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Mammalian GPCR profiling

JC John D. Chan
PC Pauline M. Cupit
GG Gihan S. Gunaratne
JM John D. McCorvy
YY Yang Yang
KS Kristen Stoltz
TW Thomas R. Webb
PD Peter I. Dosa
BR Bryan L. Roth
RA Ruben Abagyan
CC Charles Cunningham
JM Jonathan S. Marchant
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Primary GPCR screening assays were coordinated through the NIMH Psychoactive Drug Screening Program (PDSP). Methods and statistical analyzes for radioligand binding and functional assays are available in the PDSP Assay Protocol Book available online (https://pdspdb.unc.edu/pdspWeb/). Ca2+ flux assays were performed as similarly described, except experiments utilized muscarinic M1, M3, M5 receptors stably expressed in CHO cells. For experiments measuring cAMP, experiments utilized co-transfection with the cAMP split-luciferase reporter GloSenso(R)-22F plasmid (Promega, 1:1 ratio with receptor) and were read for luminescence on a TriLux Microbeta (Perkin Elmer). For Gi/o mediated cAMP inhibition, forskolin (1 µM) was used to stimulate cAMP via adenylyl cyclase activation.

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