The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex

Indirect methods that rely upon pharmacologic agents have been used by some researchers in attempts to identify the site of ligand/receptor interactions. Monensin has been shown to block transport of proteins through the trans-Golgi portion of the secretory pathway (Tartakoff, 1983) . Treatment of v-sis-expressing cells with monensin prevents autophosphorylation of mature PDGF receptors and expression of c-fos (a nuclear protein involved in cellular growth regulation), suggesting that v-sis must be transported beyond the point of monensin's inhibitory activity (past the trans-Golgi portion) in order to activate signal transduction pathways (Hannink and Donoghue, 1988) . However, monensin exerts pleiotropic effects on cations within cells, so other cellular events may have been affected in these experiments. Suramin, a potent inhibitor of proliferation of cells expressing v-sis and PDGF receptors, seems to interfere only with ligand/receptor interactions at the cell surface, reducing the level of tyrosine-phosphorylated receptors. Suramin has little or no effect on intracellular phosphorylated receptors (Fleming et al., 1989) . This suggests a requirement for cellsurface interactions between receptor and ligand for expres-sion of a transformed phenotype. However, the mechanism of action of surarnin is unclear, and there is evidence that it accumulates within endosomes (Hawking, 1978) . Thus, experiments utilizing agents such as monensin or suramin have been viewed as problematic by some researchers. Therefore, we have recently exploited more direct approaches to address this issue of intraceUular ligand/receptor interactions. The recent identification of specific targeting and retention signals makes it possible to localize v-sis protein to specific intracellular compartments. This potentially allows one to scan the secretory pathway for compartments that allow functional transforming interactions between v-sis and PDGF receptors. This represents a powerful approach for examining autocrine interactions, and can be applied to other autocrine growth factors or systems as well. ER-anchored forms of v-sis were previously constructed by this lab, using an adenovirus transmembrane protein E3/19K retention signal, DEKKMP (Nilsson et al., 1989) . These constructs prevented cell surface expression of v-sis protein as determined by immunofluorescence, and transformation was inhibited by retention of the fusion protein in the ER . In this report, we continued our analysis of autocrine transformation from within secretory pathway compartments by creating novel v-sis fusion proteins targeted to unique subcellular compartments. One signal that we chose was the cis-Golgi localization signal represented by the first transmembrane domain of the avian coronavirus E1 glycoprotein (El) of infectious bronchitis virus, which has been shown by others to target heterologous proteins such as VSV-G and c~m (a derivative of the human chorionic gonadotropin-c~ subunit) to the cis-Golgi complex (Swift and Machamer, 1991) . In addition, we chose to exploit the TGN-localization signal of the protein TGN38, contained within its transmembrane domain and cytoplasmic tail, which has been shown to retarget heterologous proteins, such as the LDL receptor and the Tac antigen, to the TGN (Bos et al., 1993; Humphrey et al., 1993) . The resulting sis fusion proteins, referred to as sis-E1 and sis-TGN38, allowed a further characterization of the site of autocrine interactions be