Monitoring the state of cholecystokinin receptor oligomerization after ligand binding using decay of time-resolved fluorescence anisotropy.
Oligomeric complexes of G protein-coupled receptors (GPCRs) are now commonly recognized and can provide a mechanism for regulation of signaling systems. Receptor oligomerization has been most extensively studied using coimmunoprecipitation and bioluminescence or fluorescence resonance energy-transfer techniques. Here, we have utilized decay of time-resolved fluorescence anisotropy of yellow fluorescent protein-labeled cholecystokinin receptor constructs to examine the state of oligomerization of this receptor in living cells. The rotational correlation times established that the cholecystokinin receptor is constitutively present in an oligomeric state that is dissociated in response to agonist occupation. In contrast, antagonist occupation failed to modify this signal, leaving the oligomeric structure intact. This dynamic technique complements the other biochemical and steady-state fluorescence techniques to establish the presence of oligomeric receptor complexes in living cells.
Harikumar
Kaleeckal G
KG
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ 85259, USA.
Miller
Laurence J
LJ
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D019281:Dimerization; D005454:Fluorescence Polarization; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D011949:Receptors, Cholecystokinin / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D008024:Ligands; D008164:Luminescent Proteins; D011949:Receptors, Cholecystokinin