THE V-SIS PROTEIN RETAINS BIOLOGICAL-ACTIVITY AS A TYPE-II MEMBRANE-PROTEIN WHEN ANCHORED BY VARIOUS SIGNAL ANCHOR DOMAINS, INCLUDING THE HYDROPHOBIC DOMAIN OF THE BOVINE PAPILLOMA-VIRUS E5-ONCOPROTEIN

Membrane-anchored forms of the v-sis oncoprotein have been previously described which are oriented as type I transmembrane proteins and whic h efficiently induce autocrine transformation. Several examples of nat urally occurring membrane-anchored growth factors have been identified , but all exhibit a type I orientation. In this work, we wished to con struct and characterize membrane-anchored growth factors with a type I I orientation. These experiments were designed to determine whether ty pe II membrane-anchored growth factors would in fact exhibit biologica l activity. Additionally, we wished to determine whether the hydrophob ic domain of the E5 oncoprotein of bovine papilloma virus (BPV) can fu nction as a signal-anchor domain to direct type II membrane insertion. Type II derivatives of the v-sis oncoprotein were constructed, with t he NH2 terminus intracellular and the COOH terminus extracellular, by substituting the NH2 terminal signal sequence with the signal-anchor d omain of a known type II membrane protein. The signal-anchor domains o f neuraminidase (NA), asialoglycoprotein receptor (ASGPR) and transfer rin receptor (TR) all yielded biologically active type II derivatives of the v-sis oncoprotein. Although transforming all of the type II sig nal/anchor-sis proteins exhibited a very short half-life. The short ha lf-life exhibited by the signal/anchor-sis constructs suggests that, i n some cases, cellular transformation may result from the synthesis of growth factors so labile that they activate undetectable autocrine lo ops. The E5 oncoprotein encoded by BPV exhibits amino acid sequence si milarity with PDGF, activates the PDGF beta-receptor, and thus resembl es a miniature membrane-anchored growth factor with a putative type II orientation. The hydrophobic domain of the E5 oncoprotein, when subst ituted in place of the signal sequence of v-sis, was indistinguishable compared with the signal-anchor domains of NA, TR, and ASGPR, demonst rating its ability to function as a signal-anchor domain. NIH 3T3 cell s transformed by the signal/anchor-sis constructs exhibited morphologi cal reversion upon treatment with suramin, indicating a requirement fo r ligand/receptor interactions in a suramin-sensitive compartment, mos t likely the cell surface. In contrast, NIH 3T3 cells transformed by t he E5 oncoprotein did not exhibit morphological reversion in response to suramin.