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Pediatric Endocrinology Section, University Children’s Hospital, Hoppe-Seyler-Street 1, D-72076 Tuebingen, Germany

(Requests for offprints should be addressed to G Binder; Email: gerhard.binder{at}med.uni-tuebingen.de)

N E Wittekindt is now at The Pennsylvania State University, 311 Wartik Building, University Park, Pennsylvania 16802, USA

Zinc (Zn²⁺) 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 Zn²⁺ 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 GH₄C₁ cells. Plasmids encoding ß -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 ß -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 Zn²⁺ 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 Zn²⁺ binding as a major pathogenetic mechanism in dominantly transmitted GH deficiency.