Abstract
ABSTRACTThis study investigates endocytosis of Saccharomyces cerevisiae α-factor receptor and the role that receptor oligomerization plays in this process. α-factor receptor contains signal sequences in the cytoplasmic C-terminal domain that are essential for ligand-mediated endocytosis. In an endocytosis complementation assay, we found that oligomeric complexes of the receptor undergo ligand-mediated endocytosis when the α-factor binding site and the endocytosis signal sequences are located in different receptors. Both in vitro and in vivo assays suggested that ligand-induced conformational changes in one Ste2 subunit do not affect neighboring subunits. Therefore, recognition of the endocytosis signal sequence and recognition of the ligand-induced conformational change are likely to be two independent events.
Highlights
G protein-coupled receptors (GPCRs) are cell-surface receptors that are present in all eukaryotic organisms
Complementation between two mutant Ste2 subunits during endocytosis Our laboratory previously has shown that when wild-type afactor receptors undergo ligand-mediated endocytosis, they cause a concomitant internalization of co-expressed endocytosisdefective receptors (Yesilaltay and Jenness, 2000)
Green fluorescent protein (GFP) was fused to the C-terminus of the truncated Ste2-T326 receptors and to the truncated binding-defective Ste2-F204S,T326 receptors (Ste2-T326-GFP and Ste2-F204S,T326-GFP, respectively) in order to monitor their position by fluorescence microscopy when they are co-expressed with full-length untagged receptors
Summary
G protein-coupled receptors (GPCRs) are cell-surface receptors that are present in all eukaryotic organisms. They mediate cellular responses to a wide variety of extracellular ligands and conditions (e.g. hormones, neurotransmitters, odorants and light), and their activity is regulated by covalent modification, by interacting proteins and by membrane protein trafficking (Jean-Alphonse and Hanyaloglu, 2011; Magalhaes et al, 2012). The a-factor receptor encoded by the STE2 gene of Saccharomyces cerevisiae is a GPCR that mediates the Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA. *Present address: Department of Cancer Biology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01655, USA.
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