In vitro characterization of ligand-induced oligomerization of the S. cerevisiae G-protein coupled receptor,Ste2p |
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Authors: | Chunhua Shi Matthew F Paige Jason Maley Michèle C Loewen |
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Institution: | 1. Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada;2. Department of Chemistry, 110 Science Place, University of Saskatchewan, Saskatoon, SK, S7N 5C9, Canada;3. Saskatchewan Structural Sciences Center, 110 Science Place, University of Saskatchewan, Saskatoon, SK, S7N 5C9, Canada;4. Department of Biochemistry, 107 Wiggins Road, University of Saskatchewan, Saskatoon, SK, S7N 5E2, Canada |
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Abstract: | BackgroundThe S. cerevisiae α-factor receptor, Ste2p, is a G-protein coupled receptor that plays key roles in yeast signaling and mating. Oligomerization of Ste2p has previously been shown to be important for intracellular trafficking, receptor processing and endocytosis. However the role of ligand in receptor oligomerization remains enigmatic.MethodsUsing functional recombinant forms of purified Ste2p, atomic force microscopy, dynamic light scattering and chemical crosslinking are applied to investigate the role of ligand in Ste2p oligomerization.ResultsAtomic force microscopy images indicate a molecular height for recombinant Ste2p in the presence of α-factor nearly double that of Ste2p alone. This observation is supported by complementary dynamic light scattering measurements which indicate a ligand-induced increase in the polydispersity of the Ste2p hydrodynamic radius. Finally, chemical cross-linking of HEK293 plasma membranes presenting recombinant Ste2p indicates α-factor induced stabilization of the dimeric form and higher order oligomeric forms of the receptor upon SDS-PAGE analysis.Conclusionsα-factor induces oligomerization of Ste2p in vitro and in membrane.General significanceThese results provide additional evidence of a possible role for ligand in mediation of Ste2p oligomerization in vivo. |
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Keywords: | AFM atomic force microscopy DLS dynamic light scattering DM N-dodecyl-β-d-maltoside GFP green fluorescent protein GPCR G protein-Coupled receptor PBS phosphate buffered saline Ste2p α-factor receptor SMCC succinimidyl trans-4-(maleimidylmethyl) cyclohexane-1-carboxylate 293E human embryonic kidney cells stably expressing the 293Epstein Barr virus nuclear antigen |
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