ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2003) 5 P70

Growth hormone (GH) insensitivity due to a mutation in the cytoplasmic domain of the GH receptor distal to Box 1

AD Milward1, L Metherell2, M Maamra1, IR Wilkinson1, C Camacho-Hubner2, MO Savage2, RJM Ross1, AJL Clark2 & SM Webb3

1Division of Clinical Sciences North, University of Sheffield, Sheffield, UK; 2Barts & the London, Queen Mary University of London, UK; 3Hospital Sant Pau, Autonomous University of Barcelona, Spain.

Background: The majority of patients with GH insensitivity have defects in the extracellular domain of the GHR. We have identified a 47yr old woman homozygous for a 22bp deletion in the cytoplasmic domain of the GHR. The patient had high GH levels, and low IGF-1 of 8 ug/L (normal 54-389 ug/L), IGFBP-3 16nmol/L (normal 61-254 nmol/L) and GHBP 6.8 percent (normal greater than 10 percent) levels. We report functional studies for this mutation (GHR1-449) which results in premature termination of the receptor downstream of Box 1 (Jak2 binding) and upstream of the STAT5 binding site. Results: In transfected HEK293 cells, GHR1-449 showed similar specific binding (total bound - nonspecific bound / total counts x 100) of iodinated GH at the cell surface compared to GHRwt (mean plus/minus sem 4.1 plus/minus1.8 and 4.2 plus/minus 1.1 percent). In HEK293 cells, GHR1-449 showed no GH stimulated activation of a Stat5 responsive luciferase reporter and when cotransfected into cells with GHRwt inhibited GH stimulated Stat5 activation. As HEK293 cells have low level endogenous GHRwt expression, further experiments were performed in CHO cells. On Western blotting, CHO cells stimulated with GH and transfected with GHRwt showed Stat5 phosporylation whilst no Stat5 phosporylation was detected in cells transfected with GHR1-449. In contrast, GH-induced Jak2 phosphorylation was detectable in both GHR1-449 and GHRwt transfected cells (10.7 plus/minus 6 and 17.2 plus/minus 5 increased OD over control), as was Stat3 phosphorylation (33 plus/minus 7 and 39 plus/minus 8). Immunohistochemistry for GHR1-449 and GHRwt showed they had a similar cellular distribution. Conclusions: The mutant receptor GHR1-449 causes GH insensitivity presumably through a failure to signal through Stat5 as this receptor shows a similar cellular distribution to GHRwt and retains some signalling capacity through Stat3.

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