The proto-oncogene Wnt-1 encodes a
cysteine-rich, secretory
glycoprotein implicated in virus-induced mouse
mammary cancer and intercellular signaling during vertebrate neural development. To attempt to correlate structural motifs of
Wnt-1 protein with its function, 12 mutations were introduced singly and in several combinations into the coding sequence of Wnt-1
cDNA by site-directed mutagenesis. Mutant alleles in a retroviral vector were tested for their ability to transform the mouse mammary epithelial cell line C57MG in two ways: by direct
infection of C57MG cells and by
infection of NIH3T3 cells that serve as donors of
Wnt-1 protein to adjacent C57MG cells in a secretion-dependent (paracrine) assay. In addition, the synthesis and secretion of
mutant proteins were monitored in multiple cell types by immunological assays. Deletion of the
signal peptide demonstrated that transformation in both direct and paracrine assays depends upon entry of
Wnt-1 protein into the endoplasmic reticulum. Changes in potential proteolytic processing sites (two basic
dipeptides and a probable
signal peptidase cleavage site) did not adversely impair
biological activity or
protein processing and uncovered a second site for cleavage by
signal peptidase. Replacement of each of the four
asparagine-linked glycosylation sites did not affect transforming activity at normal temperatures, but one glycosylation site mutant was found to be temperature-sensitive for transformation. An allele encoding a
protein that lacks all four glycosylation sites was also transformation competent. In two of four cases, substitution of
serine for a
cysteine residue impaired transforming activity at the usual temperature, and transformation was temperature sensitive in a third case, implying that at least some of the highly conserved
cysteine residues are important for Wnt-1 function.