Comparison of the Activation Kinetics of the M3 Acetylcholine Receptor and a Constitutively Active Mutant Receptor in Living Cells

Abstract

Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G q -coupled M 3 acetylcholine receptor (M 3 -AChR) with that of a constitutively active mutant receptor (M 3 -AChR-N514Y) using M 3 -AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M 3 -AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M 3 -AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-Gprotein interaction was measured by FRET between M 3 -AChRyellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-G␥ 2 . Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M 3 -AChR-N514Y reached only 12% of that of M 3 -AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged G␣ q and CFP-tagged G␥ 2 . Activation of G q was significantly slower than receptor activation and indistinguishable for the two agonists. However, G q deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agoniststimulated coupling to G q , agonist-stimulated G q activation by M 3 -AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M 3 -AChR by decreasing the rate of receptor deactivation, while having minimal effect on receptor activation.

Spatial and Temporal Aspects of Signaling by G-Protein–Coupled Receptors

“…Using again various fluorescence resonance energy transfer-based sensors, Hlavackova et al (2012) determined a sequence of activation events in a dimeric metabotropic glutamate receptor, beginning with movement of the two receptor moieties toward each other (30 milliseconds), followed by the conformational change in the transmembrane region thought to produce the active, G-protein coupling form (40 milliseconds). Surprisingly, however, the activation of G-proteins in these systems is much slower, taking 500 milliseconds for the GDP/GTP exchange that results in the active form of the G-protein, irrespective of its subtype (i.e., G i , G s , or G q ) Hein et al, 2005Hein et al, , 2006Jensen et al, 2009;Hoffmann et al, 2012). This may indicate a slow step within the process of complex formation.…”

Influence of Gαq on the Dynamics of M3-Acetylcholine Receptor–G-Protein–Coupled Receptor Kinase 2 Interaction

“…The M 3 -AChR was obtained from the Missouri S&T cDNA Resource Center (Rolla, MO). cDNAs for Ga q , Ga q -yellow fluorescent protein (YFP) (Hughes et al, 2001), Gb 1 , Gg 2 , a 2A -adrenergic receptor (a 2A -AR), Ga i1 -YFP (C351I) (Bünemann et al, 2003), Ga i1 (C351I) (Wise et al, 1997), Ga o , Gb 1 -Cer (Frank et al, 2005), M 3 -AChR-YFP (Hoffmann et al, 2012), arrestin3-YFP (Krasel et al, 2005), and GRK2 (Winstel et al, 1996) were described previously. M 3 -AChR-mTurquoise fluorescent protein (mTurq) was cloned analogously to M 3 -AChR-YFP.…”

Functional studies cast light on receptor states

“…The analogous estimates for carbachol were 1.6 × 10 7 M −1 and 5.5 × 10 3 M −1 . Because acetylcholine has tenfold-greater potency than carbachol for eliciting M 3 responses 22 , the results suggest a K act value of approximately 10 8 M −1 for acetylcholine. Nearly the same K act value was estimated for acetylcholine at the muscle-type nicotinic receptor (5 × 10 7 M −1 ) 1 using single channel analysis, suggesting that similar binding pockets have evolved for acetylcholine on muscarinic and nicotinic receptors 23 .…”