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Structural Analysis of Human Growth Hormone with Respect to the Dominant Expression of Growth Hormone (GH) Mutations in Isolated GH Deficiency Type II

Pediatric Endocrinology Section, University Children’s Hospital, D-72076 Tuebingen Germany

Address all correspondence and requests for reprints to: Dr. Gerhard Binder, University Children’s Hospital, Pediatric Endocrinology Section, Hoppe-Seyler-Strasse 1, D-72076 Tuebingen Germany. E-mail: gerhard.binder{at}med.uni-tuebingen.de.

Human GH protein consists of four -helices and contains two disulfide bridges. Isolated GH deficiency type II (IGHD II) is mainly caused by heterozygous splice site mutations of GH-1 leading to the disruption of one disulfide bridge (Cys⁵³-Cys¹⁶⁵) and to the loss of amino acids (aa) 32–71, which comprise the complete loop between -helices 1 and 2. The mutant GH protein exerts a dominant negative effect on wild-type (wt) GH secretion by unclear mechanisms. For study of the structure-function relationship of GH mutants concerning the dominant negative effect, expression vectors harboring mutated GH cDNAs were transiently cotransfected with a vector encoding wtGH (pwtGH) into GH₄C₁ cells. Plasmids encoding ß-galactosidase, luciferase, or IGF-binding protein-2 were cotransfected with pwtGH and either of the GH mutants. Compared with the control transfection with pwtGH, GH secretion was mildly decreased by coexpressing wtGH and different GH point mutants with isolated disruption of the disulfide bridge Cys⁵³-Cys¹⁶⁵. Similar results were observed with GH mutants deleted in aa 32–46 or 32–52. Deletion of more aa (32–53, 32–63, 32–69, 32–71) ascendingly decreased GH secretion and content in parallel with the increasing length of the deleted stretch. An inhibitory dose-dependent effect of del32–69GH and del32–71GH on the activity/amount of coexpressed ß-galactosidase, luciferase, and IGF-binding protein-2 was found, whereas mRNA levels were unaffected. Hence, the extent of deletion played the major role in expression of the dominant negative effect. The inhibitory effect of GH mutants on heterologously expressed, non-GH proteins suggests that the dominant negative effect is not limited to GH or to proteins of the regulated secretory pathway, but may depend on expression levels.

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				D. I Iliev, N. E Wittekindt, M. B Ranke, and G. Binder In vitro analysis of hGH secretion in the presence of mutations of amino acids involved in zinc binding J. Mol. Endocrinol., August 1, 2007; 39(2): 163 - 167. [Abstract] [Full Text] [PDF]