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      Signaling through a G Protein-coupled receptor and its corresponding G protein follows a stoichiometrically limited model.

      The Journal of Biological Chemistry

      Transfection, physiology, drug effects, Signal Transduction, metabolism, genetics, Receptors, G-Protein-Coupled, Receptor, Bradykinin B2, cytology, Kidney, Humans, GTP-Binding Proteins, GTP-Binding Protein gamma Subunits, GTP-Binding Protein beta Subunits, GTP-Binding Protein alpha Subunits, Gq-G11, Fluorescence Resonance Energy Transfer, Cell Line, Calcium, pharmacology, Bradykinin

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          Abstract

          The bradykinin receptor is a G protein-coupled receptor (GPCR) that is coupled to the Galpha(q) family of heterotrimeric G proteins. In general, a GPCR can exert intracellular signals either by transiently associating with multiple diffusing G protein subunits or by activating a G protein that is stably bound to the receptor, thus generating a signal that is limited by the stoichiometry of the complex. Here we have distinguished between these models by monitoring the association of type 2 bradykinin receptor (B(2)R) and the Galpha(q)/Gbetagamma heterotrimer in living human embryonic kidney 293 cells expressing fluorescent-tagged proteins. Stable B(2)R-Galpha(q) x Gbetagamma complexes are observed in resting cells by fluorescence resonance energy transfer from either Galpha(q)-eCFP or eCFP-Gbetagamma to B(2)R-eYFP. Stimulating the cells with bradykinin causes detachment of B(2)R from the G protein subunits as the receptor internalizes into early endosomes, with a corresponding elimination of B(2)R-G protein fluorescence resonance energy transfer because Galpha(q) and its associated Gbetagamma remain on the plasma membrane. Single point and scanning fluorescence correlation spectroscopy measurements show that a portion of B(2)R molecules diffuses with a mobility corresponding to dimers or small oligomers, whereas a second fraction diffuses in higher order molecular assemblies. Our studies support a model in which receptors are pre-coupled with their corresponding G proteins in the basal state of cells thereby limiting the response to an external signal to a defined stoichiometry that allows for a rapid and directed cellular response.

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          Journal
          17420253
          10.1074/jbc.M701558200

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