Metastable GPCR dimers trigger the basal signal by recruiting G-proteins

G-protein-coupled receptors (GPCRs) constitute the largest family of integral membrane proteins in the human genome and are responsible for various important signaling pathways for vision, olfaction, gustation, emotion, cell migration, etc. A distinct feature of the GPCR-family proteins is that many GPCRs, including the prototypical GPCR, β2-adrenergic receptor (β2AR), elicit low levels of basal constitutive signals without agonist stimulation, which function in normal development and various diseases1–3. However, how the basal signals are induced is hardly known. Another general distinctive feature of GPCRs is to form metastable homo-dimers, with lifetimes on the order of 0.1 s, even in the resting state. Here, our single-molecule-based quantification4 determined the dissociation constant of β2AR homo-dimers in the PM (1.6 ± 0.29 copies/μm2) and their lifetimes (83.2 ± 6.4 ms), and furthermore found that, in the resting state, trimeric G-proteins were recruited to both β2AR monomers and homo-dimers. Importantly, inverse agonists, which suppress the GPCR’s basal constitutive activity, specifically blocked the G-protein recruitment to GPCR homo-dimers, without affecting that to monomers. These results indicate that the G-proteins recruited to transient GPCR homo-dimers are responsible for inducing their basic constitutive signals. These results suggest novel drug development strategies to enhance or suppress GPCR homo-dimer formation.


G-protein-coupled receptors (GPCRs) constitute the largest family of integral
performed at 37˚C). The lifetime value obtained in this manner, termed  observed , was corrected for 52 the photobleaching lifetime and incidental colocalisation lifetime (this method was used for 53 evaluating all of the lifetimes investigated in the present research; see Methods), giving a dimer 54 lifetime  of 83.2 ± 6.4 ms (Fig. 1c). The dimer lifetime obtained at a time resolution of 4 ms (250 55 Hz) was 78.8 ± 10.7 ms, after corrections for photobleaching and incidental colocalisation lifetime 56 (Extended Data Fig. 3, Fig. 1c

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Using single-molecule-based "super-quantification" method previously developed by us 4 , we 65 obtained the 2-dimensional dissociation constant (2D-K D ) of 2AR homo-dimers in the PM of living 66 cells, which was 1.6 ± 0.29 copies/µm 2 (Fig. 2a, Extended Data Fig. 2b; see Methods). This 67 value is comparable to the 2D-K D of another GPCR, formyl-peptide receptor (FPR), which was 3.6 68 copies/µm 2 (in fact, this was the first determination of the 2D-K D in the PM of living cells) 4 .

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Under physiological conditions, 2AR is expressed at number densities of 16 ~ 260 copies/µm 2 , 70 depending on the cell type 8,9 . From the 2D-K D determined here, we conclude that 12 ~ 240 71 copies/µm 2 exist as dimers and 4 ~ 20 copies/µm 2 exist as monomers at any moment; i.e., 75 ~ 72 92% of 2AR molecules exist as homo-dimers at any moment, although dimers and monomers are 73 continually interconverting rapidly all the time (with a dimer lifetime of ~80 ms). In contrast, FPR is 74 mainly expressed in neutrophils, and an average of about 6,000 copies of FPR copies exist in the PM 75 of a single neutrophil 10 . Assuming that the neutrophil surface area is 1,300 µm 2 (approximated by a 76 sphere with a radius of 10 µm, providing a number density of ~4.6 FPR copies/ µm 2 ) and based on 77 the 2D-K D of FPR (3.6 copies/µm 2 ), 3,200 copies of FPR would exist as monomers and 2,800 copies 78 4 / 24 as dimers; namely, 54% of FPR molecules are dimers, which is about two-thirds of the dimer fraction 79 of 2AR. Note that, even under the conditions where the dimer population dominates (i.e., at higher 80 expression levels), the dimer lifetimes do not change and remain on the order of 0.1 s. Only the 81 monomer lifetimes are shortened at higher expression levels.

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Direct detection of β2AR dimers by the BiFC method 84 Thus far, we have detected dimers by the colocalisation of two single molecules, which reveals the 85 close encounters of molecules in the space scale of 220 nm, a distance much greater than the 86 molecular scale. Therefore, in this method, the incidental approaches of two molecules were 87 evaluated and subtracted. To ascertain the molecular-level interactions (dimers), we employed 88 YFP-based bimolecular fluorescence complementation (BiFC; meanwhile, our attempt to detect 89 FRET did not work, perhaps because the distance between the probes attached to two C-termini was 90 too far for FRET) 4,11 . In BiFC, two potentially interacting proteins are fused to the N-and C-terminal the dimer lifetime significantly (1.55x) ( Fig. 1c; Extended Data Fig. 2d). Cholesterol reportedly 107 modulates the functions of some GPCRs 13 , but here we found that mild cholesterol depletion by 108 methyl--cyclodextrin (MCD) treatment failed to affect the 2AR dimer lifetime (Fig. 1c).

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Since the inverse agonists suppressed the basal constitutive GPCR signaling, and since we 110 found that they also reduced the 2AR dimer lifetime, we next examined the dimer lifetimes of the 111 2AR mutants with high and low basal activities in the resting state (HBA and LBA mutants, 112 representing E268A and C327R mutants, respectively 14,15 ). The HBA mutant exhibited a 1.78x longer 113 dimer lifetime, and interestingly, after the inverse-agonist addition, it was reduced to the dimer 114 lifetime of the wild-type receptor ( Fig. 1c; Extended Data Fig. 2d). Meanwhile, the LBA mutant 115 exhibited a 0.74x shorter dimer lifetime than that of the wild-type receptor, and the agonist addition   Table 4). The addition of the agonist isoproterenol to the cells increased The presence of the basal constitutive activity of β2AR suggests that a stimulatory trimeric G-protein 138 might be recruited to β2AR in the resting state 17 . Therefore, we examined whether Gs and G 139 (conjugated with mGFP) are recruited to β2AR monomers or dimers, by examining their 140 colocalisations. Using L cells expressing both β2AR (ATTO594-ACP-β2AR) and Gs or G, we found 141 that, even in resting cells, Gs and G were recruited to both β2AR monomers and dimers ( Fig. 3a; 142 Supplementary Videos 1 and 2).

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The recruitment frequencies of Gs and G to 2AR monomers and dimers were measured  Table   146 3) 18 . In resting cells, both Gs and G molecules were recruited to 2AR monomers or dimers 147 equally well, suggesting that (non-stimulated) 2AR monomers and/or dimers might induce 148 intracellular Gs-dependent signals even in the resting state, which would lead to the basal 149 constitutive activity of 2AR.  Table 4). This result is quite surprising, but it suggests that the isoproterenol-induced 2AR 159 conformation enhanced its GEF activity, rather than increasing the on-rate of G protein binding to 160 2AR.

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Inverse agonists specifically block G-protein recruitment to β2AR dimers 163 Next, we examined the effects of the inverse agonists. The recruitment of Gs and G to β2AR 164 dimers was almost completely blocked, whereas that to β2AR monomers was unaffected (Fig. 3b,   165 c). This result unequivocally shows that β2AR dimers are responsible for the basal constitutive 166 activity of β2AR. Furthermore, it demonstrates that the inverse agonists work by blocking the 167 recruitment of stimulatory G-proteins to β2AR dimers, perhaps by inducing β2AR conformational 168 changes in the binding sites for G-proteins, rather than blocking the β2AR dimerisation (although, as almost completely blocked the recruitment of Gs and G to β2AR dimers, without affecting their 202 recruitment frequencies to β2AR monomers (Fig. 3b, c). The third result unequivocally shows that 203 the G proteins bound to β2AR dimers, and not monomers, in the resting state are responsible for 204 triggering the β2AR's basal constitutive activity, which is sensitive to inverse agonism. It further 205 suggests that the binding of the inverse agonists to β2AR induces conformational changes that 206 decrease the β2AR dimer affinities for G proteins (Fig. 2a)

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The present results suggest that the major functions of some of the orphan GPCRs might be 218 9 / 24 performed by the basal activities induced by their homo-dimers, as the GPCR family includes many 219 orphan receptors 3,23 . Based on the results described in this report, we strongly suggest the pursuit of 220 drug development strategies that emphasise the generation of more inverse agonists for various 221 GPCRs, as well as drugs that enhance or suppress GPCR homo-dimer formation.     (b, c) The inverse agonists, ICI-118,551 and timolol, inhibitors of 2AR basal constitutive activities, blocked the recruitment of trimeric G-proteins to 2AR homo-dimers, but not that to monomers. Gs (b) and G (c) colocalisation frequencies with 2AR monomers and homo-dimers, normalised by the number densities of Gαs (or G) and 2AR protomers (per sec per µm 4 ).