title: Arginine vasopressin secretion with mutants of wild-type and Brattleboro rats AVP gene.
abstract: Defects in peptide processing are associated with several disorders, including central diabetes insipidus (CDI). In the Brattleboro (BB) rat with CDI, the mRNA and protein of arginine vasopressin (AVP) are present in the hypothalamus, but no circulating AVP is detectable, thus suggesting a processing defect. The present study examined AVP secretion in cultured COS cells transfected with various constructs from wild-type and mutated Brattleboro AVP gene precursors. The precursor contains three exons encoding for vasopressin (VP), neurophysin (NP), and glycopeptide (GP). The Brattleboro rat has a deletion of a single base, guanine (G), in the NP coding region that leads to a frameshift, resulting 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 BB AVP gene pcBB (1.2 +/- 0.4 pg/10[-2] U beta-gal), was associated with AVP secretion from the COS cells as measured by RIA. The wild-type AVP gene without the GP coding region was associated with AVP release greater (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 secreted. Normalizing the pcBB total length with the insertion of a stop codon at the site of the normal stop codon was not associated with AVP 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 the 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 VP and intact NP, but not GP, coding regions are necessary for AVP processing and secretion; (2) decreasing the length of the NP coding region 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 BB rats is caused by the G deletion in NP coding region. This defect leads to abnormalities that contribute to the abnormal AVP processing. Specifically, the frameshift and absence of a stop codon cause a mutated extended C terminus, which, along with the mutated NP, contribute to the abnormal steps of AVP processing, transport, and secretion in the BB rat. These defects no doubt impair the folding and configuration necessary for normal processing of the AVP gene precursor.
authors.affiliation: Department of Medicine, University of Colorado Health Sciences Center, Denver 80262, USA.
authors.forename: J K
authors.initials: JK
authors.lastname: Kim
authors.forename: S N
authors.initials: SN
authors.lastname: Summer
authors.forename: W M
authors.initials: WM
authors.lastname: Wood
authors.forename: J L
authors.initials: JL
authors.lastname: Brown
authors.forename: R W
authors.initials: RW
authors.lastname: Schrier
mesh_terms: D000818:Animals; D001127:Arginine Vasopressin / Q000737:chemistry / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells / Q000378:metabolism; D003062:Codon / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D003919:Diabetes Insipidus / Q000235:genetics*; D016368:Frameshift Mutation; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011910:Rats, Brattleboro / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012636:Secretory Rate; D013728:Terminator Regions, Genetic; D014162:Transfection
chemical_list: D003062:Codon; D018076:DNA, Complementary; D011498:Protein Precursors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D001127:Arginine Vasopressin