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

Abstract

G-protein-coupled receptors (GPCRs), which constitute the largest family of cell surface receptors, were originally thought to function as monomers, but are now recognized as being able to act in a wide range of oligomeric states and indeed, it is known that the oligomerization state of a GPCR can modulate its pharmacology and function. A number of experimental techniques have been devised to study GPCR oligomerization including those based upon traditional biochemistry such as blue-native PAGE (BN-PAGE), co-immunoprecipitation (Co-IP) and protein-fragment complementation assays (PCAs), those based upon resonance energy transfer, FRET, time-resolved FRET (TR-FRET), FRET spectrometry and bioluminescence resonance energy transfer (BRET). Those based upon microscopy such as FRAP, total internal reflection fluorescence microscopy (TIRFM), spatial intensity distribution analysis (SpIDA) and various single molecule imaging techniques. Finally with the solution of a growing number of crystal structures, X-ray crystallography must be acknowledged as an important source of discovery in this field. A different, but in many ways complementary approach to the use of more traditional experimental techniques, are those involving computational methods that possess obvious merit in the study of the dynamics of oligomer formation and function. Here, we summarize the latest developments that have been made in the methods used to study GPCR oligomerization and give an overview of their application.

Keywords: FRET technology; GPCR; computational methods; oligomerization.

Figure 1. Techniques for studying oligomerization of GPCRs

The techniques used to study oligomerization of GPCRs may be divided into five categories, which are biochemical methods, biophysical methods, physiological methods, X-ray crystallography and computational methods. Biochemical approaches include Co-IP and BN-PAGE. Biophysical approaches include FRET, bioluminescence resonance energy transfer (BRET), protein-fragment complementation assays (PCA). SpIDA, single fluorescent-molecule and TIRFM are physiological methods.

Figure 2. Principles of BN-PAGE

(A) Cartoon representation of the major steps involved in BN-PAGE. (i) A mixture of proteins and protein complexes. (ii) Protein complexes are solubilized by mild non-ionic detergents (such as digitonin) and given a negative charge by adding Coomassie Blue that ‘coats’ the proteins in a similar manner to SDS, but without destroying quaternary structure. (iii) Proteins and complexes are separated by electrophoresis according to their molecular weight. (B) BN-PAGE shows that VSV-G–OX₁–eYFP migrates consistently with a predominantly dimeric structure. Samples were transferred onto a PVDF membrane and immunoblotted to detect the VSV–G tag, with two different exposures of the same samples shown. The migration of protein molecular mass markers is shown in the left-hand lane [23].

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 (<10 nm), so that upon excitation of CFP (with light at a wavelength of 433 nm), energy is transferred to YFP and causes emission (at 530 nm). (B) The principle of BRET is that energy transfer occurs between the luciferase and YFP. The reaction is initiated by addition of the substrate of luciferase, coelenterazine h, which results in the emission of light at 480 nm. If the distance between the donor and acceptor is <10 nm, energy is transferred from the luciferase to the YFP, resulting in a signal emitted by the YFP at 530 nm. (C) The principle of TR-FRET. A potential dimer pair of receptors bearing either Myc- or Flag-epitope tags is labelled with anti-Myc or -Flag antibodies against these. The antibodies are conjugated to either the ‘donor’ (Eu3+) species or the ‘acceptor’ (allophycocyanin, APC). (D) A potential dimer pair of receptors fused to SNAP and CLIP tags is treated with specific substrates for SNAP-tag and CLIP-tag, which have been linked to the ‘donor’ (Eu3+) and ‘acceptor’ (Cy5).

Figure 4. Schematic representation of PCA technology

(A) Cartoon representation of PCA. When the two receptors fused to two complementary fluorescent protein fragments (Vn and Vc) are in close proximity, fluorescence can be detected. (B) When three receptors labelled with RLuc, Vn and Vc are in close proximity, BRET will occur between RLuc and the re-assembled fluorescent protein. (C) FRET can be detected when three receptors fused to CFP, Vn and Vc are in close proximity.

Figure 5. The application of FRET spectrometry to the study of GPCR oligomerization

(A) FRET spectrometry analysis of Flp-In TM T-REx TM-293 cells expressing Myc–Y149C/A239GM₃R–Cerulean (inducible) and FLAG–M₃R–Citrine (constitutive). (i) Photomicrograph of a cell showing donor fluorescence in the presence of acceptor, acceptor fluorescence in the presence of donor and the Eapp distribution map at each pixel. (ii) Distinct configurations of donors and acceptors within a parallelogram (or rhombus)-shaped tetramer and their Eapp. (iii) Eapp histograms obtained from pixels representing the plasma membrane of the cell shown in (i) and the theoretical best fit with a sum of five correlated Gaussian peaks whose positions are given by a single adjustable parameter Ep (=0.18) via the rhombus tetramer model shown in (ii) (from Patowary et al. [107]). (B) Quantal brightness analysis in the technique of SpIDA of 5-HT_2C. Each intensity histogram was fit to a single population model with results consistent with the receptor being a monomer, a dimer and a tetramer [39].