Mutant and misfolded human growth hormone is rapidly degraded through the proteasomal degradation pathway in a cellular model for isolated growth hormone deficiency type II.
Autosomal dominant isolated growth hormone deficiency type II (IGHD II) is mainly caused by splice site mutations of the GH-1 gene, leading to deletion of amino acids 32-71 of the human growth hormone (hGH). The severe hGH deficit in IGHD II suggests a dominant negative effect of the partially deleted del(32-71)-hGH on the production, storage or secretion of normal wild-type (wt)-hGH in somatotrophic cells of the pituitary. To shed more light on the cellular and molecular basis of IGHD II, we established and analysed diverse clones of the rat somatotrophic cell line GH(4)C(1) stably expressing either wt-hGH, del(32-71)-hGH, or both proteins concomitantly. The cellular morphology of all transfected GH(4)C(1) cell clones showed moderate differences to untransfected GH(4)C(1) cells. On the molecular level, both cDNA-constructs induced transcription but, under normal culture conditions, only wt-hGH protein was found to be synthesised and secreted in readily detectable amounts. By contrast, only after inhibition of proteasomes did high amounts of del(32-71)-hGH show up. The solubility of del(32-71)-hGH in nondenaturing buffer was poor compared to wt-hGH, hinting at molecular aggregation, and several epitopes recognised by monoclonal hGH antibodies were not present on del(32-71)-hGH, confirming the assumption that del(32-71)-hGH must be severely misfolded. Expression of both proteins in Escherichia coli mirrored the findings from the GH(4)C(1) cell clones in terms of solubility and immunological reactivity. The results of the present study indicate that, in IGHD II, somatotrophs continuously have to remove misfolded del(32-71)-hGH via the proteasomal degradation pathway, suggesting a mechanism that may result in chronic cellular stress.
Kannenberg
K
K
Pediatric Endocrinology Section, University Children's Hospital, Tübingen, Germany. kai.kannenberg@med.uni-tuebingen.de
Wittekindt
N E
NE
Tippmann
S
S
Wolburg
H
H
Ranke
M B
MB
Binder
G
G
D000818:Animals; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D019382:Human Growth Hormone / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000378:metabolism; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011487:Protein Conformation; D017510:Protein Folding; D051381:Rats; D022142:Secretory Vesicles / Q000737:chemistry / Q000648:ultrastructure
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D019382:Human Growth Hormone; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde