ABSTRACT A purified cell surface receptor protein for lam- inin (Mr = 70,000) isolated from mouse fibrosarcoma cells binds to actin with specificity and high ...
Proc. Nati. Acad. Sci. USA Vol. 80, pp. 5927-5930, October 1983
Cell Biology
Connectin: Cell surface protein that binds both laminin and actin (extracellular matrix/cytoskeleton/transmembrane signaling)
SUSAN S. BROWN*, HERBERT L. MALINOFFt,
AND
MAX S. WICHAt
*Department of Anatomy and Cell Biology and tDivision of Hematology/Oncology, Department of Internal Medicine, Simpson Memorial Institute, The University of Michigan Medical School, Ann Arbor, Michigan 48109
Communicated by James V. Neel, July 15, 1983
ABSTRACT A purified cell surface receptor protein for laminin (Mr = 70,000) isolated from mouse fibrosarcoma cells binds to actin with specificity and high affinity. This binding was demonstrated both by cosedimentation of the receptor with actin and binding of the receptor to actin immobilized on nitrocellulose filters. Specificity was demonstrated by displacement of 'S-labeled receptor by unlabeled receptor. Scatchard analysis of receptor binding to actin yielded a Kd of 6 x 10-7 M. The receptor was observed to reduce the viscosity of actin filaments. It also caused the formation of bundles of parallel filaments. This observation and the stoichiometry of binding suggest that the receptor binds along the sides of actin filaments. Based on the ability of this receptor to bind both extracellular laminin and intracellular actin, we have named this protein "connectin." Connectin may be an example of a transmembrane protein that is capable of mediating the interaction of a cell with its extracellular matrix. It is now clear that the extracellular matrix influences cell locomotion, growth, and differentiation (1, 2). Although it has been postulated that these effects may be mediated by specific interactions between components of this matrix and the cell surface, the mechanisms of these interactions are unknown (3). There is also evidence that such events at the external cell surface are accompanied by the rearrangement of actin within the
cell (4, 5). We and others recently have found that a variety of cells have the ability to specifically bind the basement membrane glycoprotein laminin to their surface (6-8). Using laminin affinity chromatography, we have isolated a protein from murine fibrosarcoma cells that on reduced gels appears as a single band (or doublet) with a Mr of =70,000. This protein retains the ability to specifically bind laminin with high affinity (Kd = 2 x 10' M for laminin) (9). By iodinating intact cells, we demonstrated that this protein is present on the cell surface (9). Recently, Rao et al. (10) have isolated a protein from BL6 murine melanoma cells with similar molecular weight and laminin-binding characteristics. We now report that the fibrosarcoma cell surface laminin receptor protein also has the ability to bind actin with specificity and high affinity (Kd = 6 x 10-7 M). We postulate that this protein may be a transmembrane linker that is capable of connecting the extracellular matrix protein laminin with the cytoskeletal protein actin. Based on this function, we have named this protein "connectin." The existence of membrane proteins such as connectin may provide a mechanism for the modulation of cell behavior by the extracellular matrix. MATERIALS AND METHODS Proteins. Connectin was isolated from the Np subline of murine fibrosarcoma cells by laminin affinity chromatography as The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
- connectin I0
0
0
+ connectin 15
30 Time, Minutes
90
FIG. 1. Effect of connectin on actin viscosity. Actin (150 /g/ml) was assembled in the presence or absence of connectin at 90 pg/ml in 60 mM glycine/42 mM Tris/4 mM imidazole/0.07 mM ATP/0.07 mM dithiothreitol/100 mM KCl/0.004% Triton X-100.
described by Malinoff and Wicha (9). Cells were labeled with [35S]methionine at 50 ,uCi/ml (1,300 Ci/mol; 1 Ci = 37 GBq) of culture medium for 48 hr prior to harvesting. Approximately 3,000 cpm/,ug of connectin was obtained in this manner. Greater than 90% of these cpm were found in the pellet upon precipitation with trichloroacetic acid. Laminin was isolated from the Engelbreth-Holm-Swarm (EHS) tumor cells and purified as described (11). Actin was purified from Dictyostelium discoideum as described (12). Viscometry. Viscometry was performed with a Brookfield model LVTDCB cone plate viscometer with a CP-40 cone at 25°C and 60 rpm. Viscosity was continuously monitored and displayed on a chart recorder. All samples were adjusted to 0.004% Triton X-100 (Sigma) to increase reproducibility of readings. At time 0, KCI was added to a final concentration of 100 mM to induce assembly of actin. This was done in the presence of either connectin or connectin buffer, and 450 Al of sample was immediately loaded into the viscometer. The initial viscosity of actin with or without connectin was equal to that of buffer alone, 0.89 centipoise (1 P = 0.1 Pa-sec). Electron Microscopy. Samples were stained with 1% uranyl acetate, which was centrifuged for 5 min at 3,000 X g immediately prior to use. They were viewed at 60 kV in a Philips 400 electron microscope. Abbreviation: F-actin, filamentous actin.
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Proc. Natl. Acad. Sci. USA 80 (1983)
and counted in a Beckman LS 9000 scintillation counter. Pellets were resuspended by sonication in the original volume of buffer, and radioactivity was assayed as above. The cpm reported are corrected for a background of 40 cpm. Aliquots of supernates and pellets were analyzed on NaDodSO4/polyacrylamide gels to ensure that actin sediments whether or not connectin is present. Nitrocellulose disk assay. Binding of 'S-labeled connectin to actin immobilized on 6-mm diameter nitrocellulose filter disks (8-gm Millipore) was performed as described by Malinoff and Wicha (9). Filters were dissolved in Cellosolve (Sigma) before
Binding Assays. Sedimentation assay. Connectin was assayed for its ability to cosediment with actin as follows: Airfuge tubes (Beckman) were coated with Sigmacote (Sigma) and dried in air. Samples including [3S]methionine-labeled connectin with or without filamentous actin (F-actin) were mixed in these tubes, incubated for 10 min, and centrifuged for 20 min at 30 psi (1 psi = 6.89 kPa) in a Beckman Airfuge (A-100/30 rotor) in order to sediment the actin. These procedures were carried out at room temperature. Bovine serum albumin was added at 0.5 mg/ ml to the mixture to prevent nonspecific connectin loss. Supernatants were mixed with 2 ml of ACS scintillant (Amersham)
li
r., Ir
4;
-7
.e
B FIG. 2. Electron micrograph of actin filaments in the presence or absence of connectin. F-actin at 50 ug/ml was incubated in the presence of 150 ug of connectin per ml (A) or buffer (100 mM glycine/70 mM Tris/100 mM KCl, pH 7.5) alone (B). Note the "bundling" of actin filaments in the presence of connectin. (x 77,000.)
Cell Biology: Brown et al. Table 1. Cosedimentation of connectin with actin % in pellet With Without actin actin Protein added 'S-Labeled connectin 4 35 Exp. 1* 3 79 Exp.2* 2 2 '25I-Labeled a-lactalbumin Connectin or a-lactalbumin at 0.1 pM was incubated in the presence or absence of 10 ,M actin in 10 mM imidazole/0.2 mM ATP/0.2 mM dithiothreitol/100 mM KCl, pH 7.5/0.5 mg of bovine serum albumin per ml, and the sample was centrifuged as described. * These two experiments are representative of 10 experiments, done with different preparations of connectin, with between 30% and 80% of connectin sedimenting with actin.
assay to eliminate quench. Connectin was stored in polypropylene tubes to reduce binding to plastic or glass surfaces. As a control, "2I-labeled F-actin was bound to nitrocellulose and washed extensively to demonstrate quantitative binding.
RESULTS Viscometry. When actin was polymerized in the absence of connectin, the viscosity at 90 min was 11.6 dl/g. Addition of connectin during the polymerization resulted in an 86% reduction to 1.6 dl/g (Fig. 1). Electron Microscopy. The addition of connectin (150 ,ug/ml) to F-actin (50 Ag/ml) resulted in the formation of bundles of parallel filaments, with few individual filaments seen (Fig. 2A). Such bundles were not seen in the absence of connectin (Fig. 2B) or in the presence of connectin at 15 tkg/ml. Others also have observed a viscosity decrease concomitant with bundle formation (e.g., ref. 13); presumably, a tangled meshwork of individual filaments is more viscous than a bundle. Binding Assays. A cosedimentation assay was used to assess the binding of connectin to actin. In each experiment, we measured the cosedimentation of 3S-labeled connectin in the presence or absence of F-actin. In different preparations of connectin, between 4% and 30% of connectin sedimented in the absence of F-actin. This may represent aggregated connectin; in some experiments, it was removed by presedimenting the connectin before it was mixed with F-actin. In the presence of F-actin, an additional 25-76% of the connectin sedimented (with or without presedimentation), resulting in a total sedimentation of 30-80% of added connectin (Table 1). Eighty-five percent of the connectin in the actin pellet resedimented with actin when the pellet was resuspended by Table 2. Binding of connectin to proteins immobilized on nitrocellulose disks Addition of 35S-labeled connectin unlabeled Protein bound, ng on disk connectin 412 Actin No 253 423 174 F-actin (30 ,Ag) or laminin (10 Ag) was spotted on nitrocellulose disks and, after blocking bound sites with bovine serum albumin, the disks were incubated with 600 ng of 3S-labeled connectin (3,000 cpm/,ug) with an equimolar amount-of unlabeled connectin or bovine serum albumin alone. Data represent the mean of triplicates with a standard deviation
