Methods used to study the oligomeric structure of G-protein-coupled receptors.

Methods to study oligomers of GPCRs Biochemical methods Co-IP and Western blotting Co-IP and Western blotting are probably one of the first techniques that provided direct evidence for β 2 -AR that it can form homodimers [ 12 ]. It has been used to detect oligomeric complexes of a number of GPCRs, such as β 2 -AR [ 12 ], DOR [ 31 ] and mGluR 5 [ 15 ] receptors. Using epitope tags and appropriate antibodies, the β 2 -AR was found to form homodimers and furthermore, these could be stabilized by agonists, whereas inverse agonists treatment was found to stabilize monomeric species. This suggests the possibility that interconversion between monomeric and dimeric forms may be important for biological activity [ 12 ]. Co-IP studies have also revealed that the adenosine receptor A 2 AR, dopamine receptor D 2 R and mGluR 5 receptors can form higher order oligomers [ 32 ], whereas the OX 1 receptor can form homodimers and even higher order oligomers [ 23 ]. When this approach is used to study GPCR quaternary structure, the cells containing the putative oligomers must first be lysed and solubilized. An antibody raised against one of the receptors or against epitope sequences such as V5, His or Myc fused to one of the receptors is used to separate the oligomer from the mixture of other proteins present in the lysate by, for instance the addition of protein G or A sepharose. This binds the antibody and allows separation by centrifugation. The oligomer may then be resolved by SDS/PAGE and the second receptor detected by Western blotting with a second antibody raised against the second receptor or an epitope tag to which it is fused [ 33 ]. Co-IP can be effective for detecting GPCR dimers and oligomers, both homodimers/oligomers and heterodimers/oligomers. However, a significant restriction to the use of Co-IP in the study of oligmerization in native systems is the paucity of specific and high-affinity antibodies to GPCRs themselves. Therefore, the ability to differentially epitope-tag GPCRs has been central to the use of Co-IP, though this does tend to limit the use of the technique to heterologously expressed receptors [ 22 ]. A further drawback of this methodology is that the sample lysis and solubilization required for releasing the protein of interest from its insoluble membrane environment, is a step that may, by itself, lead to artificial protein–protein associations being formed or possibly destroying existing associations. However, as an early and classic method, the Co-IP plus Western blotting approach is still used for detecting receptor–receptor interactions in vivo [ 33 ].

Biochemical methods Co-IP and Western blotting

Co-IP and Western blotting are probably one of the first techniques that provided direct evidence for β 2 -AR that it can form homodimers [ 12 ]. It has been used to detect oligomeric complexes of a number of GPCRs, such as β 2 -AR [ 12 ], DOR [ 31 ] and mGluR 5 [ 15 ] receptors. Using epitope tags and appropriate antibodies, the β 2 -AR was found to form homodimers and furthermore, these could be stabilized by agonists, whereas inverse agonists treatment was found to stabilize monomeric species. This suggests the possibility that interconversion between monomeric and dimeric forms may be important for biological activity [ 12 ]. Co-IP studies have also revealed that the adenosine receptor A 2 AR, dopamine receptor D 2 R and mGluR 5 receptors can form higher order oligomers [ 32 ], whereas the OX 1 receptor can form homodimers and even higher order oligomers [ 23 ]. When this approach is used to study GPCR quaternary structure, the cells containing the putative oligomers must first be lysed and solubilized. An antibody raised against one of the receptors or against epitope sequences such as V5, His or Myc fused to one of the receptors is used to separate the oligomer from the mixture of other proteins present in the lysate by, for instance the addition of protein G or A sepharose. This binds the antibody and allows separation by centrifugation. The oligomer may then be resolved by SDS/PAGE and the second receptor detected by Western blotting with a second antibody raised against the second receptor or an epitope tag to which it is fused [ 33 ]. Co-IP can be effective for detecting GPCR dimers and oligomers, both homodimers/oligomers and heterodimers/oligomers. However, a significant restriction to the use of Co-IP in the study of oligmerization in native systems is the paucity of specific and high-affinity antibodies to GPCRs themselves. Therefore, the ability to differentially epitope-tag GPCRs has been central to the use of Co-IP, though this does tend to limit the use of the technique to heterologously expressed receptors [ 22 ]. A further drawback of this methodology is that the sample lysis and solubilization required for releasing the protein of interest from its insoluble membrane environment, is a step that may, by itself, lead to artificial protein–protein associations being formed or possibly destroying existing associations. However, as an early and classic method, the Co-IP plus Western blotting approach is still used for detecting receptor–receptor interactions in vivo [ 33 ].

Biophysical methods FRET

FRET is a non-invasive and non-destructive method for studying protein–protein interactions. The principle of resonance energy transfer was first illustrated in the late 1940s by Förster [ 40 ]. FRET is a physical phenomenon in which energy transfers from one excited fluorescent protein (the donor) to another (the acceptor) in a non-radiative (dipole–dipole) manner [ 41 ], assuming certain conditions that are required for FRET to occur are met [ 42 , 43 ]. One key parameter is that there must be an overlap between the emission spectrum of the donor molecule and the excitation spectrum of the acceptor molecule, whereas another is that the donor and the acceptor must be in close proximity, generally less than 100-Å apart [ 44 ]. GFP from the crystal jelly Aequorea victoria and its variants such as CFP/YFP were the fluorophores most commonly used in early analyses for labelling potential ‘partner’ proteins of interest and we now refer to the use of these as classical FRET analyses ( Figure 3 A). Previously, in some applications, YFP has been replaced with a fluorescein arsenical hairpin binder (FlAsH) sequence, the binding of which by an inherently non-fluorescent ligand generates a fluorescent species that can function as an energy acceptor for the donor CFP [ 45 , 46 ]. Figure 3 Principles of FRET, BRET and time-resolved FRET (TR-FRET) ( A ) The principle of FRET using CFP and YFP as a donor-acceptor pair. FRET occurs when the donor (CFP, shown in cyan) and the acceptor (YFP, shown in yellow) are in close proximity (