title: Effect of zinc binding residues in growth hormone (GH) and altered intracellular zinc content on regulated GH secretion.
abstract: Endocrine cells store hormones in concentrated forms (aggregates) in dense-core secretory granules that are released upon appropriate stimulation. Zn(2+) binding to GH through amino acid residues His18, His21, and Glu174 are essential for GH dimerization and might mediate its aggregation and storage in secretory granules. To investigate whether GH-1 gene mutations at these positions interfere with this process, GH secretion and intracellular production were analyzed in GC cells (rat pituitary cell line) transiently expressing wt-GH and/or GH Zn mutant (GH-H18A-H21A-E174A) in forskolin-stimulated vs nonstimulated conditions. Reduced secretion of the mutant variant (alone or coexpressed with wt-GH) compared with wt-GH after forskolin stimulation was observed, whereas an increased intracellular accumulation of GH Zn mutant vs wt-GH correlates with its altered extracellular secretion. Depleting Zn(2+) from culture medium using N,N,N',N'-tetrakis(2-pyridylemethyl)ethylenediamine, a high-affinity Zn(2+) chelator, led to a significant reduction of the stimulated wt-GH secretion. Furthermore, externally added Zn(2+) to culture medium increased intracellular free Zn(2+) levels and recovered wt-GH secretion, suggesting its direct dependence on free Zn(2+) levels after forskolin stimulation. Confocal microscopy analysis of the intracellular secretory pathway of wt-GH and GH Zn mutant indicated that both variants pass through the regulated secretory pathway in a similar manner. Taken together, our data support the hypothesis that loss of affinity of GH to Zn(2+) as well as altering intracellular free Zn(2+) content may interfere with normal GH dimerization (aggregation) and storage of the mutant variant (alone or with wt-GH), which could possibly explain impaired GH secretion.
authors: PhD, Division of Pediatric Endocrinology, Diabetology, and Metabolism, University Children's Hospital, Inselspital, CH-3010 Bern, Switzerland. vibor.petkovic@dkf.unibe.ch.
authors: Vibor
authors: V
authors: Petkovic
authors: Maria Consolata
authors: MC
authors: Miletta
authors: Andrée
authors: A
authors: Eblé
authors: Daniel I
authors: DI
authors: Iliev
authors: Gerhard
authors: G
authors: Binder
authors: Christa E
authors: CE
authors: Flück
authors: Primus E
authors: PE
authors: Mullis
mesh_terms: D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D002470:Cell Survival; D003470:Culture Media; D005021:Ethylamines; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D011725:Pyridines; D051381:Rats; D013997:Time Factors; D015032:Zinc / Q000008:administration & dosage / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D003470:Culture Media; D005021:Ethylamines; C511824:N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine; D011725:Pyridines; D013006:Growth Hormone; D015032:Zinc