The concentration of sphingosine 1-phosphate (S1P) in plasma or serum is much higher than the half-maximal concentration of the sphingolipid needed to stimulate its receptors. Nevertheless, the inositol phosphate response to plasma or serum mediated by Edg-3, one of the S1P receptors, which was overexpressed in Chinese hamster ovary cells, was much smaller than the response expected from the total amount of S1P in these samples. The inositol phosphate response to exogenous S1P was markedly attenuated in the presence of charcoal-treated low-S1P serum. The inhibitory effect was lost by boiling but not by dialysis of the serum. The inhibitory action of the serum was specific to S1P and was associated with the trapping of exogenous S1P; the inositol phosphate response to P(2)-purinergic agonists was somewhat enhanced by the charcoal-treated serum. Among the components of plasma or serum, lipoproteins such as low-density and high-density lipoproteins showed a stronger activity for trapping S1P than lipoprotein-deficient serum. Consistent with this observation, we detected a 15-100-fold higher amount of S1P per unit amount of protein in lipoproteins than in the lipoprotein-deficient serum. Thus even though the protein content of the lipoprotein fraction contributes to only 4% of the total protein content of plasma or serum, more than 60% of S1P is distributed in this fraction. These results suggest that the tight binding of S1P to the components of serum or plasma, including lipoproteins, may interfere with the S1P binding to its receptors and thereby attenuate the lipid-receptor-mediated actions in the cells.

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Fos family proteins form stable heterodimers with Jun family proteins, and each heterodimer shows distinctive transactivating potential for regulating cellular growth, differentiation, and development via AP-1 binding sites. However, the molecular mechanism underlying dimer specificity and the molecules that facilitate transactivation remain undefined. Here, we show that BAF60a, a subunit of the SWI⅐SNF chromatin remodeling complex, is a determinant of the transactivation potential of Fos/Jun dimers. BAF60a binds to a specific subset of Fos/Jun heterodimers using two different interfaces for c-Fos and c-Jun, respectively. Only when the functional SWI⅐SNF complex is present, can c-Fos/c-Jun (high affinity to BAF60a) but not Fra-2/JunD (no affinity to BAF60a) induce the endogenous AP-1-regulated genes such as collagenase and c-met. These results indicate that a specific subset of Fos/Jun dimers recruits SWI⅐SNF complex via BAF60a to initiate transcription.Transcription factor AP-1, which plays pivotal roles in cell growth, differentiation, development, and tumor formation, is composed of Fos family proteins (Fos; c-Fos, Fra-1, Fra-2, and FosB) and Jun family proteins (Jun; c-Jun, JunB, and JunD). The members of both families form dimers through a leucine zipper structure; Jun family members can form low-affinity homodimers and high affinity heterodimers with the Fos family (1, 2). However, members of the Fos family do not form stable homodimers. Although these hetero-and homodimers bind to similar sites on DNA (TGAC/GTCA, AP-1 binding sites) through the basic domains of both proteins, each dimer has a distinct transcriptional regulatory function that can be modulated either positively or negatively (3). For example, transcriptional activity of the c-Fos/c-Jun dimer is much higher than the Fra-2/c-Jun dimer. Although functional interactions between some members of the Fos/Jun family of proteins and adaptor proteins such as CREB-binding protein (CBP) or TATA-binding protein (TBP) have been reported (4, 5), crucial proteins that recognize the dimer specificity and/or facilitate the induction of transcriptional initiation were largely unknown. Therefore, we proposed here to isolate such a crucial protein using a yeast two-hybrid system and have demonstrated that BAF60a (6), a component of the SWI⅐SNF chromatin remodeling complex, can select specific Fos/Jun dimers and function as a determinant of transcriptional activation via AP-1 binding sites. EXPERIMENTAL PROCEDURESPlasmid Construction-The Gal4DBD-c-Jun (25-187) fusion construct (pDBLeu-cJ(25-187)) was generated by inserting the 0.49 kilobase pair EheI fragment of the rat c-jun gene into the MscI restriction endonuclease cleavage site within the open reading frame of Gal4DBD. For the construction of template DNA for in vitro translation, we first generated a starter plasmid from pGEM2-475/Jun-D, the translation initiation site of which has a Kozak consensus sequence with a unique NcoI site that is preceded by a fragment 475 sequence for an efficient translational ...

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