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.

Abstract

Abstract. Membrane-anchored forms of the v-sis oncoprotein have been previously described which are oriented as type I transmembrane proteins and which efficiently induce autocrine transformation. Several examples of naturally occurring membrane-anchored growth factors have been identified, but all exhibit a type I orientation. In this work, we wished to construct and characterize membrane-anchored growth factors with a type II orientation. These experiments were designed to determine whether type II membrane-anchored growth factors would in fact exhibit biological activity. Additionally, we wished to determine whether the hydrophobic domain of the E5 oncoprotein of bovine papilloma virus (BPV) can function as a signal-anchor domain to direct type II membrane insertion.Type II derivatives of the v-sis oncoprotein were constructed, with the NH2 terminus intracellular and the COOH terminus extracellular, by substituting the NH2 terminal signal sequence with the signal-anchor domain of a known type II membrane protein. The signal-anchor domains of neuraminidase (NA), asialoglycoprotein receptor (ASGPR) and transferrin receptor (TR) all yielded biologically active type II derivatives of the v-sis oncoprotein. Although transforming all of the type II signal~anchor-sis proteins exhibited a very short half-life. The short half-life exhibited by the signal~anchor-sis constructs suggests that, in some cases, cellular transformation may result from the synthesis of growth factors so labile that they activate undeteetable autocrine loops.The E5 oncoprotein encoded by BPV exhibits amino acid sequence similarity with PDGF, activates the PDGF B-receptor, and thus resembles a miniature membrane-anchored growth factor with a putative type II orientation. The hydrophobic domain of the E5 oncoprotein, when substituted in place of the signal sequence of v-sis, was indistinguishable compared with the signal-anchor domains of NA, TR, and ASGPR, demonstrating its ability to function as a signal-anchor domain. NIH 31"3 cells transformed by the signal/anchor-sis constructs exhibited morphological reversion upon treatment with suramin, indicating a requirement for ligand/receptor interactions in a suramin-sensitive compartment, most likely the cell surface. In contrast, NIH 31"3 cells transformed by the E5 oncoprotein did not exhibit morphological reversion in response to suramin.

Growth factor PDGF‐B/v‐ sis confers a tumorigenic phenotype to human tumor cells bearing PDGF receptors but not to cells devoid of receptors: Evidence for an autocrine, but not a paracrine, mechanism

“…PDGF expression was analyzed on the protein level by immunoprecipitation studies ( Fig. 1) using an anti-serum which has been determined to be specific for the PDGF-Blv-sis product, intracellular processing intermediates and secreted PDGF-B/ v-sis Xu et al, 1993). A secreted product of approximately 16 kDa was observed in the conditioned medium of T98G cells.…”

“…It follows, therefore, that the endogenous receptors are sufficient to mediate a substantially greater mitogenic response than that observed for the parental cells. However, since growth in vitro was not completely inhibited by addition of anti-PDGF-B antibody, we cannot rule out that a proportion of the self-stimulated growth derives from intracel-Mar activation of PR-6, as has been observed previously (Xu et al, 1993;Harsh et al, 1990 and references therein).…”

Profound Ligand-Independent Kinase Activation of Fibroblast Growth Factor Receptor 3 by the Activation Loop Mutation Responsible for a Lethal Skeletal Dysplasia, Thanatophoric Dysplasia Type II

“…The TDII construct and activation loop mutants were derived as follows. The FGFR3 sequence between the ScaI site at amino acid 608 and the SphI site at amino acid 732 was reconstructed in the pSP64 vector (Promega) by using three long pairs of complementary oligonucleotides which were synthesized and purified as described previously (33). The first pair extended from the ScaI site to a novel EcoRV site at amino acid 641, the second pair created a silent AgeI site at amino acid 658 and extended to a novel silent BamHI site at amino acid 691, and the third pair extended from the BamHI site to the SphI site.…”

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

“…Cleavage of the propeptide region of v-sis is not required for its activity, as demonstrated initially in previous work from this laboratory in which the Lys-Arg cleavage site of v-sis was mutated to Asn-Ser with no change in biological activity (Saner et al, 1986). In subsequent studies from our lab, the KR to NS mutation has been incorporated into a variety of membrane-anchored derivatives (Hannink and Donoghue, 1986b;Lee and Donoghue, 1992;Xu et al, 1993), including sis-G and sis-G-ER-, with no effect on biological activity. This is an important point, as the constructs that are retained in the early Golgi, sis-E1 and sis-El-G, would not be expected to undergo this cleavage process.…”