J. Cell Biol.
© The Rockefeller University Press
0021-9525/97/12/1583/13 $2.00
Volume 139, Number 6, December 15, 1997 1583-1595

Muscle 1D Integrin Reinforces the Cytoskeleton-Matrix Link: Modulation of Integrin Adhesive Function by Alternative Splicing

Alexey M. Belkin,^* S. Francesco Retta, Olga Y. Pletjushkina,^§ Fiorella Balzac, Lorenzo Silengo, Reinhard Fassler, Victor E. Koteliansky,^¶ Keith Burridge,^* and Guido Tarone^**

^* Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, North Carolina 27599;  Department of Biology, Genetics and Medical Chemistry, University of Torino, 10126 Torino, Italy; ^§ Belozersky Institute of Physico-Chemical Biology, Moscow State University, 117311 Moscow, Russia;  Max-Planck-Institute for Biochemistry, Martinsried D-82152, Germany; ^¶ Biogen Inc., Cambridge, Massachusetts 02142; ^** Department of Psychology, University of Rome, Italy; and  Institute of Biology, University of Palermo, 90133 Palermo, Italy

Abstract

Materials and Methods

Results

Discussion

Footnotes

Acknowledgements

References

Abstract

INTEGRINS are a large family of transmembrane heterodimeric receptors that play a key role in cell adhesion to extracellular matrix (Hynes, 1992). Integrin receptors serve a dual purpose, linking extracellular matrix to the actin cytoskeleton and providing bidirectional transmission of signals between the extracellular matrix and the cytoplasm (Schwartz et al., 1995; Yamada and Miyamoto, 1995; Burridge and Chrzanowska-Wodnicka, 1996). At least two major actin-binding proteins, talin and -actinin, are thought to interact directly with the cytoplasmic domain of several  subunits, providing a link to the actin cytoskeleton (Horwitz et al., 1986; Otey et al., 1990; Hemler et al., 1994). Among integrins, 1 is typically the most abundant and ubiquitously expressed subunit associated with a number of  subunits to form distinct heterodimers. These interact with a variety of extracellular matrix and cell adhesion molecules (Hynes, 1992). The entire structure of the 1 integrin cytoplasmic domain is critical for integrin- cytoskeleton interaction (Hayashi et al., 1990; LaFlamme et al., 1992; Reszka et al., 1992; Ylanne et al., 1993; Lewis and Schwartz, 1995).

Integrin functions within the cell can be regulated at different levels. These include cell type-specific biosynthesis of certain integrin heterodimers, maturation and processing of the receptors, as well as their transport to the cell surface (Hynes, 1992). Another level of control of integrin function is through regulation of the ligand-binding affinity of integrins on the cell surface. This type of regulation involves conformational changes within integrins. The conformational state of the extracellular domains (activation) of integrins is regulated via their cytoplasmic tails and is referred to as inside-out signaling (Ginsberg et al., 1992; O'Toole et al., 1994; Schwartz et al., 1995). Thus, deletions or mutations of certain residues in the cytoplasmic domains of  and  subunits can either increase or inhibit the ligand-binding activity of integrin receptors (Takada et al., 1992; O'Toole et al., 1994, 1995). The activation state of integrins can also be controlled by some lipid metabolites (Hermanowski-Vosatka et al., 1992; Smyth et al., 1993) and small GTP-binding proteins (Zhang et al., 1996; Hughes et al., 1997). Finally, functional properties of integrin receptors can be modulated by alternative splicing involving their cytoplasmic tails.

So far, four cytoplasmic domain variants of the 1 integrin subunit have been described. Besides the major 1A isoform, characteristic for all known cell types except red blood cells and terminally differentiated striated muscles, two minor cytoplasmic domain isoforms of 1 integrin, 1B and 1C, have been characterized (Altruda et al., 1990; Languino and Ruoslahti, 1992). Although their functions remain uncertain, it has been speculated that 1B can serve as a negative regulator of cell adhesion during development, whereas 1C can strongly inhibit cell growth (Balzac et al., 1993, 1994; Meredith et al., 1995). The alternatively spliced sequences of 1B and 1C have no homology to the major 1A isoform and are unable to localize to cell-matrix adhesion sites apparently because of impaired interaction with the actin cytoskeleton (Balzac et al., 1993; Meredith et al., 1995). Interestingly, 1B and 1C variants have been found only in humans, whereas the fourth 1 isoform, 1D, is highly conserved at least throughout vertebrate evolution, suggesting an important role for this muscle-specific variant (van der Flier et al., 1995; Zhidkova et al., 1995; Baudoin et al., 1996; Belkin et al., 1996).

1 integrin is localized at junctional structures of striated muscles (Bozyczko et al., 1989). Expression of the 1 integrin subunit as well as the ligand occupation of 1-containing heterodimers is essential for myodifferentiation and the formation of sarcomeric cytoarchitecture (Menko and Boettiger, 1987; Volk et al., 1990). Integrin-mediated cytoskeleton-matrix linkage has to be distinct in muscle cells because of high tensile forces transmitted across the membrane and enhanced stability of muscle adhesive structures. This implies a modified function for 1 integrin in muscles. This function is now attributed primarily to 1D cytoplasmic domain variant, which is a major 1 isoform that completely displaces 1A integrin in differentiated striated muscles (Belkin et al., 1996). Its cytoplasmic domain is highly homologous to that of 1A, including conservation of both NPXY motifs involved in the regulation of ligand-binding affinity (Tamkun et al., 1986; Argraves et al., 1987; Zhidkova et al., 1995; van der Flier et al., 1995). 1D accumulates at all major cell-matrix adhesion sites both in skeletal muscle fibers and cardiomyocytes. 71D is a predominant integrin in adult skeletal and heart muscle tissues (Belkin et al., 1996). However, other  subunits, including 5 and 6A, can pair with 1D in developing heart muscle (Brancaccio et al., 1997). The data obtained so far lead to the suggestion that 1D integrin plays a crucial role in linking the subsarcolemmal cytoskeleton to the surrounding extracellular matrix in muscle tissues (Belkin et al., 1996; Fassler et al., 1996).

Upon transfection into nonmuscle cells, 1D is targeted to focal adhesions, proving that the muscle-specific isoform of 1 integrin is able to interact with the nonmuscle cytoskeleton as well (Belkin et al., 1996). To get an insight in the functional properties of this integrin, we have expressed human 1D and 1A cytoplasmic domain isoforms in CHO cells and the mouse GD25 cell line. GD25 cells lack endogenous 1 integrin as a consequence of gene inactivation (Wennerberg et al., 1996). Here we report that the expression of 1D integrin in nonmuscle cells leads to a conversion of cellular phenotype. The observed alterations in cell morphology, inhibition of spreading and motility, as well as an increase in the ligand-binding affinity, fibronectin matrix assembly and contractility, are caused by an enhanced association of 1D integrin with both the actin cytoskeleton and extracellular matrix ligands. 1D-mediated enhancement of actin-membrane attachment is, at least in part, due to a higher affinity interaction of this integrin with the focal adhesion protein, talin. The altered structure of the 1D cytoplasmic domain causes a conformational change of its ectodomain via inside-out signaling mechanisms, leading to activation of ligand binding. Reinforcement of the cytoskeleton-matrix association by 1D reflects a key role for this integrin as a cytoskeleton-matrix linker, strengthening adhesive structures in muscle tissues.

Materials and Methods

Antibodies and Reagents

The following antibodies against 1 integrin were used in this study: TS2/ 16 mAb to human 1 subunit, which activates ligand binding by 1-containing heterodimers was a gift from Dr. M. Hemler (Dana-Farber Cancer Institute, Boston, MA) (Hemler et al., 1984; Arroyo et al., 1992); function-blocking P4C10 mAb against human 1 integrin (Carter et al., 1990) was from GIBCO BRL (Gaithersburg, MD); 102DF5 mAb against human 1 integrin (Ylanne and Virtanen, 1989); 12G10 mAb, which reacts with activated (high affinity conformation for ligand binding) human 1 (Mould et al., 1995); 9EG7 mAb reacting with ligand-, Mg²⁺-, Mn²⁺-induced, Ca²⁺- inhibited epitope on human 1 integrin subunit (Bazzoni et al., 1995); A1A5 mAb against human 1 integrin (Hemler et al., 1984), conjugated with fluorescein and rabbit polyclonal antibody against human 1 integrin (Belkin et al., 1990). 7E2 mAb against hamster 1 integrin and inhibitory PB1 mAb against intact hamster 51 heterodimer were generous gifts from Dr. R. Juliano (University of North Carolina, Chapel Hill, NC) (Brown and Juliano, 1985, 1988). Isoform-specific antibodies against 1A and 1D integrins were described earlier (Belkin et al., 1996).

Blocking anti-mouse v H9.2B8 mAb (Moulder et al., 1991) was obtained from PharMingen (San Diego, CA). Rabbit polyclonal antibodies against v, 3, and 5 cytoplasmic domains were described earlier (Defilippi et al., 1992; Balzac et al., 1994). mAb 8d4 against talin was obtained from Sigma Chemical Co.(St. Louis, MO) and mAb 1682 against -actinin was from Chemicon International, Inc. (Temecula, CA). Rabbit polyclonal antibody against platelet myosin II, cross-reacting with nonmuscle myosin, was a gift from Dr. R. Adelstein (National Institutes of Health, Bethesda, MD). Rabbit polyclonal antibody against human plasma fibronectin (Fn) was provided by Dr. L.B. Chen (Dana-Farber Cancer Institute).

Human plasma Fn was from GIBCO BRL. Recombinant, 12-kD cell-binding Fn fragment corresponded to the tenth Arg-Gly-Asp-containing (cell-binding), type III Fn repeat. Cytochalasin D was from Sigma Chemical Co.. Digitonin was purchased from Sigma Chemical Co. and purified by dissolving in water, filtering, and lyophilization before use. ³⁵S-Translabel and methionine- and cysteine-free medium were from ICN Biomedicals Inc. (Costa Mesa, CA). Na¹²⁵I and [³²P]orthophosphate were from Dupont-NEN (Boston, MA).

Expression Constructs, Transfection, and Cell Culture

Human full-length cDNAs encoding 1A integrin or 1D integrin in SV40-based expression vector pECE (Ellis et al., 1986), were transfected into CHO cells or 1-minus GD25 cell line (Wennerberg et al., 1996), and transfectants were selected as described (Belkin et al., 1996). More than 95% of cells in each population expressed human 1 integrin; the expression levels of the transfected 1A and 1D integrins were very similar and comparable to the level of the endogenous hamster 1 integrin subunit in CHO cells and close to the levels of the endogenous v and 3 integrins in GD25 transfectants. CHO transfectants were cultured in Ham's F12 medium with 10% FBS, and GD25 transfectants were cultured in DME plus 10% FBS.

Morphological Analysis and Spreading

For analysis of cell phenotype, CHO transfectants were plated and cultured for 1 d on Fn-coated dishes. Phase-contrast photographs of live 1A-CHO and 1D-CHO cells were taken on an inverted microscope. The outlines of randomly chosen cells not in contact with other cells were analyzed by the computer Tracer V1.0 software (Dunn and Brown, 1986). The spread area, cell perimeter, and two morphometric parameters of cell shape, cell dispersion and elongation, were calculated as characteristics of cell spreading and polarization. For analysis of the time course of spreading, 1A- and 1D-transfected CHO and GD25 cells were plated in serum-free medium on Fn, laminin, vitronectin, or on immobilized mAb TS2/16 to human 1 integrin (Balzac et al., 1994; Belkin et al., 1996). After specific periods of time, cells were fixed with formaldehyde, stained with Coomassie brilliant blue (Balzac et al., 1994), and then photographed.

Measurements of the Ligand-Binding Affinity

The binding of the ¹²⁵I-labeled Fn(III)10 fragment to 1A-CHO, 1D-CHO, 1A-GD25, and 1D-GD25 cells in suspension was quantified as described (O'Toole et al., 1990; Wu et al., 1995). Since CHO cells express the endogenous 51, and 1-minus GD25 cells express v3 as a major Fn-binding integrin (Wennerberg et al., 1996), inhibitory mAbs PB1 against hamster 51 or H9.2B8 against mouse v were used for CHO and GD25 cells, respectively. In some experiments, blocking P4C10 mAb against human 1 integrin was used in combination with either PB1 mAb (for CHO cells) or H9.2B8 mAb (for GD25 cells). Cells (0.2 ml of 5 × 10⁶ cells/ml) in Tyrode's buffer were incubated with specified concentrations of ¹²⁵I-labeled Fn(III)10 fragment (sp act 0.12 mCi/nM) for 30 min at 37°C either alone or in the presence of 10 µg/ml of purified TS2/16 mAb, which activates human 1 integrins. Coincubation with an excess of unlabeled Fn(III)10 fragment (0.5 mg/ml) was used to determine and subtract the nonspecific background binding. 50-µl aliquots were layered on 0.3 ml of 20% sucrose in Tyrode's buffer and centrifuged for 3 min at 12,000 rpm. Radioactivity associated with the cell pellet was determined in a gamma counter.

Flow Cytometry

Cell surface expression of the transfected human 1A or 1D integrins in CHO and GD25 transfectants was assessed with 102DF5 and TS2/16 mAbs, whose binding to the 1 subunit is conformation independent. Their expression levels were compared to those of the endogenous hamster 1 integrin (examined with 7E2 mAb). 12G10 mAb reacting with activated human 1 integrin subunit (Mould et al., 1995) and conformation-specific 9EG7 anti-human 1 mAb (Bazzoni et al., 1995) were used either in the absence of Mn²⁺ ions or in the presence of 1 mM Mn²⁺. Fluorescein- labeled, affinity-purified donkey anti-mouse IgG (Chemicon International, Inc.) was used as secondary antibody.

Fn Matrix Assembly Assays

1A-CHO, 1D-CHO, as well as 1A-GD25 and 1D-GD25 cells did not assemble Fn matrix well when confluent cell monolayers were cultured in growth medium containing 1% FBS for 2 d. To boost the formation of Fn matrix, exogenous human plasma Fn was added at 200 nM concentration for 2 d to the confluent cell monolayers grown on glass coverslips. Inhibitory mAbs PB1 against hamster 51 and H9.2B8 against mouse v were used to block Fn matrix assembly by the endogenous Fn-binding integrins in CHO and GD25 cells, respectively. Activating TS2/16 and inhibitory P4C10 mAbs were used for the transfected human 1A and 1D integrins. After 2 d, cell monolayers were fixed and stained with anti-Fn antibody. Stained cells were observed using a Zeiss epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY) and representative fields were photographed using equal exposure lengths on Kodak T-Max 400 film (Eastman Kodak, Rochester, NY).

To quantitate Fn incorporation into deoxycholate-insoluble matrix, confluent 1A-CHO, 1D-CHO, 1A-GD25, and 1D-GD25 cultures were incubated for 2 d with 100, 200, or 300 nM of ¹²⁵I-labeled Fn (sp act 0.08 mCi/nM) in growth medium containing 1% FBS and blocking and activating mAbs as specified above. Deoxycholate-insoluble fraction was obtained from cell monolayers as described (McKeown-Longo and Mosher, 1985; Wu et al., 1993, 1995).¹²⁵I-labeled Fn incorporated into the deoxycholate-insoluble extracellular matrix was analyzed by reducing SDS-PAGE (6% running gel) and autoradiography. Iodinated Fn bands were cut out and counted in a gamma counter.

Migration Assays

Migratory properties of 1A-CHO, 1D-CHO, 1A-GD25, and 1D-GD25 cells were examined by a wound closure assay and time lapse videomicroscopy. For the wound closure assay, confluent cell monolayers grown on Fn-coated coverslips were wounded by dragging a sterile 1-mm pipette tip across the monolayer to create cell-free fields (Romer et al., 1994). 2 d later, glass coverslips were fixed with formaldehyde, stained with Coomassie blue, and then photographed.

For time lapse videomicroscopy, 1A- and 1D-transfected CHO and GD25 cells were plated on plastic dishes coated with 10 µg/ml of human plasma Fn. Five to six cells were scanned per field in eight different fields, every 20 min for 4 h. The displacement of the cell center as a function of time was calculated for each cell using nonoverlapping time intervals. To block the endogenous Fn receptors, PB1 mAb was used for CHO transfectants and H9.2B8 mAb for GD25 transfectants. TS2/16 was used as the activating mAb and P4C10 as the blocking mAb for the transfected human 1A and 1D integrins.

Analysis of the Association of 1A and 1D Integrins with  Subunits

1D-CHO cells as well as 1A- and 1D-GD25 transfectants were lysed in buffer containing 1% Triton X-100 in 50 mM TrisCl, 150 mM NaCl, pH 7.5, and protease inhibitors. Each lysate was clarified by centrifugation, divided into four equal parts, and the transfected human 1 integrins were immunoprecipitated using TS2/16 mAb, whereas 3, 5, and v subunits were immunoprecipitated with antibodies against cytoplasmic domains of these integrins. The resulting immunoprecipitates were run on 10% gel and blots were probed with the isoform-specific antibodies against 1A or 1D integrins.

Localization of the Transfected 1A and 1D Integrins and the Endogenous 1A and v Subunits and Analysis of their Association with the Actin Cytoskeleton

To localize the transfected and the endogenous 1 integrins, as well as the endogenous v integrins in the transfectants, cells cultured on Fn-coated coverslips were fixed with formaldehyde and permeabilized with 0.5% Triton X-100 in PBS. CHO transfectants were costained with fluorescein-labeled A1A5 mAb to human 1 and rhodamine-labeled 7E2 mAb to hamster 1 integrin. GD25 cells were double stained with mouse fluorescein-labeled A1A5 mAb and rabbit anti-v antibody followed by rhodamine-labeled donkey anti-rabbit antibody (Chemicon International Inc.). Stained cells were observed using epifluorescence with a Zeiss Axiophot microscope and photographed using Kodak T-Max 400 film.

To study whether the solubility of integrins in digitonin correlates with their cytoskeletal association, ³⁵S-labeled 1A-CHO and 1D-CHO cells, either untreated or treated for 1 h with 10 µM of cytochalasin D, were fractionated into soluble and cytoskeleton-associated fractions by sequential extraction at 4°C with 0.1% digitonin in 50 mM Pipes, 1 mM MgCl₂, 1 mM EGTA, 1 mM EDTA, pH 6.9, and then with radioimmunoprecipitation assay (RIPA) buffer (50 mM TrisCl, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, and 0.1% SDS, pH 7.5). Both buffers contained 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 0.5 mM PMSF as protease inhibitors. The transfected 1A and 1D integrins were immunoprecipitated from digitonin- and RIPA-soluble fractions using TS2/16 mAb. ³⁵S-labeled 1 immunoprecipitates were run on 10% gels and analyzed by autoradiography.

To assess the association of the transfected and the endogenous 1 integrin subunits and the endogenous v integrins with the actin cytoskeleton, ³⁵S-labeled CHO and GD25 transfectants were sequentially extracted with digitonin and RIPA buffers as described above. Immunoprecipitation of the transfected human 1A and 1D integrins from both cellular fractions was performed with TS2/16 mAb. 7E2 mAb antibody was used for the endogenous hamster 1A integrin. Rabbit anti-v antibody was used to immunoprecipitate the endogenous v3/v5 integrins from GD25 transfectants. ³⁵S-labeled 1 and v immunoprecipitates were analyzed by SDS-PAGE on 10% gels and subsequent autoradiography.

Analysis of the Association of 1A and 1D Integrins with Talin and -Actinin by Coimmunoprecipitation

To compare the association of 1A and 1D integrins with talin and -actinin, 5 × 10⁶ transfected cells were incubated in suspension with 10 µg of either purified TS2/16 mAb, 12G10 mAb, or 7E2 mAb at 4°C for 30 min on a rotator. In the case of 12G10 mAb, cells were either preincubated for 5 min with 1 mM Mn²⁺ or used in the absence of Mn²⁺. Cells were centrifuged (1,000 rpm, 3 min) and the pellets were extracted for 3 min on ice with buffer containing 0.5% digitonin in 50 mM Pipes, 1 mM MgCl₂, 1 mM EGTA, 1 mM EDTA, pH 6.9, with 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 0.5 mM PMSF. Under these conditions ~80-90% of cellular 1 integrins was extracted. Cell extracts were centrifuged (12,000 rpm, 30 min, 4°C) and the resulting supernatants incubated at 4°C for 45 min with donkey anti-mouse IgG immobilized on protein A-Sepharose beads. Immunoprecipitates were washed with the same buffer, boiled in SDS sample buffer, and then run on 10% gels. Proteins were transferred onto Immobilon membranes (Millipore Corp., Bedford, MA) and blotted with either rabbit polyclonal antibody to human 1 integrin, 8d4 mAb against talin, or 1682 mAb against -actinin. To verify the equal amount of 1A and 1D isoforms in the immunoprecipitates, the blots were stripped and reprobed with the isoform-specific antibodies against 1A and 1D (Belkin et al., 1996).

Interaction of Talin and -Actinin with 1A and 1D Cytoplasmic Domain Peptides

Talin was purified from human platelets as described earlier (Collier and Wang, 1982). -Actinin purification from chicken gizzards was performed as described (Otey et al., 1990). Talin and -actinin were iodinated using ¹²⁵I and Iodobeads (Pierce Chemical Co., Rockford, IL). The proteins were labeled to a specific activity of 1.2 × 10⁶ cpm/µg for talin and 7.5 × 10⁶ cpm/µg for -actinin.

Full-length cytoplasmic domain peptides of 1A and 1D integrins (Belkin et al., 1996) were iodinated using Iodogen method. Both peptides were initially tested for their binding to the microtiter wells in the range of 1-150 µM concentrations in buffer containing 50 mM TrisCl, 150 mM NaCl, pH 7.5. Since they bound similarly to the wells, saturating 50 µM concentration of 1A and 1D peptides was used in subsequent experiments to immobilize them on plastic 96-well microtiter plates for 1 h at 37°C. After blocking with 2% BSA in 50 mM TrisCl, 150 mM NaCl, wells with the bound peptides were incubated with 1 nM of ¹²⁵I-talin or ¹²⁵I- -actinin and 1 nM to 1 µM concentrations of unlabeled talin or -actinin in the same buffer with 0.1% BSA for 4 h at 37°C. After the incubations, wells were washed three times with the same buffer and bound radioactivity was measured in a gamma counter. Nonspecific background was determined and subtracted for talin and -actinin binding to BSA-coated wells.

Measurements of Cellular Contractility and Myosin Light Chain Phosphorylation

Silicone rubber substrata for assessing cellular contractility were made as described previously (Harris et al., 1980; Danowski, 1989). The UV glow discharge polymerization was used in combination with gold-palladium coating (Chrzanowska-Wodnicka and Burridge, 1996). 5- and 12-s polymerization was used for CHO and GD25 transfectants, respectively. Cells were plated on the cross-linked rubber substrata in growth medium with 10% FBS and photographed on the next day.

Possible changes in myosin light chain phosphorylation were examined as described (Chrzanowska-Wodnicka and Burridge, 1996). Briefly, subconfluent 1A-CHO and 1D-CHO cells cultured on Fn-coated, 35-mm dishes, were labeled for 4 h with 20 µCi/ml of ³⁵S-Translabel and 100 µCi/ml of [³²P]orthophosphate in phosphate-free medium. Cells were washed with PBS and equal amounts of material, as judged by ³⁵S-incorporated radioactivity, were taken for immunoprecipitation with antimyosin antibody, followed by protein A-Sepharose beads. Immunoprecipitates were washed, boiled in SDS sample buffer, and then run on 15% polyacrylamide gel. Phosphorylated myosin light chain bands were visualized by autoradiography using three sheets of aluminum foil to block traces of ³⁵S radiation.

Results

1D Integrin Alters Cell Morphology and Inhibits Spreading

The levels of surface expression of the transfected human 1A and 1D, measured with mAbs 102DF5 and TS2/16, were very close to each other in both CHO and GD25 transfectants (Table I). They were also similar to the levels of the endogenous 1A in CHO transfectants and v integrins in GD25 cells (see Fig. 9, I and J). No difference in association of the transfected 1A and 1D with endogenous  subunits was found in the two types of transfectants (Fig. 7).

Table I. Expression Levels and Activation States of Transfected Human 1A and 1D Integrins in CHO and GD25 Transfectants [View Table]

To determine possible effects of 1A and 1D integrin expression on cell morphology, CHO transfectants were grown on Fn for 1 d (Fig. 1, A and B). 1D-CHO cells appear more rounded with fewer cytoplasmic extensions at their periphery than their 1A-transfected counterparts. Statistical analysis of the cell shape was performed for randomly chosen 1A-CHO and 1D-CHO cells (Dunn and Brown, 1986). This demonstrated that spread areas were similar for 1A and 1D transfectants, but the average cell perimeter was ~20% lower for 1D-CHO cells (Table II). Two cell shape parameters, dispersion and elongation, representing measures of cell multipolarity and bipolarity, respectively, were significantly lower for 1D transfectants. These data indicated that pseudopodial activity and cell polarization were reduced in CHO cells expressing 1D integrin.

Table II. Cell Shape Parameters of 1A-CHO and 1D-CHO Cells [View Table]

To analyze the time course of spreading, 1A and 1D transfectants were plated on purified Fn (Fig. 1, C-F). Spreading of CHO cells expressing 1D integrin on Fn was significantly delayed compared with 1A transfectants. When both 1A and 1D transfectants were plated on TS2/16 mAb specific for human 1 integrin, 1D-CHO were less spread than 1A-CHO cells (Fig. 1, G and H). Notably, 1D transfectants also spread more slowly on laminin, vitronectin, and 7E2 mAb against hamster 1 integrin (data not shown), indicating that inhibition of spreading was not limited to a certain type of substrate. Likewise, spreading of 1D-GD25 cells on both Fn and vitronectin was significantly delayed compared with the spreading of 1A-GD25 cells (Fig. 1, I-L). These observations suggested that retardation of spreading was a general phenomenon of 1D expression.

Constitutive Activation of the 1D Integrin Ectodomain Increases Ligand Binding

To characterize the affinity states of 1A and 1D integrins, the binding of the Fn(III)10 fragment to 1A- and 1D-transfected CHO and GD25 cells in suspension was examined. In the presence of function-blocking mAbs PB1 and H9.2B8, which inhibited endogenous Fn-binding hamster 51 and mouse v3 integrins, respectively, 1D transfectants exhibited a significant increase in binding the soluble ligand compared with 1A-expressing cells (Fig. 2, A and B). TS2/16 mAb, which activates human 1 integrin, markedly increased Fn(III)10 fragment binding to both 1A-CHO and 1A-GD25 cells, whereas the ligand binding by 1D transfectants in the presence of TS2/16 mAb remained unaffected (Fig. 2, A and B). Blocking P4C10 mAb against human 1 integrin completely abolished the binding of Fn(III)10 fragment to all types of transfectants (Fig. 2, A and B), therefore proving its specificity.

To further assess the difference in the conformation of the ectodomains of the transfected 1A and 1D, a flow cytometry analysis was used with mAb 12G10. This antibody recognizes preferentially a Mn²⁺- and ligand-induced conformation of the human 1 integrin subunit (Mould et al., 1995; Mould, 1996). The amount of 12G10 mAb bound to the cell surface was significantly higher for 1D-expressing CHO and GD25 cells, than for 1A-expressing cells. Moreover, the binding of 12G10 mAb was almost unchanged for 1D-CHO and 1D-GD25 cells in the presence of Mn²⁺, whereas the 12G10 mAb binding to both types of 1A transfectants was dramatically increased by Mn²⁺ (Table I). Another mAb, 9EG7, whose binding to human 1 integrin is stimulated by ligands and Mn²⁺, but inhibited by Ca²⁺ (Bazzoni et al., 1995), also reacted more strongly with 1D-GD25 compared with 1A-GD25 cells (Table I). Together, these results showed that in 1D- expressing cells in suspension the majority (~77-88%) of 1D integrins were constitutively activated on the cell surface, whereas only ~27-44% of the transfected 1A integrins were present in the activated state.

1D Enhances Fn Matrix Assembly

Fn biosynthesis and secretion levels in CHO and GD25 cells were not altered by 1A or 1D expression and appeared to be identical for each pair of transfectants (data not shown). Fn matrix assembly by 1A- and 1D-transfected cells was analyzed with the exogenous Fn by both immunofluorescence and measurements of ¹²⁵I-Fn incorporation into deoxycholate-insoluble matrix (McKeown-Longo and Mosher, 1985; Wu et al., 1993, 1995). Using different concentrations of the exogenous Fn, we consistently observed an enhanced Fn matrix assembly by 1D transfectants compared to their 1A-expressing counterparts (Fig. 3 A). In our subsequent experiments we used 200 nM of exogenous Fn for matrix assembly studies with the transfectants. There was no difference in Fn matrix assembly between 1A and 1D transfectants in the presence of blocking P4C10 mAb against human 1 integrin, showing that the observed effects can be ascribed specifically to the expressed 1D (Fig. 3 B).

In the presence of blocking anti-hamster 51 integrin mAb PB1 (for CHO cells) or inhibitory anti-mouse v integrin mAb H9.2B8 (for GD25 cells), 1D transfectants assembled more abundant meshwork of Fn fibrils, than 1A-expressing counterparts (Fig. 4, A, B, E, F, and I, a, b, e, and f). Activating mAb TS2/16 significantly increased Fn matrix assembly by 1A-CHO and 1A-GD25 cells, but did not change the levels of assembly for 1D-transfected CHO and GD25 cells (Fig. 4, C, D, G, H, and I, c, d, g, and h). Quantitation of ¹²⁵I-Fn incorporated into the extracellular matrix showed a five- to sixfold increase in Fn assembly by 1D compared with 1A integrin in CHO and GD25 cells (Fig. 4, I and J). Interestingly, whereas mAb TS2/16 caused two- to threefold increase in Fn matrix assembly by 1A integrin for both types of transfectants, these levels appeared still much lower than those exhibited by 1D integrin (Fig. 4, I and J).

1D Integrin Inhibits Cell Migration

Initially, migratory properties of 1A- and 1D-transfected CHO and GD25 cells were analyzed using a monolayer wounding assay. In 2-d wound closure experiments, 1D transfectants exhibited significantly slower migration rates than 1A-expressing cells (Fig. 5, A-D). When migratory behavior of 1A and 1D transfectants was analyzed on Fn substrate by time lapse videomicroscopy, 1D-expressing cells migrated three- to fourfold slower than 1A-transfected cells. Again, blocking P4C10 mAb against human 1 integrin completely abolished the effects of 1D on cell migration (Fig. 5 E).

Migration of 1A- and 1D-expressing cells was also examined on Fn by time lapse videomicroscopy in the presence of blocking mAbs PB1 for CHO and H9.2B8 for GD25 transfectants (Fig. 6). In both cases when the endogenous Fn-binding integrins were inhibited, the mean cell speed of 1D transfectants appeared to be drastically reduced compared to that of 1A-expressing cells. Activating TS2/16 mAb significantly decreased migration mediated by 1A integrin but did not alter the migratory behavior of 1D-expressing cells. The migration rates of 1A-transfected CHO and GD25 cells treated with TS2/16 mAb still exceeded substantially the migration rates of 1D-transfectants.

Interaction of 1A and 1D Integrins with  Subunits in CHO and GD25 Transfectants

Many of the observed properties of 1D including enhanced ligand binding, elevated Fn matrix assembly, and decreased cell motility could be explained by different mode of association of 1A and 1D integrins with  subunits in the transfected cells. Therefore, we examined  subunit association for 1A and 1D in CHO and GD25 transfectants. Among various 1-associated  subunits, 3, 5, and very small amount of v were detected in both types of transfectants by immunoprecipitation (Fig. 7). Immunoblotting of the corresponding immunoprecipitates from 1D-CHO (Fig. 7, A and B), 1A-GD25 (Fig. 7, C and D), and 1D-GD25 (Fig. 7, E and F) cells showed that equal amounts of 1D and 1A integrins were associated with 3 and 5 subunits in CHO and GD25 transfectants.

Cytoskeletal Association of 1A and 1D Integrins Correlates with Their Insolubility in Digitonin

To determine whether there is a difference in cytoskeletal association between 1A and 1D integrins, we designed a method of sequential extraction using digitonin and RIPA buffers for ³⁵S-labeled cell cultures, followed by integrin immunoprecipitation from both cellular fractions. To test whether integrin insolubility in digitonin is determined by the mode of integrin-cytoskeleton association, we compared 1 integrin immunoprecipitates from digitonin and RIPA fractions of untreated and cytochalasin D-treated 1A- and 1D-transfected CHO cells (Fig. 8). Treatment of cultured cells with cytochalasin D shifted almost all 1A and the majority of 1D to the digitonin-soluble fraction. These experiments demonstrated that insolubility of 1 integrins in digitonin depends on their association with the actin cytoskeleton.

1D Integrin Displaces the Endogenous 1A and v Subunits from Focal Adhesions and Associates Strongly with the Digitonin-insoluble Cytoskeleton

Since 1D and 1A integrins have structurally different cytoplasmic domains, and the two types of transfectants displayed dissimilar phenotypes, we next attempted to compare cytoskeletal interactions of the 1A and 1D isoforms. To localize the transfected and the endogenous 1 integrins in CHO transfectants, cells grown on Fn were double stained with anti-human 1 and anti-hamster 1 mAbs. In 1A-CHO cells, both the transfected and the endogenous 1A subunits colocalized at focal adhesions (Fig. 9, A and C). 1D integrin was prominently localized at focal adhesions of 1D-CHO cells. Surprisingly, no endogenous 1A integrin was detected in focal adhesions of 1D transfectants grown on Fn or other extracellular matrix proteins (Fig. 9, B and D; and data not shown). Similarly, both the transfected 1A and 1D integrins were targeted to focal adhesions of GD25 transfectants on Fn (Fig. 9, E and F). The endogenous v subunit of Fn-binding v3 integrin in 1A-GD25 cells was at least partially colocalized with 1A at sites of cell-matrix contact (v does not pair with 1 integrins in GD25 cells; Wennerberg et al., 1996). In contrast, 1D displaced the endogenous v integrins from focal adhesions (Fig. 9, G and H). Therefore, the displacement of 1A and v integrins from focal adhesions by expressed 1D was a general phenomenon, suggesting a considerably stronger association of 1D integrin with the actin cytoskeleton.

To define biochemically the modes of 1A and 1D interaction with the cytoskeleton, both the transfected 1A and 1D, as well as the endogenous 1A and v integrins were immunoprecipitated from soluble and cytoskeleton-associated fractions of ³⁵S-labeled transfectants grown on Fn (Fig. 9, I and J). In 1A-CHO cells, the transfected 1A was equally distributed between the soluble and the cytoskeletal fractions, wheras the majority of the endogenous hamster 1A subunit was associated with the cytoskeleton. In contrast, 1D was found exclusively in the cytoskeletal fraction, whereas almost all the endogenous 1A integrin was present in the soluble fraction of 1D-CHO cells, displaced from the cytoskeleton (Fig. 9 I). Cytoskeletal associations of the transfected 1A and 1D integrins in GD25 transfectants were similar to those observed in CHO transfectants. Again, much of the transfected 1A integrin was digitonin soluble. However, the majority of 1D integrin was digitonin insoluble, whereas most of the endogenous v3/v5 integrins were displaced from their association with the cytoskeleton by the transfected 1D (Fig. 9 J).

Differential Interaction of 1A and 1D Integrins with Talin and -Actinin

At least two cytoskeletal proteins, talin and -actinin, are known to interact in vitro with the cytoplasmic domain of 1A integrin (Horwitz et al., 1986; Otey et al., 1990). To identify cytoskeletal proteins, associated preferentially with the 1D integrin subunit in vivo, antibody clust