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First published online 4 July 2006
doi: 10.1242/jcs.03036

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Disulfide bonds determine growth hormone receptor folding, dimerisation and ligand binding

Department of Cell Biology, Institute of Biomembranes, University Medical Center Utrecht, Heidelberglaan 100, G02.525, 3584 CX Utrecht, The Netherlands

View larger version (28K): [in a new window]   Fig. 1. Schematic representation of the ß-sheet and disulfide bond organisation of the N-terminal portion of the extracellular domain of the GHR. The three-stranded ß-sheet, with strands A,B,E, is indicated in dark grey. The four-stranded ß sheet, with strands D,C,F,G, is indicated in light grey. Disulfide bonds are connected with lines. Bond 1 is C38-C48, bond 2 is C83-C94 and bond 3 is C108-C122.

View larger version (71K): [in a new window]   Fig. 2. Folding of the wild-type GHR. Transiently transfected ts20 cells were pulse-labelled for 10 minutes and chased for the indicated times. (A) Postnuclear lysates were split in equal amounts and either immunoprecipitated with anti-GHR-T (left panel), or isolated with btGH and streptavidin beads (right panel). Samples were subjected to reducing SDS-PAGE. p, 110-kDa precursor form; m, 130-kDa mature form. (B) After immunoprecipitation, samples were treated with EndoH (+) or mock treated (-). All samples were immunoprecipitated with anti-GHR-T and subjected to reducing SDS-PAGE. (C) Cells were pre-incubated with MG132 for 1 hour and subsequently treated with MG132 during pulse and chase periods (+), or mock treated (-). Samples were immunoprecipitated with anti-GHR-T and subjected to reducing SDS-PAGE. (D) After immunoprecipitation, samples were subjected to reducing (+) and non-reducing (-) SDS-PAGE. Relative molecular mass standards are indicated.

View larger version (48K): [in a new window]   Fig. 3. Maturation efficiency of wild-type and mutant GHRs. (A) Folding and maturation of wild type (WT) and mutated GHRs transiently transfected in ts20 cells. Cells were pulse labelled for 10 minutes and chased for 15 or 180 minutes (as indicated). Samples were immunoprecipitated with anti-GHR-T and subjected to reducing SDS-PAGE. p, 110-kDa precursor form; m, 130-kDa mature form; A, GHR(C38A-C48A); B, GHR(C83A-C94A); C, GHR(C108A-C122A); AB, AC, BC and ABC are combinations of mutations A,B and/or C. Relative molecular mass standards are shown on the right. (B) Quantification of the maturation efficiencies of the wild-type and mutant receptors. The amount of radioactivity was determined using ImageQuant (Molecular Dynamics). The amount of mature GHR species (130 kDa) was divided by the amount of precursor (110 kDa) and mature GHR species at 180 and 240 minutes chase and represented as a percentage relative to the maturation rate of wtGHR. The values represent the mean ± s.d. of at least two different experiments.

View larger version (60K): [in a new window]   Fig. 4. Ligand-binding capacities of the mutant receptors. (A) Transiently transfected ts20 cells were pulse-labelled for 10 minutes and chased for 90 or 240 minutes as indicated. Samples were split into equal amounts and immunoprecipitated (IP) with anti-GHR-T or isolated with btGH and streptavidin beads (btGH) and subjected to reducing SDS-PAGE. Receptor species are indicated as in Fig. 3. p, 110-kDa precursor form; m, 130-kDa mature form. Relative molecular mass standards are shown on the right. (B) Quantification of the ligand binding capacity of wild-type and mutated receptors. The amount of radioactivity was determined using ImageQuant (Molecular Dynamics). Precursor form intensities of the wild-type and mutant receptors after btGH pull-down were divided by precursor form intensities of the wild-type and mutant receptors after immunoprecipitation and represented as a percentage. The values represent the mean ± s.d. of at least two different experiments.

View larger version (37K): [in a new window]   Fig. 5. Dimerisation of the GHR mutations. Full-length (FL) wild-type (WT) and mutant GHRs were transiently transfected in ts20 cells, either together with GHR(1-369; HA-6xHis-Myc) (369-HA) bearing the different mutations or with empty vector (EV). 369-HA receptors were also co-transfected with EV. After lysis, full-length receptors were immunoprecipitated with anti-GHR-C and separated by reducing SDS-PAGE. (A) Immunoblotting of lysates with anti-HA antibody. Transfection combinations (+) are indicated. Mutant GHRs as indicated in Fig. 3. (B) Immunoblotting of anti-GHR-C immunoprecipitates with anti-HA antibody. (C) Efficiency of immunoprecipitation was determined by reprobing the blot shown in B with anti-GHR antibody Mab5. M, mix of lysate from ts20 cells transiently transfected with full-length GHR (mutants) and empty vector with lysate from ts20 cells transiently transfected with 369-HA GHR (mutants) and empty vector; m, mature GHR; p, precursor GHR; IP, immunoprecipitation; WB, immunoblotting. Relative molecular mass standards are shown on the left.

View larger version (27K): [in a new window]   Fig. 6. Cell-surface appearance of the mutant receptors. Cells transiently expressing empty vector, wtGHR (WT) or the GHR mutants were surface stained with monoclonal antibody Mab5 and subsequently with Cy5-conjugated goat-anti-mouse IgG. Living cells were gated and analysed by flow cytometry. WtGHR and the mutants are indicated with open histograms and shown as an overlay with the negative control, empty vector transfected cells (solid histogram). Receptor species are indicated as in Fig. 3. The data shown are representative of three independent experiments.

View larger version (32K): [in a new window]   Fig. 7. Signalling capacities of the mutant receptors. Transiently transfected ts20 cells expressing wtGHR (WT), GHR(C108S) or GHR(C108A-C122A) (C) were treated for 15 minutes with 8 nM btGH (GH) or btGHA (GHA) at permissive temperature (30°C) and lysed. (A) Immunoblotting of lysates with anti-GHR-C. After lysis, btGH(A)-GHR complexes were isolated with streptavidin beads and subjected to reducing SDS-PAGE. (B) Immunoblotting of complexes with anti-GHR-C. (C) The same blot as in B immunoblotted with anti-phosphotyrosine (anti-PY) antibody. m, mature GHR; p, precursor GHR; IP, immunoprecipitation; WB, immunoblotting. Relative molecular mass standards are shown on the left.

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