Nuclear forms of parathyroid hormone-related peptide are translated from non-AUG start sites downstream from the initiator methionine

PTH-related peptide (PTHrP), normally a secreted protein, exerts at least part of its biological functions via intracrine actions at the level of the cell nucleus/nucleolus. To gain insight into the mechanism whereby this peptide accesses the cytosol for subsequent nuclear import, we characterized its nuclear forms. In transfected COS-1 cells, three nuclear PTHrP species were produced, which were larger than the mature form of the protein but smaller than prepro-PTHrP and comprised both the amino- and carboxyl-terminal regions of the peptide. This suggested that nuclear PTHrP proteins contain part of the prepropeptide and Likely arise from alternate initiation of translation at downstream non-AUG codons within the signal sequence.Transient expression of two PTHrP forms, one in which the unique initiator ATG was mutated to a noninitiator ATC codon, and another encompassing an engineered N-linked glycosylation site, generated peptides of size comparable to nuclear PTHrP proteins. These were inefficiently targeted to the ER, bypassed ER transit, and exclusively localized to nucleoli. Using a polyclonal antiserum against the proregion of PTHrP, nuclear PTHrP species were shown to harbor the propeptide sequence. Hence, our data argue that nuclear PTHrP forms result from translation initiated at alternate, internal codons. This may constitute the first example of translation initiation at downstream non-AUG codons in a mammalian protein.