We have utilized a number of well-defined, simple, synthetic promoters (upstream factor binding sites and TATA elements) to analyze the activation mechanisms of the human cytomegalovirus immediate-early (IE) proteins. We found that the 86-kDa IE protein (known as IEP86, IE2,g5aa, or ppUL122a) can recognize and activate a variety of simple promoters, in agreement with the observation that it is a promiscuous activator. However, in the comparison of otherwise identical promoters IEP86 does have preferences for specific TATA elements (hsp70 > adenovirus E2 > simian virus 40 early) and specific upstream transcription factor binding sites (CAAT > SPI Tef-1 > ATF; no activation with AP1 or OCT). In contrast, the 72-kDa IE protein (known as IEP72, IE1491aa, or ppUL123) alone did not significantly activate the simple promoters under our experimental conditions. However, each promoter activated by IEP86 was synergistically affected by the addition of IEP72. In addition, the 55-kDa IE protein (IEP55, a splice variant form of IE2, IE2425aa, or ppUL122b) repeatedly had a negative effect, downregulating the activation of promoters caused by IEP86 and the synergy of IEP86 and IEP72. We show that the ability of IEP86 to activate many simple promoters correlates not only with its previously described ability to interact with the TATA-binding protein (TBP) (B. A.

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The early proteins of simian virus 40 (SV40) large T and small t antigen (T/t antigen) can each cause the transcriptional activation of a variety of cellular and viral promoters. We showed previously that simian cellular DNA-binding factors (the Band A factors) bind to sequences within the SV40 late promoter which are important for transcriptional activation in the presence of the SV40 early proteins. Band A factors isolated from simian cells which produce T/t antigen (COS cells or SV40-infected CV-1 cells) have altered binding properties in comparison with the factors from normal simian cells (CV-1). This suggests that the transcriptional activation mediated by T/t antigen may be due to either modification of existing factors or induction of new members of a family of factors. We have purified the Band A factors from both COS and CV-1 cells and have determined the binding site by methylation interference and DNase protection footprinting. The COS cell factors have altered chromatographic properties on ion-exchange columns and have higher-molecular-weight forms than the CV-1 cell factors. Major forms of the CV-1 factors migrate between 20 and 24 kilodaltons, while the COS factors migrate between 20 and 28 kilodaltons. The binding sites for the factors from CV-1 and COS cells are similar, covering a rather broad region within the 72-base-pair repeat comprising the AP-1 site and the two-octamer binding protein (OBP100/Oct 1) sites, OBP I and OBP H. Specific binding competition analyses indicate that the two general regions within the binding site (the AP-1-OBP II site and the OBP I site) each retain partial binding ability; however, the factors bind best when the two regions are adjacent in a relatively specific spatial arrangement. The binding site for the Band A factors corresponds very well to sequences necessary for the activation of the late promoter as defined by deletion and base substitution mutagenesis studies (

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Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI- 1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell- bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.