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    • Albert Einstein College of Medicine: Doctoral Dissertations
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    • Albert Einstein College of Medicine (AECOM)
    • Albert Einstein College of Medicine: Doctoral Dissertations
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    Control of yeast alpha-factor receptor localization and signaling

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    Date
    1998
    Author
    Shah, Anjani A.
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    Abstract
    The {dollar}\alpha{dollar}-factor receptor is a yeast G protein-coupled receptor that confers response to the mating pheromone, {dollar}\alpha{dollar}-factor. The role of highly conserved residues W295 and A296 situated at the end of the seventh hydrophobic domain of the receptor was tested. Substituted receptors exhibited altered ligand sensitivity and constitutive ubiquitination which suggested the equilibrium between inactive and active receptor states was shifted towards the activated state. Ligand-binding assays, cell fractionation and immunofluorescence microscopy revealed that substituted receptors were poorly expressed at the cell surface despite the fact that total protein levels of substituted receptors were similar to wild-type levels. An endocytosis block led to a disproportionate increase of A296D substituted receptor on the cell surface compared to wild-type receptor suggesting that A296D receptor was internalized more rapidly from and delivered more slowly to the plasma membrane than was wild-type receptor. Our results are most consistent with a model where residues W295 and A296 serve to keep the receptor in an inactive conformation. An active receptor conformation in addition to stimulating receptor endocytosis, may also inhibit efficient receptor transport to the plasma membrane.;The {dollar}\alpha{dollar}-factor receptor ({dollar}\alpha{dollar}-FR) is ubiquitinated and internalized in response to ligand binding. We tested the ability of an activated receptor to stimulate ubiquitination of an unactivated receptor. The {dollar}\alpha{dollar}-FR was ubiquitinated upon treatment of cells with an {dollar}\alpha{dollar}-factor antagonist only when a mutant receptor which could be activated by the antagonist was co-expressed. This result indicated that ubiquitination could be stimulated in trans. I showed that activation of the heterotrimeric G protein was not required for activation of receptor ubiquitination. Furthermore, an R233P/R234G substitution in the third intracellular loop of {dollar}\alpha{dollar}-FR that blocked activation of G protein did not block ubiquitination of the receptor. I propose that stimulation of {dollar}\alpha{dollar}-FR ubiquitination is a multi-step event initiated by residues distinct from those required for activation of G protein and distinct from the region of the receptor which receives the ubiquitin modification.;The role of Sst2 in modulating receptor signaling was studied. Sst2 is a member of the RGS family of GAP proteins. We showed that only in the absence of Sst2 did cell surface receptor levels determine the amount of response conferred to {dollar}\alpha{dollar}-factor. These and other results suggested that Sst2 may play a role in stabilizing a receptor/G protein complex.
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    https://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9821063
    https://hdl.handle.net/20.500.12202/3752
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    • Albert Einstein College of Medicine: Doctoral Dissertations [1674]

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