Abstract
Members of the cytokine receptor family have a consensus WSXWS sequence (WS motif) in the extracellular domain. With the interleukin-2, erythropoietin, and prolactin receptors, alteration of the WS sequence disrupts ligand binding and receptor signaling. The structural basis for these effects is unclear. To examine the role of the WS equivalent sequence (Y222GEFS226) in the function of the growth hormone receptor, each residue was mutated to alanine or to the WS consensus sequence. Although we used stable cell lines expressing all of these mutants, we show only three mutants, Y222A, G223A, and S226A, which display lower ligand affinity. Using conformation-specific monoclonal antibodies, we show that Y222A and S226A receptors have structural perturbations, which result in decreased signal transduction. This was shown by a decreased ability of growth hormone to stimulate protein synthesis and to transactivate the c-fos promoter with these mutants. The crystal structure of the ligand-occupied extracellular domain of growth hormone receptor indicates that Tyr222 and Ser226 have important interactions within the second beta-barrel domain, providing a structural basis for our results. The WS segment is not involved in sequence-specific accessory protein interaction, as mutation of residues Gly223, Glu224, and Phe225 does not alter receptor function.
Highlights
From the Department of PhysiologylPharrnacology and §The Centre for Molecular Biology and Biotechnology, The University of Queensland, St
Studies on the WS motif in interleukin-2(271, TyP’ pied extracellular domain of growth hormone receptorerythropoietin (28, 291, and prolactin [30] receptors have demindicates that and S e P 6have important interac- onstrated that this region is critical for maintaining ligand tions within the second P-barrel domain, providing a binding but is involved in signal transduction
Determination of Binding Constants-Cells containingstably expressed rGHR weregrown to confluence in six-well plates. These were of the rabbitgrowth hormonereceptor, each was mutated to anincubated for 16 ha t 12 "C with 200,000 cpm of 12sII-labelerdecombinant alanine. Inaddition, these residues were converted to thecon- bovine growth hormone in the presence of 12 different concensensus sequence (WSXWS)to explore why this motif differs in trations of unlabeled recombinant bGH in 1ml of serum-free binding the growth hormone receptor (GHR)
Summary
Studies on the WS motif in interleukin-2(271, TyP’ pied extracellular domain of growth hormone receptorerythropoietin These were of the rabbitgrowth hormonereceptor, each was mutated to anincubated for 16 ha t 12 "C with 200,000 cpm of 12sII-labelerdecombinant alanine Inaddition, these residues were converted to thecon- bovine growth hormone (bGH) in the presence of 12 different concensensus sequence (WSXWS)to explore why this motif differs in trations of unlabeled recombinant bGH in 1ml of serum-free binding the GHR. Fluence in 100-mm Petri dishes and serum-starvedfor 30 min in 10ml of PBS, 0.16bovine serum albumin. "'I-bGH (lo6cpm) was added to the dishes in the abseonrcepresence of 5 pglml cold bGH and incubated
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
