The G protein coupled receptor (GPCR) GP133 is one of ~600 human orphan GPCRs that have been cloned whose ligands remain unidentified. GP133 is structurally related to four GPCRs found in the cellular slime mold D. discoideum that bind the extracellular agonist cyclic adenosine monophosphate (cAMP), resulting in the production of intracellular cAMP. A hemagglutinin (HA) tag was inserted in-frame in the GP133 coding sequence corresponding to the receptor's extracellular, N-terminal region. To confirm the receptor's cell surface expression, COS-7 cells transfected with HA-tagged GP133 will be immunofluorescently visualized utilizing anti-HA antibodies. Western immunoblotting will also be used to localize and quantitate GP133 expression, utilizing the same antibodies. Successful GP133 surface expression in the transfected cells will be indicated both by fluorescent staining localized to only the plasma membrane and high levels of GP133 in only the plasma membrane subcellular fraction.

Plasma membrane-specific expression of human G-protein coupled receptor GP133

247th National Meeting (2014)

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