In vitro analysis of hGH secretion in the presence of mutations of amino acids involved in zinc binding.
Zinc (Zn(2+)) 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(2+) 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 beta -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 beta -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(2+) 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(2+) binding as a major pathogenetic mechanism in dominantly transmitted GH deficiency.
Iliev
Daniel I
DI
Pediatric Endocrinology Section, University Children's Hospital, Tuebingen, Germany.
Wittekindt
Nicola E
NE
Ranke
Michael B
MB
Binder
Gerhard
G
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D005822:Genetic Vectors / Q000235:genetics; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D011863:Radioimmunoassay; D014162:Transfection; D015032:Zinc / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D050505:Mutant Proteins; D019382:Human Growth Hormone; D001616:beta-Galactosidase; D015032:Zinc