Processing of Frameshifted Vasopressin Precursors

Abstract

Biosynthesis of the vasopressin (VP) prohormone in magnocellular neurones of the hypothalamo-neurohypophysial system comprises endoplasmic reticulum (ER) transit, sorting into the regulated secretory pathway and subsequent processing in the individual proteins VP, neurophysin and a glycoprotein. These processes are severely disrupted in the homozygous diabetes insipidus (di/di) Brattleboro rat, which expresses a mutant VP precursor due to a single nucleotide deletion in the neurophysin region of the VP gene resulting in VP deficiency. Previous studies have shown the presence of additional frameshift mutations in VP transcripts, in solitary magnocellular neurones of the di/di rat due to a GA dinucleotide deletion resulting in two different mutant VP precursors with partly restored reading frame. Frameshifted VP precursors are also expressed in several magnocellular neurones in wild-type rats. In this study, we determined if the +1 frameshifted precursors from di/di and wild-type rats can lead to biosynthesis of the hormone VP. Therefore, eukaryotic expression plasmids containing the frameshifted VP cDNAs were transiently expressed in peptidergic tumour cell lines, and cells were analysed by reversed phase high-performance liquid chromatography and specific radioimmunoassays, and by immunofluoresence. Neuro2A neuroblastoma cells expressing the +1 frameshifted precursors of di/di rats retained products in the cell body. Only precursor or insignificant quantities of neurophysin-immunoreactive products were detected. In contrast, in AtT20 cells, frameshifted VP precursors were at least partly processed to yield the VP peptide, indicating that they have access to the regulated secretory pathway. Comparison between the two cell lines showed a very slow ER transit of the wild-type prohormone combined with inefficient processing in Neuro2A cells. The results show that mutant precursors can reach the regulated secretory pathway if ER transport is sufficiently rapid as in the case of AtT20 cells. This suggests that the di/di rat may regain the capacity to biosynthesize authentic VP through these +1 frameshifted precursors in magnocellular neurones.

Trafficking of the Vasopressin and Oxytocin Prohormone Through the Regulated Secretory Pathway

“…This is due to a +1 frameshift later in life in the neurophysin domain further downstream which restores the reading frame, but leaves a region of 13–22 residues in the C ‐terminal domain of neurophysin altered. Remarkably, these frameshifted precursors are still properly processed and sorted by a neuroendocrine cell, such as AtT20 cells ( 24).…”

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“…However, the vasopressin was packaged abnormally in clumps rather than the usual dispersed pattern indicating cytoplasmic secretory granules. Subsequently, Evans et al (2000) provided evidence that solitary magnocellular neurons of the di/di rat can regain the capacity to biosynthesise authentic vasopressin through C1 frameshifted precursors, but probably with impaired ER transit and sorting into the regulated secretory pathway (Fig. 6).…”