Processing of frameshifted vasopressin precursors

Area of Science:

• Neuroendocrinology
• Molecular Biology
• Cell Biology

Background:

• Vasopressin (VP) prohormone biosynthesis involves ER transit, sorting, and processing in magnocellular neurones.
• The diabetes insipidus (di/di) Brattleboro rat has disrupted VP precursor processing due to a gene deletion, leading to VP deficiency.
• Previous studies identified frameshift mutations in VP transcripts in di/di rats, creating altered VP precursors.

Purpose of the Study:

• To investigate if +1 frameshifted VP precursors from di/di and wild-type rats can lead to the biosynthesis of the hormone VP.
• To determine the cellular mechanisms governing the processing and secretion of these mutant VP precursors.

Main Methods:

• Transient expression of eukaryotic plasmids containing frameshifted VP cDNAs in peptidergic tumour cell lines (Neuro2A and AtT20).
• Analysis of cells using reversed-phase high-performance liquid chromatography, specific radioimmunoassays, and immunofluorescence.
• Comparison of precursor processing and ER transit times between different cell lines.

Main Results:

• In Neuro2A cells, +1 frameshifted precursors from di/di rats were retained in the cell body with minimal neurophysin-immunoreactive product.
• In AtT20 cells, frameshifted VP precursors were processed to yield VP peptide, indicating access to the regulated secretory pathway.
• ER transit of the wild-type prohormone was slow in Neuro2A cells, with inefficient processing observed.

Conclusions:

• Mutant VP precursors can access the regulated secretory pathway if ER transport is sufficiently rapid, as demonstrated in AtT20 cells.
• The di/di rat might regain the capacity to biosynthesize authentic VP via these +1 frameshifted precursors.
• Cellular context significantly influences the processing efficiency of mutant VP precursors.