial Weibel-Palade bodies(51) , platelet-dense granules(52) , and the major histocompatibility complex class II compartment(53) . The CD63 molecule can be rapidly delivered to the cell surface after cells are treated (activated) with various stimuli(41, 54) .

Immunofluorescence staining of and showed that both were evenly distributed on the cell surface. Colocalization of CD63 and in cell footprints suggests that the cell-surface fraction of the CD63 antigen may be more likely to associate with integrins. However, we cannot exclude intracellular association because it is possible that conditions used for cell permeabilization might not be optimal for detection of integrins in intracellular granules. The CD63 protein and/or mRNA is present in many tissues(55) , with high expression in the kidney, especially in the glomerulus(56, 57) . The integrin is also expressed in kidney glomeruli(58) , and both and are widely expressed on many cell and tissue types (59, 60, 61, 62) , as well as on nearly all cultured adherent cell lines. Consistent with this, we found that -CD63 and -CD63 complexes could be detected in cells of different tissue origins (lymphoid, mesenchymal, epithelial). Thus, the possibility exists that integrin-CD63 association could be widespread and of general importance for cellular physiology.

Because we have not yet found a cell line lacking CD63, we have been unable to carry out definitive transfection experiments to address the functional implications of CD63- and CD63- associations. Nonetheless, we hypothesize that such associations might specifically affect signaling through and integrins and could be important for cell proliferation, migration, or cell-cell adhesion. The colocalization of CD63 with integrin in footprints left by cells on the different extracellular matrix substrates ( Fig. 9and preliminary results) suggests that the function(s) of the complexes could be related to the adhesive properties of integrins. In this regard, CD63 and other members of the TM4 protein family may modulate cell proliferation(63) , cell motility (64) , and cell-cell adhesion(31, 65) . Likewise, various integrins (including ) play key roles in regulating cell proliferation(24, 66) , cell migration(8, 67) , and cell-cell adhesion(68, 69) .()Another possibility is that CD63 association with and might modulate the recycling of these integrins. Notably, it has been reported that has a reduced rate of endocytic recycling compared to and integrins(70) .

In several experiments, a similar pattern of additional proteins was coprecipitated by antibodies to both CD63 and (e.g.Fig. 7). These results suggest that association of and integrins with CD63 could possibly occur within the context of a larger multiprotein complex so that instead of a direct interaction, these proteins are connected with one another via other proteins present in the complex. In this respect, our preliminary experiments show that other members of the TM4 family can interact with both CD63 and / integrins. Also, it is interesting to note that another member of the TM4 family, the CD9 antigen, was suggested to associate with the integrin on platelets (71) and with VLA-4 and VLA-5 integrins on leukocyte cell lines(72) .

In conclusion, we have identified novel protein-protein associations that are highly specific for a subset of integrins and highly reproducible in many experiments from multiple cell types. Awareness of these CD63- and CD63- interactions now provides the opportunity to gain new insights into the functional properties of , , and CD63.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM46526, the Dana-Farber/Sandoz Drug Discovery Program, and a fellowship from the Sanofi Foundation for Thrombosis Research (Paris) (to G. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dana-Farber Cancer Institute, Rm. M613, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3410; Fax: 617-632-2662.

The abbreviations used are: mAb, monoclonal antibody; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; FBS, fetal bovine serum.

G. Bazzoni and M. E. Hemler, submitted for publication.

J. Weitzman, A. Chen, and M. E. Hemler, submitted for publication.


ACKNOWLEDGEMENTS

We thank Drs. R. Juliano and A. Mercurio for integrin-transfected cell lines; Drs. John McDonald, V. Quaranta, and R. Hynes for polyclonal antisera; A. Mercurio for cDNA; Drs. K. Skubitz, K. Nieuwenhuis, and E. Wayner for monoclonal antibodies; and R. Pasqualini for help during the initial stages of hybridoma production.


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