GPCR homo-oligomerization

Abstract

G protein-coupled receptors (GPCRs) are an extensive class of trans-plasma membrane proteins that function to regulate a wide range of physiological functions. Despite a general perception that GPCRs exist as monomers an extensive literature has examined whether GPCRs can also form dimers and even higher-order oligomers, and if such organization influences various aspects of GPCR function, including cellular trafficking, ligand binding, G protein coupling and signalling. Here we focus on recent studies that employ approaches ranging from computational methods to single molecule tracking and both quantal brightness and fluorescence fluctuation measurements to assess the organization, stability and potential functional significance of dimers and oligomers within the class A, rhodopsin-like GPCR family.

Graphical abstract

Figure 1

Determination of receptor oligomeric structure by Spatial Intensity Distribution Analysis (SpIDA). (a) Constructs for determination of monomeric quantal brightness: upper panel, monomeric enhanced green fluorescent protein (mEGFP) is linked to the plasma membrane via a myristoylation/palmitoylation motif: lower panel A GPCR tagged at the intracellular C-terminal tail with mEGFP. (b) Constructs are expressed in, for example Flp-In T-REx-293 cells, and confocal microscope images obtained. (c) Images are opened in the SpIDA software (https://neurophotonics.ca/software), regions of interest selected and analysed for protein density and quantal brightness. (d) Average quantal brightness for mEGFP is determined using the myristoylation/palmitoylation-linked construct shown in (a). This generates a value of monomeric equivalent unit. (e) Measurements of the labelled GPCR yield an average quantal brightness value which can then be compared to the monomeric quantal brightness to determine oligomeric organization. See [4,16] for further details.

Figure 2

Effect of antagonist treatment on the serotonin 5-HT_2C receptor quaternary organization. SpIDA showing measures of individual regions of interest plotted as receptor number (density.μm²) versus monomeric equivalent units in Flp-In T-REx-293 cells expressing 5-HT_2C-mEGFP [14,20]. (a) untreated, (b), (c) and (d) Cells treated with SB-221284 (2,3-dihydro-5-(methylthio)-N-3-pyridinyl-6-(trifluoromethyl)-1H-indole-1-carboxamide) (75 nM), SDZ SER 082 fumarate ((+)-cis-4,5,7a,8,9,10,11,11a-octahydro-7H-10-methylindolo[1,7-bc][2,6]-naphthyridine fumarate) (5 μM) or S32212 hydrochloride (N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-1,2-dihydro-3-H-benzo[e]indole-3-carboxamide) (1 μM) respectively for 24 hours. Chemical structures for each ligand are shown. Each of these ligands has antagonist activity at the 5-HT_2CR and was used at a concentration calculated to be 10 × K_i. In each case treatment with the ligand results in predominantly monomeric status of the receptor