Processing of frameshifted vasopressin precursors

Biosynthesis of the vasopressin (VP) prohormone in magnocellular neurones o f the hypothalamo-neurohypophysial system comprises endoplasmic reticulum ( ER) transit, sorting into the regulated secretory pathway and subsequent pr ocessing in the individual proteins VP, neurophysin and a glycoprotein. The se processes are severely disrupted in the homozygous diabetes insipidus (d i/di) Brattleboro rat, which expresses a mutant VP precursor due to a singl e nucleotide deletion in the neurophysin region of the VP gene resulting in VP deficiency. Previous studies have shown the presence of additional fram eshift mutations in VP transcripts, in solitary magnocellular neurones of t he di/di rat due to a GA dinucleotide deletion resulting in two different m utant VP precursors with partly restored reading frame. Frameshifted VP pre cursors are also expressed in several magnocellular neurones in wild-type r ats. 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. Therefor e, eukaryotic expression plasmids containing the frameshifted VP cDNAs were transiently expressed in peptidergic tumour cell lines, and cells were ana lysed by reversed phase high-performance liquid chromatography and specific radioimmunoassays, and by immunofluoresence. Neuro2A neuroblastoma cells e xpressing the +1 frameshifted precursors of di/di rats retained products in the cell body. Only precursor or insignificant quantities of neurophysin-i mmunoreactive products were detected. In contrast, in AtT20 cells, frameshi fted VP precursors were at least partly processed to yield the VP peptide, indicating that they have access to the regulated secretory pathway. Compar ison between the two cell lines showed a very slow ER transit of the wild-t ype prohormone combined with inefficient processing in Neuro2A cells. The r esults show that mutant precursors can reach the regulated secretory pathwa y 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 authe ntic VP through these +1 frameshifted precursors in magnocellular neurones.