A fluorescence resonance energy transfer-based sensor indicates that receptor access to a G protein is unrestricted in a living mammalian cell.
Fluorescence recovery after photobleaching of muscarinic receptors and G protein subunits tagged with cyan or yellow fluorescent protein showed that receptors and G proteins were mobile and not immobilized on the cell membrane. The cyan fluorescent protein-tagged Galpha and yellow fluorescent protein-tagged Gbeta subunits were used to develop sensors that coupled selectively with the M2 and M3 muscarinic receptors. In living Chinese hamster ovary cells, imaging showed that sensors emitted a fluorescence resonance energy transfer signal that was abrogated on receptor activation. When sequentially activated with highly expressed muscarinic receptors and endogenous receptors expressed at low levels, sensor molecules were sensitive to the sequence of activation and the receptor numbers. The results distinguish between models proposing that receptor and G protein types interact freely with each other on the cell membrane or that they function as mutually exclusive multimolecular complexes by providing direct support for the former model in these cells.
Azpiazu
Inaki
I
Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Gautam
N
N
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D018679:Cholinergic Agonists / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D043585:Receptor, Muscarinic M2 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D043587:Receptor, Muscarinic M3 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology; D014162:Transfection
D018679:Cholinergic Agonists; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D008164:Luminescent Proteins; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011993:Recombinant Fusion Proteins; D012701:Serotonin; D002217:Carbachol; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11