Accumulation |
Ca2+ or cAMP
accumulation |
Binding/coupling |
Proximity |
Activation |
Receptor conformation and G protein activation e.g., GTPase
activity, GTPγS binding, or recruitment of downstream
effectors. |
Dissociation |
G protein dissociation/rearrangement |
Phosphorylation |
Incorporation of 32P in a specific
protein. Two-dimensional phosphopeptide mapping after orthophosphate
labelling, MS-based identification of phosphorylation sites,
Phosphosite-specific antibody assays or arrays |
Recruitment |
Subcellular redistribution |
Signaling |
Downstream cellular second messenger levels |
Internalization |
ELISA, Flow cytometry, diffusion-enhanced resonance
energy transfer (DERET), enhanced bystander BRET (ebBRET), (confocal)
microscopy, antibody-feeding experiments, real-time internalization by
SNAP-tagged receptors |
Tissue phenotype |
Blood vessel contraction, cell migration, hormone
secretion, heart rate, force of contraction, glandular secretion
etc. |
Transcription |
Differential gene arrays after agonism, qPCR, and gene
reporter assay. |