ARGININE-VASOPRESSIN SECRETION WITH MUTANTS OF WILD-TYPE AND BRATTLEBORO RATS AVP GENE

Defects in peptide processing are associated with several disorders, i ncluding central diabetes insipidus (CDI). In the Brattleboro (BE) rat with CDI, the mRNA and protein of arginine vasopressin (AVP) are pres ent in the hypothalamus, but no circulating AVP is detectable, thus su ggesting a processing defect. The present study examined AVP secretion in cultured COS cells transfected with various constructs from wild-t ype and mutated Brattleboro AVP gene precursors. The precursor contain s three exons encoding for vasopressin (VP), neurophysin (NP), and gly copeptide (GP). The Brattleboro rat has a deletion of a single base, g uanine (G), in the NP coding region that leads to a frameshift, result ing in the loss of normal stop codon. The wild-type pcVP (22.0 +/- 5.2 pg/10(-2) U beta-galactosidase [beta-gal]), but not the mutated BE AV P gene pcBB (1.2 +/- 0.4 pg/10(-2) U beta-gal), was associated with AV P secretion from the COS cells as measured by RIA. The wild-type AVP g ene without the GP coding region was associated with AVP release great er (47.4 +/- 13.5 pg/10(-2) U beta-gal, n = 5, P < 0.05, versus pcVP) than the pcVP with intact VP, NP, and GP coding regions. However, the wild-type AVP gene with VP coding region alone was not processed and s ecreted. Normalizing the pcBB total length with the insertion of a sto p codon at the site of the normal stop codon was not associated with A VP secretion (3.0 +/- 1.4 pg/10(-2) U beta-gal). However, insertion of a stop codon so that the pcBB length equaled the length of VP and NP coding regions of the wild type was associated with AVP secretion (13. 5 +/- 4.0 pg/10(-2) U beta-gal) When a stop codon was inserted into th e wild-type NP coding region at the same site as the G deletion in the pcBB, the AVP secretion was significantly lower (15.1 +/- 5.0 pg/10(- 2) U beta-gal) than pcVP with VP + NP but no GP coding regions (47.4 /- 13.5 pg/10(-2) U beta-gal, n = 5, P < 0.05). In summary, (1) both V P and intact NP, but not GP, coding regions are necessary for AVP proc essing and secretion; (2) decreasing the length of the NP coding regio n diminishes but does not abolish AVP processing and secretion; and (3 ) shortening of the pcBB length with a stop codon at a site comparable to wild-type VP + NP allows AVP secretion, albeit less than with wild -type gene precursor. Thus, the CDI in BE rats is caused by the G dele tion in NP coding region. This defect leads to abnormalities that cont ribute to the abnormal AVP processing. Specifically, the frameshift an d absence of a stop codon cause a mutated extended C terminus, which, along with the mutated NP, contribute to the abnormal steps of AVP pro cessing, transport, and secretion in the BE rat. These defects no doub t impair the folding and configuration necessary for normal processing of the AVP gene precursor.