Background: Mutations in the GHR result in extreme short stature (Laron's syndrome). We have studied mutations from two patients with Laron's syndrome. Patient 1 (GHRextra) has a mutation in the GHR extracellullar domain due to a 108bp inframe pseudoexon between exons 6 and 7. Patient 2 (GHRintra) has a 22bp deletion in exon 10 of the GHR leading to premature termination of the GHR upstream of the intracellular STAT5 binding site.
Aim: To compare GHR structure and function.
Methods: GHR mutant and wild type (GHRwt) cDNAs were cloned into mammalian expression vectors, and experiments performed after transient transfection of cDNA into HEK293 and CHO cells. Receptor expression was studied by radiolabelled GH binding studies and FACS analysis. GHR signalling was measured using the STAT5/STAT3 responsive reporter plasmid LHRE-TK-luc.
Results: GHRextra showed low specific binding of GH at the cell surface compared to GHRintra and GHRwt (1.0 plus/minus 0.2 vs 4.1 plus/minus 1.8 and 4.2 plus/minus 1.1 percent). Low binding of GHRextra was shown to be due to reduced receptor expression by FACS analysis using an antibody with an epitope distant to the mutation. In HEK293 cells, GHRextra showed greater induction of the LHRE luciferase reporter to GHRwt (24.7 plus/minus 0.7 vs 10.85 plus/minus 2.15 fold induction luciferase activity), whilst GHRintra showed no signalling and inhibited GHRwt signalling. In CHO cells GHRintra showed some induction of the LHRE at high concentrations of GH (2.27 plus/minus 0.20 fold induction with GH 500 nanograms/millilitre).
Conclusions: GHRextra has a 36aa insertion in the proposed dimerisation domain of the GHR. This insertion results in reduced expression of receptor at the cell surface but the receptor appears to retain signalling activity through STAT5. GHRintra shows good cell surface expression but as expected has reduced signalling, although at high concentrations of GH signalling may occur through STAT3.
08 - 11 Apr 2002
British Endocrine Societies