Fluorescence Correlation Spectroscopy Analysis of Serotonin, Adrenergic, Muscarinic, and Dopamine Receptor Dimerization: The Oligomer Number Puzzle

Abstract

The issue of G protein-coupled receptor (GPCR) oligomer status has not been resolved. Although many studies have provided evidence in favor of receptor-receptor interactions, there is no consensus as to the exact oligomer size of class A GPCRs. Previous studies have reported monomers, dimers, tetramers, and higher-order oligomers. In the present study, this issue was examined using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH) analysis, a sensitive method for monitoring diffusion and oligomer size of plasma membrane proteins. Six different class A GPCRs were selected from the serotonin (5-HT 2A ), adrenergic (a 1b -AR and b 2 -AR), muscarinic (M 1 and M 2 ), and dopamine (D 1 ) receptor families. Each GPCR was C-terminally labeled with green fluorescent protein (GFP) or yellow fluorescent protein (YFP) and expressed in human embryonic kidney 293 cells. FCS provided plasma membrane diffusion coefficients on the order of 7.5 Â 10 29 cm 2 /s. PCH molecular brightness analysis was used to determine the GPCR oligomer size. Known monomeric (CD-86) and dimeric (CD-28) receptors with GFP and YFP tags were used as controls to determine the molecular brightness of monomers and dimers. PCH analysis of fluorescence-tagged GPCRs revealed molecular brightness values that were twice the monomeric controls and similar to the dimeric controls. Reduced x 2 analyses of the PCH data best fit a model for a homogeneous population of homodimers, without tetramers or higher-order oligomers. The homodimer configuration was unaltered by agonist treatment and was stable over a 10-fold range of receptor expression level. The results of this study demonstrate that biogenic amine receptors freely diffusing within the plasma membrane are predominantly homodimers.

G Protein–Coupled Receptor Oligomerization Revisited: Functional and Pharmacological Perspectives

“…As with the results obtained with TIR-FM, the homodimer configuration was unaltered by agonist treatment (Calebiro et al, 2013;Herrick-Davis et al, 2013). Unlike the fast dynamic behavior of GPCR monomers/dimers shown in TIR-FM experiments (Hern et al, 2010;Kasai et al, 2011;Calebiro et al, 2013), however, the dimers were stable over a 10-fold range of receptor expression levels (Herrick-Davis et al, 2013). This is also in line with other findings that suggest much more stable interactions, such as higher-order dopamine D 2 receptor oligomers, over a high range of receptor expression (Guo et al, 2008) and stable b 2 -adrenoceptor tetramers in phospholipid vesicles (Fung et al, 2009).…”

“…The recent FCS with a particle counting histogram approach by HerrickDavis et al (2013) also provides support for homodimers being the predominant, and perhaps only, species for several GPCRs, including a 1B -adrenoceptor, b 2 -adrenoceptor, serotonin 5-HT 2A and 5-HT 2c , muscarinic acetylcholine M 1 and M 2 , and dopamine D 1 receptors. As with the results obtained with TIR-FM, the homodimer configuration was unaltered by agonist treatment (Calebiro et al, 2013;Herrick-Davis et al, 2013). Unlike the fast dynamic behavior of GPCR monomers/dimers shown in TIR-FM experiments (Hern et al, 2010;Kasai et al, 2011;Calebiro et al, 2013), however, the dimers were stable over a 10-fold range of receptor expression levels (Herrick-Davis et al, 2013).…”

“…In summary, although a pattern of similar interfaces of GPCR homomers seems to be emerging, different interfaces can be found in different oligomers and even in different conformations of the same oligomer. Although agonists did not modify the dynamics of b 2 -adrenoceptor oligomerization in TIR-FM experiments (Calebiro et al, 2013) or the stability of b 2 -adrenoceptor oligomers in FCS-photon-counting histogram experiments (Herrick-Davis et al, 2013) or in FRET experiments in receptors into reconstituted phospholipid vesicles, an inverse agonist did modify the stability of b 2 -adrenoceptor oligomers in this latter preparation (Fung et al, 2009). Thus, selected ligands (which can stabilize specific conformations of the receptor) may still modify GPCR oligomeric interfaces and, therefore, the dynamics of receptor oligomerization.…”

Probing the pharmacology of G protein-coupled receptors with fluorescent ligands

“…The use of photon counting histogram (PCH) analysis may go some way to addressing this, as a doubling in mass (i.e. monomer to dimer) theoretically results in a doubling in molecular brightness, which is detectable (Herrick-Davis et al, 2013;Muller et al, 2000). PCH analysis has been used for GPCRs tagged with full length fluorescent proteins (Herrick- Davis et al, 2013;Kilpatrick et al, 2012) but as yet not been used with fluorescent ligands.…”

“…monomer to dimer) theoretically results in a doubling in molecular brightness, which is detectable (Herrick-Davis et al, 2013;Muller et al, 2000). PCH analysis has been used for GPCRs tagged with full length fluorescent proteins (Herrick- Davis et al, 2013;Kilpatrick et al, 2012) but as yet not been used with fluorescent ligands. FCS and other fluctuation based techniques in conjunction with fluorescent ligands can therefore be used to characterise populations of receptors within membrane microdomains, and FCS has provided important information on GPCR mobility, activation and oligomeric states.…”