In vitro characterization of ligand-induced oligomerization of the S. cerevisiae G-protein coupled receptor, Ste2p.
BACKGROUND
The S. cerevisiae alpha-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.


METHODS
Using 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.


RESULTS
Atomic force microscopy images indicate a molecular height for recombinant Ste2p in the presence of alpha-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 alpha-factor induced stabilization of the dimeric form and higher order oligomeric forms of the receptor upon SDS-PAGE analysis.


CONCLUSIONS
alpha-factor induces oligomerization of Ste2p in vitro and in membrane.


CONCLUSIONS
These results provide additional evidence of a possible role for ligand in mediation of Ste2p oligomerization in vivo.
Shi
Chunhua
C
Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada.
Paige
Matthew F
MF
Maley
Jason
J
Loewen
Michèle C
MC
D001704:Biopolymers; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D003902:Detergents; D006801:Humans; D008024:Ligands; D008027:Light; D008301:Maleimides / Q000737:chemistry; D000072235:Mating Factor; D008823:Micelles; D018625:Microscopy, Atomic Force; D010455:Peptides / Q000737:chemistry; D051964:Receptors, Mating Factor / Q000819:agonists / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000819:agonists / Q000737:chemistry* / Q000235:genetics; D012542:Scattering, Radiation; D012996:Solutions
D001704:Biopolymers; D003432:Cross-Linking Reagents; D003902:Detergents; D008024:Ligands; D008301:Maleimides; D008823:Micelles; D010455:Peptides; D051964:Receptors, Mating Factor; D011994:Recombinant Proteins; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D000072235:Mating Factor; C025285:N-(4-carboxycyclohexylmethyl)maleimide N-hydroxysuccinimide ester