title: A 36 residues insertion in the dimerization domain of the growth hormone receptor results in defective trafficking rather than impaired signaling.
abstract: Growth hormone insensitivity syndrome (GHIS) has been reported in a family homozygous for a point mutation in the GH receptor (GHR) that activates an intronic pseudoexon. The resultant GHR (GHR1-656) includes a 36 amino-acids insertion after residue 207, in the region known to be important for homodimerization of GHR. We have examined the functional consequences of such an insertion in mammalian cells transfected with the wild type (GHRwt) and mutated GHR (GHR1-656). Radio-ligand binding and flow cytometry analysis showed that GHR1-656 is poorly expressed at the cell surface compared with GHRwt. Total membrane binding and Western blot analysis showed no such difference in the level of total cellular GHR expressed for GHR1-656 vs GHRwt. Immunofluorescence showed GHR1-656 to have different cellular distribution to the wild type receptor (GHRwt), with the mutated GHR being mainly perinuclear and less vesicular than GHRwt. Western blot analysis showed GH-induced phosphorylation of Jak2 and Stat5 for both GHR1-656 and GHRwt, although reduced Stat5 activity was detected with GHR1-656, consistent with lower levels of expression of GHR1-656 than GHRwt at the cell surface. In conclusion, we report that GHIS, due to a 36 amino-acids insertion in the extracellular domain of GHR, is likely to be explained by a trafficking defect rather than by a signalling defect of GHR.
authors.affiliation: Division of Clinical Sciences (North), University of Sheffield, Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7AU, UK.
authors.forename: M
authors.initials: M
authors.lastname: Maamra
authors.forename: A
authors.initials: A
authors.lastname: Milward
authors.forename: H Zarkesh
authors.initials: HZ
authors.lastname: Esfahani
authors.forename: L P
authors.initials: LP
authors.lastname: Abbott
authors.forename: L A
authors.initials: LA
authors.lastname: Metherell
authors.forename: M O
authors.initials: MO
authors.lastname: Savage
authors.forename: A J L
authors.initials: AJ
authors.lastname: Clark
authors.forename: R J M
authors.initials: RJ
authors.lastname: Ross
mesh_terms: D000596:Amino Acids / Q000235:genetics; D015153:Blotting, Western; D002462:Cell Membrane; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique / Q000379:methods; D005786:Gene Expression Regulation / Q000235:genetics; D006720:Homozygote; D006801:Humans; D053614:Janus Kinase 2; D046150:Laron Syndrome / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008297:Male; D010766:Phosphorylation; D017354:Point Mutation / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D011986:Receptors, Somatotropin / Q000235:genetics*; D050799:STAT5 Transcription Factor / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection
chemical_list: D000596:Amino Acids; D011518:Proto-Oncogene Proteins; D011986:Receptors, Somatotropin; D050799:STAT5 Transcription Factor; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2