Effect of zinc binding residues in growth hormone (GH) and altered intracellular zinc content on regulated GH secretion

摘要

Endocrine cells store hormones in concentrated forms (aggregates) in dense-core secretory granules that are released upon appropriate stimulation. Zn2+ 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/orGHZn mutant (GH-H18A-H21AE174A) 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 Zn2+ chelator, led to a significant reduction of the stimulated wt-GH secretion. Furthermore, externally added Zn2+ to culture medium increased intracellular free Zn2+ levels and recovered wt-GH secretion, suggesting its direct dependence on free Zn2+ 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 Zn2+ as well as altering intracellular free Zn2+ content mayinterfere with normalGHdimerization (aggregation)andstorage of themutantvariant (alone or with wt-GH), which could possibly explain impaired GH secretion.