Signaling through a G Protein-coupled receptor and its corresponding G protein follows a stoichiometrically limited model.
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.
Philip
Finly
F
Department of Physiology and Biophysics, Stony Brook University Medical Center, Stony Brook, New York 11794-8661, USA.
Sengupta
Parijat
P
Scarlata
Suzanne
S
D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology; D043782:Receptor, Bradykinin B2 / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014162:Transfection
D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D043782:Receptor, Bradykinin B2; D043562:Receptors, G-Protein-Coupled; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D001920:Bradykinin; D002118:Calcium