Miscleavage at the presequence of rat preprolactin synthesized in pituitary cells incubated with a threonine analog

We have shown previously that processing of preprolactin to prolactin in isolated rat pituitaries is inhibited by the threonine analog, β-hydroxynorvaline (Hnv), presumably because of its substitution for the threonine at the cleavage site. Here, we show by amino-terminal sequence analyses that Hnv altered the site at which preprolactin was processed such that about half of the prolactin produced had three extra amino acids at the amino terminus. The miscleaved prolactin was secreted into the medium, and there was a delay of approximately 5 min between the initial appearance of prolactin and the formation of miscleaved prolactin. This lag period suggests that miscleaved prolactin did not represent an intermediate in the normal processing of preprolactin but resulted from cleavage of completed preprolactin chains containing Hnv. These results show that modification of the structure of a preprotein can alter the site of its cleavage. One site that appears to be critical for correct cleavage is the final amino acid residue in the prepeptide. The data also indicate that complete removal of the presequence is not required for secretion of a protein.