Principal methods used to identify GPCR-interacting proteins and phosphorylated residues.

GPCR-interacting proteins (1–4) and phosphorylated residues (5).

1. GeneticY2HHighly suitableEasy to perform.
Inexpensive.Loss of spatial-temporal information.
Membrane-anchored proteins cannot be investigated.
Performed in yeast.
MYTHHighly suitableEasy to perform.
Membrane-anchored proteins can be investigated.Loss of spatial-temporal information.
Soluble proteins cannot be investigated. 
Performed in yeast.
MaMTHHighly suitableEasy to perform.
Membrane anchored proteins can be investigated.
Performed in mammalian cells.Loss of spatial-temporal information.
Soluble proteins cannot be investigated.
KISSPossibleSensitive enough for studying interaction dynamic.Loss of spatial-temporal information.
Both membrane and cytosolic proteins can be investigated.Proteins involved in the STAT3 cascade cannot be investigated.
2. BiophysicalBRET/FRETNot suitablePrecise spatial-temporal information.
High sensitivity.
Possibility to study interactions in living cells.Generation of fusion proteins.
Relies on the proximity and relative orientation between donor and acceptor.
Fluorescent lifetime microscopySuitableMore accurate than intensity-based FRET.Data analysis more laborious than intensity-based FRET.
3. BiochemicalPLANot suitablePrecise spatial information (single-molecule resolution).
Possibility to perform in ex-vivo models.Relies on antibodies.
High cost.
Not easy to scale up in large studies.
BioIDSuitablePrecise spatial information.Not well suited for studying interaction dynamic (fluorescent signal is delayed).

Several interactions in parallel.  Possibility to perform in living cells.
NanoBitSuitablePrecise spatial information.
Several interactions in parallel.
Possibility to perform in living cells.
4. ProteomicCo-IPHighly suitablePurification of protein complexes in living cells and tissues.Rely on antibodies.
Loss of spatial-temporal information.
Lysis conditions might influence results.
Pull-downHighly suitableCan prove direct interaction.Loss of spatial-temporal information.
In vitro binding assays.
Fusion of the receptor on the beads might alter receptor conformation.
BioIDHighly suitableCan detect weak and transient interactions in living cells.Fusion of the biotin to the receptor might alter its targeting or functions.
5. Phosphorylation[32P]SuitableVery sensitive.Radioactive method.
Cannot give information on the number of phosphorylated residues nor their position.
LC-MSHighly suitableCan pinpoint phosphorylated residues.Can yield false negatives.
Not quantitative unless combined with very expensive isotope tags.
MutagenesisSuitableCheap and easy.Indirect method.
Based on functional data in living cells.
Can pinpoint phosphorylated residues.Mutagenesis of the C-terminus can impair expression and/or localization of the receptor.
Labor-intensive in case of multiple phosphosites.
Not quantitative.
Phospho-antibodiesSuitableDirect and indirect.
Can be used in any cell line.
Semiquantitative and qualitative.Time consuming and expensive for the generation of the antibodies.
Useless with low affinity antibodies.
Cannot give information on contiguous phosphorylated residues.
  • BiFC, bimolecular fluorescent complementation assay; BioID, proximity-dependent biotin identification; KISS, kinase substrate sensor; MaMTH, mammalian membrane two-hybrid assay; MYTH, membrane yeast two-hybrid assay; PLA, proximity ligation assay; Y2H, yeast two-hybrid assay.