CELL REGULATION, Vol. 2, 805-817, October 1991 Laminin-binding integrin a7,1: functional characterization and expression in normal and malignant melanocytes Randall H. Kramer,*t$ Mai P. Vu,* increased attachment to laminin exhibit much Yao-Fen Cheng,* Daniel M. Ramos,* higher metastatic potential in lung colonization Rupert Timpl,§ and Nahid Waleh 11 assays (reviewed in Liotta et aL, 1986). Fur- *Departments of Stomatology and Anatomy thermore, the presence of intact laminin will en- and the tCardiovascular Research Institute hance lung colonization (Barsky et aL., 1984). University of California These results and others strongly suggest that San Francisco, California 94143 melanoma cells interact with laminin-rich base- §Max-Planck-lnstitut fur Biochemie ment membrane and that this interaction facil- Martinsried, Germany itates their vascular arrest and colonization of IISRI International distant sites. Menlo Park, California 94025 During tissue invasion, metastatic melanoma cells interact with different types of extracellular matrix, including the interstitium and basement A novel integrin, a,#,, that specifically binds with membranes. It is expected, then, that these high affinity to laminin has been identified on mel- malignant cells will express surface adhesion anoma cells. This complex was purified from both receptors with diverse ligand specificities human and murine melanoma cells by laminin-af- (Ruoslahti and Giancotti, 1989). The integrin finity chromatography, and the a7 subunit was re- family of adhesion receptors consists of a large covered after gel electrophoresis. N-terminal amino number of heterodimer receptors that appear acid sequence analysis of the a7 subunit from both to mediate many of the cell-extracellular matrix human and mouse cells verifies that this integrin interactions for melanoma and other cell types is distinct from other a chains in the #I family, al- (Hynes, 1987; Ruoslahti and Pierschbacher, though strikingly similar to the as subunit. By using 1987; Albelda and Buck, 1990; Hemler, 1990). specific proteolytically derived fragments of lami- The combination of individual a and : integrin nin, it was determined that the a7f1 complex binds subunits into complexes allows for a large num- selectively to the E8 region, which represents part ber of potential receptor structures (Ruoslahti, of the long arm of laminin. In contrast, the receptor 1991). Because the ligand-binding specificity of failed to bind to the P1 fragment, which contains each individual heterodimer is determined by the intersection of the short arms of laminin. Al- contributions from both subunits, the multiplic- though the a7,,1 complex was commonly expressed ity of combinations provides for considerable in melanoma cells, this integrin was not detected diversity of function. Of the integrin receptors in normal melanocytes, suggesting that a7 expres- so far identified, many are involved in interac- sion may be associated with malignant transfor- tions between cells and extracellular matrix, mation. These results establish the existence of a whereas some also mediate cell-cell adhesion novel integrin that binds to the E8 domain of laminin (Springer, 1990). and appears to mediate cell adhesion to this ligand. Multiple cell attachment sites have been identified in laminin (Timpl et al., 1990). The long- arm fragment E8 of laminin produced by elas- Introduction tase digestion has been shown to be the binding site for both the ai3f1 (Gehlsen et aL., 1989) and Receptors for laminin have been implicated in the a611 (Aumailley et aL, 1990b; Hall et aL., melanoma metastasis. Liotta and collaborators 1990; Sonnenberg et aL., 1990) integrins. The have shown that melanoma cells selected for a1fl1 integrin appears to bind to the cross region of laminin represented by the P1 fragment : Corresponding author. (Forsberg et aL., 1990; Hall et aL., 1990). The 0V,3 © 1991 by The American Society for Cell Biology 805 R.H. Kramer et al. integrins are important in metastasis and in- vasion. For example, RGD peptides, to which I several integrins bind, block invasion through the extracellular matrix (Gehlsen et al., 1988) -A- - and inhibit tumor metastasis (Humphries et al., ] ,~> 1988). Giancotti and Ruoslahti (1990) found that la... W j o overexpression of the fibronectin receptor (a5fll) 13. induced major changes in cell behavior. The ov- ,, _ erexpressor cells migrated less than control J] ct cells. Furthermore, the overexpressor cells, in contrast to the control cells, did not grow well l7 in soft agar and failed to form tumors in nude mice. Similarly, cells deficient in this receptor were more tumorigenic (Schreiner et al., 1991). I These results suggest that levels of integrin expression can directly modulate not only cell adhesion and migration but also cell growth. 48 - Previously, we reported that human mela- 1 L. noma cells express a unique af31 integrin com- 41 3 4 5 plex that bound to laminin yet was biochemically distinct from all known a subunits (Kramer et Figure 1. Cell-surface proteins eluted from laminin- al., 1989b). In the present study, we have pu- Sepharose column. 1251-labeled Kl 735 melancDma cells were rified the human receptor as well as the mouse extracted with starting buffer, and the extracct was applied hom to a laminin-Sepharose column as describei!din Materials ologue, and because the a subunit has a and methods. After a washing with starting tbuffer, the col- unique N-terminal amino acid sequence it is re- umn was eluted with 250 mM NaCI (lanes 1--3) followed by ferred to as aC7. We also have established that elution with 10 mM EDTA (lanes 4-5). Fracttions were an- the a7f1 complex binds to the E8 fragment of alyzed by SDS-PAGE under nonreducing (left panel) and re- ducing (right panel) conditions. EDTA eluted laminin. Furthermore, the receptor is not de- -120 kDa (lane 4) with a minor band (a7) at 125 kDa (non- tectable in human melanocytes, suggesting that reducing conditions), which after reduction miigrated at 130 its expression may be linked to the transformed kDa (00) and 95 kDa (a7). phenotype. Results integrin also binds to laminin (Kramer et al., 1 990b). In addition, a cryptic Arg-Gly-Asp(RGD)- Purification and N-terminal amino acid dependent site for aC%3 is generated in the P1 sequence of thea7 subunit fragment (Aumailley et aL, 1 990a; Sonnenberg Ligand-affinity chromatography on laminin- et al., 1990). The a2f1 integrin also binds laminin Sepharose was used to purify the novel human (Elices and Hemler, 1989; Languino et a/., 1989; and mouse melanoma laminin receptor (al1) Kramer et al., 1990), although the specific bind- essentially as described previously (Kramer et ing site on laminin for this receptor has not as al., 1989b). We now provide descriptive evi- yet been defined. dence that this complex possesses a new a Various studies have attempted to compare subunit, a7. The a731 complex is heavily ex- integrin profiles of normal and malignant cells. pressed on both the human MeWo and mouse From these few early studies it seems that ma- Kl 735 melanoma cell lines. Chromatography on lignant transformation can induce certain laminin-Sepharose columns of detergent ex- changes in integrin expression, but further work tracts of 1251 surface-labeled Kl 735 cells (Figure is needed to define how universal these alter- 1) yielded material that was resistant to elution ations are and how they specifically influence with a 0.25 M salt wash but was quantitatively the cell phenotype. For example, certain tumor recovered by elution with EDTA. As previously cells display a reduced level of f1 integrin shown (Kramer et al., 1 989b), the a7 subunit ex- expression (Peltonen et al., 1989; Plantefaber hibits an apparent molecular mass of about and Hynes, 1989), whereas other tumor cells 120-125 kDa and comigrates with the f31 subunit show an increase in f1 expression after malig- under nonreducing conditions in sodium do- nant transformation (Dedhar and Saulnier, decyl sulfate-polyacrylamide gel electrophoresis 1990). However, other studies demonstrate that (SDS-PAGE). However, after reduction a7 loses 806 CELL REGULATION The melanoma laminin-binding integrin a7fl1 a disulfide-linked 30-kDa fragment (Kramer et A al., 1989b) and migrates at 90-1 00 kDa. The ,B integrin profile of human MeWo mel- anoma cells was determined by subjecting de- tergent extracts of 1251-labeled cells to immu- -a2-6 noprecipitations using monoclonal antibodies , # * -, .0 , against specific integrin a subunits. The a!7 sub- 3 ]&-a7 unit was readily visible as a sharp band in re- I ducing gels after immunoprecipitation with an- tibody to the fI subunit (Figure 2). In contrast, immunoprecipitation with antibodies to a1, a22, a3, a5, and a6 failed to recover the a7 subunit; instead the expected individual a subunits with the associated f,B subunit were recognized by B each antibody. Under reducing conditions, a33, a5, a6, and a7 subunits showed the anticipated increase in electrophoretic mobility as a result -.a-f3-a2 of loss of the C-terminal peptide disulfide-linked -Y - :1 a3-6 fragment; the f,B subunit showed a character- istic decrease in mobility under reducing con- ditions. In the case of a2, a5, and a6 subunits, more than one specific antibody was tested. The MeWo cells were found to express moderate levels of al, a2, a3, and a5 but only minor amounts of a6 and a,4 (not shown). a1 a2 a2a3 a a5aa5a6a6a6 01 For large-scale purifications, extracts from subcutaneous tumors of the mouse Kl 735 mel- anoma or alternatively, extracts from cultured Figure 2. The a7t,0 integrin complex is not cross-reactive melanoma were used as with antibodies specific for the a subunits of other fl in- MeWo cells, starting tegrins. Human MeWo melanoma cells were surface labeled material. Analysis of the EDTA-eluted fractions with 1251 and the detergent extract was subjected to im- in silver- or Coomassie blue-stained SDS-PAGE munoprecipitation with antibody to the indicated a subunits gels gave similar protein band patterns as or to the 3,B subunit.