Fos and Jun proteins form a tight complex which binds specifically to the AP1 recognition sequence, a palindromic DNA element also referred to as the TPA responsive element (TRE). To elucidate the mechanism of Fos‐Jun interaction with the TRE we have performed UV cross‐linking studies using oligonucleotides where thymines were replaced with bromouracil. Our results indicate that both Fos and Jun directly contact the TRE but that the interaction of Fos and Jun with thymines in structurally equivalent positions in the two half sites of the TRE is different. In addition, we have carried out a comprehensive mutagenesis study of the TRE by introducing all possible point mutations plus thymine‐‐‐‐uracil substitutions into the palindromic TRE core sequences and the adjacent nucleotides on both sides. The results of this analysis clearly show that the palindromic TRE is asymmetrical with respect to binding of Fos‐Jun. We also show that a Fos protein complex with a homodimeric DNA binding site binds considerably less efficiently to TRE mutants with a perfect dyad symmetry compared with the binding to the wild‐type TRE. This demonstrates that the asymmetrical recognition of the TRE is not due to the heterodimeric nature of the Fos/Jun complex but directly related to an asymmetry in the TRE sequence. The methyl groups of all four thymine residues within the TRE seem to be functionally crucial since thymine‐‐‐‐uracil substitutions strongly reduce or abolish binding to Fos/Jun. The relevance of structurally equivalent methyl groups in the TRE core sequence is different, lending further support to the conclusion that the TRE is asymmetrical.

Abstract. The locomotion of murine myoblasts over the extracellular matrix components iaminin and fibronectin was analyzed using quantitative videomicroscopy, and the organization of the cytoskeleton was observed in parallel immunofluorescence studies. Cells plated on the laminin-nidogen complex locomoted twice as fast as on laminin alone. The main form of translocation on laminin was a jerky cycle of prolonged lamellipod extension followed by rapid (",,200-<500/~m h -~) movement of the cell body into the extended lamellipod. The locomotionstimulating activity of laminin resides in the elastase digestion fragment E8, part of the laminin long arm, while the El-4 fragment containing the three short arms is inactive.Myoblasts moved poorly over fibronectin irrespective of whether high, intermediate, or low coating concentrations were used (~5,000-~10 fmol cm-2). In contrast, the locomotory responses both to laminin and to E8 peaked sharply at coating concentrations *20-50 fmol cm -2 and decreased at higher concentrations. This response corresponds to that expected for a haptotactic stimulant. When cells locomoted over a mixed substrate of laminin and fibronectin, the fibronectin effects appeared to predominate.The cytoskeleton has been implicated in many cellular motile processes. Within 6 h on fibronectin many cells expressed vinculin-containing focal contacts, elaborated stress fibers and had periodically organized ot actinin, whereas on laminin, most cells showed diffuse vinculin and c~ actinin and a fine meshlike actin cytoskeleton. We conclude that the poor locomotion of cells over fibronectin is because of the cytoskeletal stabilization it induces.

Modulation of 5'-nucleotidase activity by the extracellular matrix proteins fibronectin, laminin and their fragments has been studied in plasma membrane preparations as well as in intact BCS-TC2 and Rugli cells. The ectoenzyme on plasma membranes is activated by laminin; fibronectin inhibits the AMPase activity on BCS-TC2 plasma membranes but no inhibitory effect is found in plasma membrane preparations from Rugli cells. These effects are dependent on the preincubation time and protein concentration. When the effect of the extracellular matrix proteins is studied on intact cells, both BCS-TC2 and Rugli cells show similar behaviour. A decrease in the enzyme activity is observed in the presence of fibronectin. The AMPase inhibitory activity is located on its 40 kDa fragment. No inhibitory activity is found in other fibronectin fragments, including the 140 kDa fragment which contains the RGDS cell-adhesion sequence. Laminin and its E1-4 and E8 fragments are able to activate the ecto-5'-nucleotidase activity of both BCS-TC2 and Rugli cells. The effect of the E1-4 fragment on intact cells is greater than that observed for the E8 fragment and uncleaved laminin. Our results suggest a bifunctional role for 5'-nucleotidase as ectoenzyme and cell receptor for extracellular matrix proteins.

The 5-AMPase activity of the ectoenzyme 5-nucleotidase has been measured in a variety of cell lines, using intact cells. Human cell types showed two orders of magnitude higher enzyme activity than mouse cell lines. The ectoenzyme is inhibited by adenosine 5-(alpha, beta-methylene) diphosphate and Concanavalin A. A different extent of 5-nucleotidase lectin inhibition was observed in the studied cell lines, suggesting that the corresponding ectoenzymes are glycoproteins with a different type or degree, or both, of glycosylation. The 5-nucleotidase activity increased during subculture and decreased after cell transformation. Generally, the 5-nucleotidase activity was two- to five-fold higher in monolayer than in suspension cell culture. A relation between cell growth and 5-AMPase activity was also observed. Enzyme activity increased at the end of the lag phase (glioblastoma cells) or during the exponential phase (the other two cell lines). After confluence, the activity decreased to the initial or even lower range of activity. Observed activity variations with cell proliferation correlate with modifications of 5-AMPase activity during subculture.

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