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 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 undetectable autocrine loops. The E5 oncoprotein encoded by BPV exhibits amino acid sequence similarity with PDGF, activates the PDGF beta-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 3T3 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 3T3 cells transformed by the E5 oncoprotein did not exhibit morphological reversion in response to suramin.
Xu
Y F
YF
Department of Chemistry, University of California, San Diego, La Jolla 92093-0322.
Meyer
A N
AN
Webster
M K
MK
Lee
B A
BA
Donoghue
D J
DJ
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001483:Base Sequence; D052688:Bovine papillomavirus 1 / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006133:Growth Substances / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase / Q000096:biosynthesis / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D020575:Oncogene Proteins v-sis; D021382:Protein Sorting Signals / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000378:metabolism; D011990:Receptors, Transferrin / Q000096:biosynthesis / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000096:biosynthesis / Q000378:metabolism*; D013498:Suramin / Q000494:pharmacology; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000378:metabolism*
D037263:Asialoglycoprotein Receptor; D004268:DNA-Binding Proteins; C069814:E1 protein, Bovine papillomavirus; D006133:Growth Substances; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D020575:Oncogene Proteins v-sis; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D015682:Retroviridae Proteins, Oncogenic; D014764:Viral Proteins; D013498:Suramin; D009439:Neuraminidase