14‐3‐3 Proteins Within the Hypothalamic‐Neurohypophyseal System of the Osmotically Stressed Rat: Transcriptomic and Proteomic Studies

The hypothalamic‐neurohypophyseal system (HNS) mediates neuroendocrine responses to dehydration through the actions of the antidiuretic hormone vasopressin (VP) and the natriuetic peptide oxytocin (OT). VP and OT are synthesised as separate prepropeptide precursors in the cell bodies of magnocellular neurones in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus, the axons of which innervate the posterior pituitary gland (PP). Dehydration evokes a massive release of both peptides into the circulation, and this is accompanied by a function‐related remodelling of the HNS. Microarray studies on mRNAs differentially expressed in the SON revealed that transcripts encoding the Ywhag and Ywhaz isoforms of the 14‐3‐3 family of regulatory proteins, are increased in the rat SON by 3 days of water deprivation; findings that we have confirmed by the real‐time polymerase chain reaction. Because there is no necessary proportionality between transcript and protein abundance, we next examined Ywhag and Ywhaz translation products throughout the HNS in parallel with 14‐3‐3 post‐translational modification, which is known to be an important determinant of functional activity. Both proteins are robustly expressed in the SON in VP‐ and OT‐containing neurones, but the abundance of neither changes with dehydration. However, the total level of Ywhaz protein is increased in the neurointermediate lobe of the pituitary (NIL, which includes the PP), in parallel with a basic post‐translationally modified isoform, suggesting transport from the cell bodies of the SON of newly‐synthesised protein and changes in its activity. The level of an acidic, probably phosphorylated, Ywhag isoform is down‐regulated in the SON by dehydration, although total levels are unchanged. Finally, based on the presence of a phosphorylated 14‐3‐3 binding motif, we have identified a 14‐3‐3 binding partner, proteasome subunit, beta type 7, in the NIL. Thus, we suggest that, through complex transcriptional, and post‐translational processes, 14‐3‐3 proteins are involved in the regulation or mediation of HNS plasticity following dehydration.

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