TABLE 1

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

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

Classification	Method	Screening	Advantages	Disadvantages
1. Genetic	Y2H	Highly suitable	Easy to perform. Inexpensive.	Loss of spatial-temporal information.
				Membrane-anchored proteins cannot be investigated.
				Performed in yeast.

	MYTH	Highly suitable	Easy to perform. Membrane-anchored proteins can be investigated.	Loss of spatial-temporal information. Soluble proteins cannot be investigated.  Performed in yeast.

	MaMTH	Highly suitable	Easy to perform. Membrane anchored proteins can be investigated. Performed in mammalian cells.	Loss of spatial-temporal information.
				Soluble proteins cannot be investigated.

	KISS	Possible	Sensitive enough for studying interaction dynamic.	Loss of spatial-temporal information.
	KISS	Possible	Both membrane and cytosolic proteins can be investigated.	Proteins involved in the STAT3 cascade cannot be investigated.

2. Biophysical	BRET/FRET	Not suitable	Precise 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.
2. Biophysical
	Fluorescent lifetime microscopy	Suitable	More accurate than intensity-based FRET.	Data analysis more laborious than intensity-based FRET.

3. Biochemical	PLA	Not suitable	Precise 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.


	BioID	Suitable	Precise spatial information.	Not well suited for studying interaction dynamic (fluorescent signal is delayed).
	BioID	Suitable	Several interactions in parallel. Possibility to perform in living cells.
	NanoBit	Suitable	Precise spatial information. Several interactions in parallel. Possibility to perform in living cells.
	NanoBit	Suitable

4. Proteomic	Co-IP	Highly suitable	Purification of protein complexes in living cells and tissues.	Rely on antibodies.
				Loss of spatial-temporal information.
				Lysis conditions might influence results.

	Pull-down	Highly suitable	Can prove direct interaction.	Loss of spatial-temporal information.
				In vitro binding assays. Fusion of the receptor on the beads might alter receptor conformation.

	BioID	Highly suitable	Can detect weak and transient interactions in living cells.	Fusion of the biotin to the receptor might alter its targeting or functions.

5. Phosphorylation	[³²P]	Suitable	Very sensitive.	Radioactive method.
	[³²P]	Suitable	Very sensitive.	Cannot give information on the number of phosphorylated residues nor their position.

	LC-MS	Highly suitable	Can pinpoint phosphorylated residues.	Can yield false negatives. Not quantitative unless combined with very expensive isotope tags.
	LC-MS	Highly suitable	Can pinpoint phosphorylated residues.
	Mutagenesis	Suitable	Cheap 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-antibodies	Suitable	Direct 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.