In vitro analysis of hGH secretion in the presence of mutations of amino acids involved in zinc binding

Abstract

Zinc (Zn 2C ) binding by human GH through amino acid residues His18, His21, and Glu174 has been described as a prerequisite for GH dimerization and storage in secretory granules. Our aim was to investigate in vitro whether disturbed Zn 2C binding of mutant GH inhibits wild-type GH (wtGH) secretion and contributes to the pathogenetic mechanisms involved in dominantly transmitted isolated GH deficiency type II. Seven expression vectors harboring mutated human GH cDNAs were constructed in which nucleotide triplets encoding histidine or glutamine at positions 18, 21, and 174 were mutated to triplets encoding alanine: H18A, H21A, G174A, H18A-G174A, H21A-G174A, H18A-H21A, and H18A-H21A-G174A. These vectors were transiently cotransfected with a vector encoding wtGH or were singly transfected into rat pituitary GH 4 C 1 cells. Plasmids encoding b-galactosidase were cotransfected. 48h after transfection, GH in media and cell extracts was measured using a GH-specific RIA, and results were normalized for transfection efficiency by means of b-galactosidase activity. In comparison with the control transfection (wtGH/wtGH set at 100%), GH secretion remained unaffected when coexpressing wtGH and any of the GH mutants in which Zn 2C binding was partially or completely prevented. When these mutants were singly expressed, the amount of GH in both media and cell extracts was decreased by about 50% when compared with cells expressing only wtGH. Our in vitro data do not support the hypothesis of disturbed Zn 2C binding as a major pathogenetic mechanism in dominantly transmitted GH deficiency.

Effect of Zinc Binding Residues in Growth Hormone (GH) and Altered Intracellular Zinc Content on Regulated GH Secretion

“…wt-GH and/or different Zn 2ϩ -binding GH mutants were transiently expressed, and GH secretion and intracellular GH expression were analyzed in nonstimulated conditions after 48 hours of incubation. Both wt-GH secretion and intracellular GH accumulation remained unaffected when wt-GH was coexpressed with any of the Zn 2ϩ -binding GH mutants (including also double and triple mutants) (20). Interestingly, extracellular secretion and intracellular production of the GH mutants singly expressed in GH 4 C 1 cells was about 50% lower when compared with that of wt-GH, suggesting the role of Zn 2ϩ -binding residues in GH stability (20).…”

“…Cloning of human wt-GH in pcDNA3.1 (Ϫ) neo vector (Invitrogen) and generation GH Zn mutant (GH-H18A-H21A-E174A) was made as previously described (20).…”

“…Furthermore, Iliev et al (20) reported that disturbed Zn 2ϩ binding of GH mutants (with alanine substitutions at positions H18, H21, and E174 introduced in various combinations) led to their reduced extracellular secretion and intracellular production compared with wt-GH (reduction of 50% or more) when singly expressed in GH 4 C 1 cells. Moreover, cotransfection with wt-GH and any of the Zn 2ϩ binding GH mutants (including also double and triple mutants) revealed that both wt-GH secretion and production remained unaffected, suggesting that disturbed Zn 2ϩ binding is unlikely to be the cause of dominantly transmitted GH deficiency (GHD).…”

The Role of Zinc Dynamics in Growth Hormone Secretion

“…The potential contribution of high-affinity Zn 2+ -binding residues in GH to the pathogenetic mechanisms involved in dominantly transmitted isolated GH deficiency type II was further studied by Iliev et al [38]. The production and extracellular secretion of wt -hGH transiently transfected in GH 4 C 1 cells (rat pituitary tumour cells) was compared to that of GH mutants in which the amino acids that bind Zn 2+ with high affinity were mutated to alanine in various combinations (Zn 2+ -binding GH mutants).…”

From Endoplasmic Reticulum to Secretory Granules: Role of Zinc in the Secretory Pathway of Growth Hormone

“…Importantly, in this study it was also demonstrated that Zn 2+ binding to GH would enhance the stability of the stored form (GH denaturation measured by guanidine hydrochloride at pH 8) and that the Zn 2+ -GH complex was more stable to denaturation when compared to monomeric GH [18]. In order to investigate further whether disturbed Zn 2+ binding of GH mutants (in which single, double or triple alanine substitutions at positions His18, His21 and Glu174 have been introduced) inhibits wt-GH secretion or production or if wt-GH and/or different GH mutants were transiently expressed in GH 4 C 1 cells [26]. The measurement of constitutive GH secretion (i.e.…”