Fusion with Promiscuous Gα₁₆ Subunit Reveals Signaling Bias at Muscarinic Receptors

Abstract

A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous α-subunit of G₁₆ protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα₁₆ limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα₁₆ mediated pathway more specifically. Our data demonstrated agonist-specific activation of G₁₆ pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards Gα₁₆ pathway at the M₂ receptor and at the same time impaired Gα₁₆ signaling of iperoxo at M₅ receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.

1. Introduction

G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins that transmit signals into a cell through heterotrimeric G-proteins. GPCRs represent the primary target for drug development with potential application in essentially all clinical fields. They mediate a broad range of physiological processes by driving multiple intracellular effectors through various classes of G-proteins. Individual GPCRs preferentially couple to the particular class of G-proteins but they can also successfully activate others [1,2,3]. This coupling promiscuity was observed in both artificial systems with over-expressed GPCRs and native cells [4,5]. Besides G-proteins, GPCR can couple with β-arrestins which desensitize and scaffold G-protein-driven signaling pathways [6]. The multiplicity of signaling leads to the high complexity of the functional response of GPCRs to agonist stimulation.

Structurally different agonists induce specific changes in the GPCRs leading to stabilization of agonist-specific conformations that can lead to non-uniform agonist-specific modulation of signaling pathways. This preferential orientation of the signaling of a GPCR towards a subset of its signal transducers is termed signaling bias [7]. An agonist biased to a particular G-protein pathway may promote therapeutically desired signaling while simultaneously avoiding side effects mediated by activation of others, especially in conditions with well-understood pathophysiology [8,9,10]. For example, melanocortin receptor 4 (MC4R) agonist melanotan II produces its anorectic effects through coupling to G_q/11 and its adverse cardiovascular effects through G_s coupling, suggesting potential therapeutic benefit in obesity for G_q/11-biased ligands [11].

The accurate evaluation of agonist bias regarding individual G-protein pathways is crucial for preclinical drug development. However, it is a difficult task given the high complexity of GPCRs signaling. The molecular crosstalk that can occur among downstream effector molecules may bring in further complexity [12]. One of the most challenging tasks is to develop a suitable technique for the analysis of the signaling pathway of interest with high sensitivity and sufficient selectivity that is free from the interference of other signaling pathways. Measurement of second messengers struggles with molecular crosstalk of signaling pathways. The analysis of coupling of GPCRs with individual G-proteins is difficult due to the presence of others that interact concurrently with a given signaling pathway, especially in studies of non-preferential signaling pathways [13] or in studies of signaling pathways mediated by individual isoforms of given G-protein.

Muscarinic signaling is implicated in numerous pathologic events, such as the promotion of carcinoma cell growth, early pathogenesis of neurodegenerative diseases in the central nervous system of Alzheimer’s and Parkinson’s, schizophrenia, drug addiction, pain, and also in some internal diseases, e.g., asthma or overactive bladder [14,15]. As of now, no affinity-based selective agonists of individual muscarinic receptors have been discovered, due to the high homology of the orthosteric binding site among individual muscarinic subtypes [16,17,18].

Selective targeting on the G_i/o versus G_q/11 mediated pathway by biased agonist could be a way to achieve selectivity to even or odd muscarinic subtypes [19]. Moreover, agonists biased to individual isoforms of G-proteins could lead to tissue-specific activation of mAChRs, due to the predominant expression of some G-proteins in specific tissues (e.g., G_o in the central nervous system or G₁₆ in hematopoietic cells) [10,20]. We have focused on variations in the G₁₆ signaling pathway that was not studied so far, is rare and may lead to very specific effects (e.g., tissue-specific activation). We have analyzed variation in the G₁₆ signaling profile among individual subtypes of muscarinic receptors.

To reveal and properly quantify putative agonist bias to certain G-proteins and their isoforms, especially non-preferential ones, among individual subtypes of muscarinic receptors, a system that is sufficiently sensitive and specific is required. Furthermore, 1:1 Gα-receptor stoichiometry would simplify the analysis and interpretation of found agonist bias. We assume that fusion proteins of muscarinic receptors with Gα subunit of interest could serve as a convenient tool to screen agonist bias towards the particular Gα among individual subtypes of muscarinic receptors. Importantly, we expect that tight fusion of a receptor with a particular Gα prevents the coupling of other competing Gα to the receptor. If so, the signaling of a pathway of interest can be selectively analyzed. Fusion proteins of GPCR and α-subunit of G-protein were used to study individual G-protein mediated pathways in several studies [21,22,23,24,25,26]. We validate our assumptions on an example of fusion proteins of individual muscarinic receptors and non-canonical promiscuous Gα₁₆ subunit.

Muscarinic signaling via non-canonical G₁₆ G-protein may play a relevant physiological role. At the protein level, G₁₆ expression is only detected in highly specific cell types (hematopoietic and epithelial cells) characterized by a high rate of cell turnover [27]. G₁₆ mediated signaling may play role in immune response [28] and tumor cell growth [29].

Promiscuous Gα₁₆ efficiently couples to any subtype of muscarinic receptor. That leads to the phospholipase C-activation, resulting in the formation of inositol phosphates. We performed a binding and functional analysis of these constructs using eleven structurally different muscarinic agonists. We demonstrated agonist-specific activation of non-canonical G₁₆ pathway varying among individual subtypes. Additionally, we compared the signaling of agonists oxotremorine and iperoxo at Gα₁₆_fused, Gα₁₆ co-transfected, and wild types of M₂ and M₅ muscarinic receptors and revealed signaling bias of oxotremorine towards Gα₁₆ pathway at the M₂ receptor and at the same time impaired Gα₁₆ signaling of iperoxo at M₅ receptors.

2. Results

2.1. Fusion Proteins

2.1.1. Description of Fusion Proteins

Fusion proteins (denoted M₁_Gα₁₆ through M₅_Gα₁₆) were constructed from individual subtypes of muscarinic receptors M₁–M₅ and α-subunit of G₁₆ G-protein. The α-subunit was tightly connected to the C-terminus of the respective receptors as described in the Methods. Palmitoylation sites at helix 8 of receptors, as well as at N-terminus of Gα₁₆, were preserved to ensure their anchoring to the membrane. Complete sequences of fusion proteins are shown in Supplementary Materials.

2.1.2. Homology Models of Fusion Proteins

To test whether fusion proteins respect the natural arrangement of the receptor and G-protein α-subunit that allows their successful coupling with no serious occurring disturbance to the structure arrangement, we have built homology models of M₁_Gα₁₆ and M₂_Gα₁₆ fusion proteins. Homology modeling resulted in a good model free of unusual structural features. Overlays of fusion proteins with cryo-EM of M₁ + Gα₁₁ (6OIJ) and M₂ + Gα_o (6OIK) receptor-G-protein complexes [30] are shown in Figure 1. The stability of structure was verified by running molecular dynamics (MD) of fusion proteins in complex with G-protein βγ-dimer in membrane/water system. Analysis of MD trajectories by Simulation Quality Analysis tools of Maestro confirmed the stability of the structures (Supplementary Materials Figure S3). No structural rearrangements occurred during 120 ns of MD. Insertion of the C-terminus of Gα₁₆ to G-protein binding site at the receptor located between transmembrane helix 3 and 6 corresponds to the insertion of Gα₁₁ at M₁ and Gα_o at M₂. At the M₁ receptor, the position of the C-terminus of Gα₁₁ and Gα₁₆ are practically identical. However, at M₂, Gα₁₆ is inserted under a sharper angle than Gα_o. Insertion of the C-terminus of Gα₁₆ instead of Gα₁₁ or Gα_o to the G-protein binding site did not induce any major change in the receptor conformation.

2.1.3. Affinity of [³H]NMS for Gα₁₆ Fused Receptors

To confirm that fusion with Gα₁₆ indeed did not influence receptor conformation as indicated by molecular modeling, we measured the binding of radiolabeled [³H]NMS to all Gα₁₆_fused receptors. The affinity of [³H]NMS to fusion proteins was determined in saturation binding experiments and calculated according to Equation (1). The fusion of muscarinic receptors with Gα₁₆ subunit did not affect the binding affinity of [³H]NMS at any fusion protein. The determined affinity of [³H]NMS to fused and wt receptors, as well as their expression level in CHO cells, is summarized in (Supplementary Materials Table S1).

2.2. Lack of Coupling of Gα₁₆_Fused Receptors with Endogenous G-Proteins

Muscarinic receptors are able to activate multiple G-proteins. Preferentially, muscarinic receptors M₁, M₃, and M₅ couple with Gα_q/11 and M₂ and M₄ receptors with Gα_i/o G-proteins. All muscarinic subtypes efficiently activate non-canonical promiscuous G-protein (G₁₆) followed by activation of the appropriate signaling pathway (phospholipase C-activation and generation of IPX). Based on molecular models, we expected that fusion of muscarinic receptors with Gα₁₆ subunit would sterically prevent coupling of endogenously expressed G-proteins. To this end, we analyzed changes in cAMP level, mediated by endogenous G_i/o and G_s proteins, after activation of wt and Gα₁₆_fused M₂ and M₄ receptors by agonist carbachol. Basal level of cAMP was determined in presence of 10 µM adenylate cyclase activator forskolin. Values of basal level determined as % of incorporated radioactivity varied in the range of 2.5–3% and are the same in cells expressing wt and fused receptors. Level of cAMP was calculated as fold over basal level. We demonstrate that tight fusion with Gα₁₆ prevented the coupling of preferential Gα_i/o and non-preferential G_s to M₂ and M₄ receptors. While carbachol stimulated accumulation of IP_X at all Gα₁₆_fused receptors (Supplementary Materials Table S3), at M₂_Gα₁₆ and M₄_Gα₁₆, did not change the level of cAMP, whereas at wt M₂ and M₄, carbachol inhibited cAMP synthesis via preferential G_i/o G-proteins at submicromolar concentrations and stimulated it via non-preferential G_s G-proteins at micromolar concentrations (Figure 2). Thus, the fusion proteins pass the signal solely through the fused Gα subunit.

2.3. Binding and Functional Analysis of Gα₁₆ Fused Receptors

Eleven structurally different agonists, varying in the binding mode to muscarinic receptors, potency, and efficacy to activate muscarinic receptors (arecoline, carbachol furmethide, iperoxo, McN-A343, N-desmethylclozapine, oxotremorine, pilocarpine, xanomeline, JR-6, and JR-7), were used for pharmacological evaluation of the fusion proteins. Structures of tested agonists are shown in Supplementary Materials Figure S2.

2.3.1. Binding Affinity of Tested Agonists to Gα₁₆ Fused Muscarinic Receptors

The affinity of tested agonists to fused proteins was assayed in competition experiments with 1nM [³H]NMS, calculated according to Equation (4), and is summarized in

Table 1. Affinities of muscarinic agonists to Gα₁₆_fused receptors Affinities of muscarinic agonists are expressed as negative logarithms of inhibition constants (K_I) of [³H]NMS binding to individual subtypes of muscarinic receptors fused with Gα₁₆-subunit. They were calculated according to Equation (4) from IC₅₀ values obtained by fitting Equation (2) or (3) to data from competition experiments with [³H]NMS. Values are means ± SD from three independent experiments performed in quadruplicates.

	M1_Gα16	M2_Gα16	M3_Gα16	M4_Gα16	M5_Gα16
Arecoline	5.19 ± 0.06	4.68 ± 0.03	5.17 ± 0.08	4.68 ± 0.01	5.16 ± 0.04
Carbachol	4.87 ± 0.01	4.62 ± 0.01	4.77 ± 0.02	4.61 ± 0.02	4.72 ± 0.01
Furmethide	5.79 ± 0.01	4.69 ± 0.04	5.27 ± 0.03	4.71 ± 0.01	5.25 ± 0.02
Iperoxo (high)	8.35 ± 0.12	n.d.	n.d.	n.d.	n.d.
Iperoxo (low)	6.20 ± 0.08	5.83 ± 0.03	6.06 ± 0.04	5.96 ± 0.03	6.99 ± 0.02
McN-A-343	4.24 ± 0.04	6.54 ± 0.04	5.14 ± 0.02	6.41 ± 0.02	5.34 ± 0.06
NDMC	7.06 ± 0.01	6.51 ± 0.04	6.75 ± 0.02	6.40 ± 0.01	6.77 ± 0.03
Oxotremorine	6.61 ± 0.01	5.70 ± 0.04	6.24 ± 0.03	5.86 ± 0.02	6.16 ± 0.03
Pilocarpine	5.26 ± 0.02	4.52 ± 0.01	4.92 ± 0.02	4.54 ± 0.03	4.88 ± 0.04
Xanomeline	7.29 ± 0.01	6.82 ± 0.02	7.19 ± 0.04	7.04 ± 0.03	7.06 ± 0.02
JR-6 (high)	n.d.	n.d.	n.d.	6.73 ± 0.28	n.d.
JR-6 (low)	4.97 ± 0.07	5.74 ± 0.10	5.07 ± 0.04	5.29 ± 0.21	5.44 ± 0.05
JR-7	4.34 ± 0.05	5.17 ± 0.07	4.22 ± 0.06	4.82 ± 0.03	4.46 ± 0.04

n.d., not determined.

. All agonists completely inhibited [³H]NMS binding to fused proteins. All tested agonists displayed only low-affinity binding, except for iperoxo at M₁_Gα₁₆ and JR6 at M₄_Gα₁₆. Affinities of low-affinity binding of tested agonists (carbachol, oxotremorine, pilocarpine, JR6, and JR7) to wt and Gα₁₆_fused muscarinic receptors were compared. Data are summarized in Supplementary Materials Table S2. The affinity of carbachol was slightly lower at all Gα₁₆_fused receptors than at corresponding wt. The decrease in affinity was observed also for pilocarpine, JR7, and oxotremorine (except oxotremorine at M₁ and JR7 at M₂). On the other hand, JR6 had a higher affinity at all Gα₁₆_fused receptors, especially at M₂_Gα₁₆ affinity of JR6 was 23-times higher than at wt M₂.

2.3.2. Functional Response of Gα₁₆_Fused Muscarinic Receptors to Agonists

The fusion with non-canonical promiscuous G-protein Gα₁₆ couples all subtypes of muscarinic receptors to phospholipase C-activation and generation of IP_X. The level of IP_X was measured by radio-chromatographic separation. Basal level (in absence of agonist) varied in range of 2–3% of incorporated radioactivity and was the same in cells expressing wt and individual fused receptors. Level of IP_X in presence of individual concentrations of tested agonists was calculated as fold over basal level. Parameters of accumulation of IP_X as a functional response of fused proteins to stimulation by a tested agonist, EC₅₀ and E′_MAX, are summarized in (Supplementary Materials Table S3). To calculate the coefficient of operational efficacy τ of functional response of individual Gα₁₆_fused receptors to tested agonists, the system E_MAX was determined from functional responses to the agonists carbachol, oxotremorine, and pilocarpine according to the procedure described recently [31]. Then, the τ value was used for the calculation of the equilibrium dissociation constant K_A. The values of τ and K_A calculated according to Equation (6) are summarized in

Table 2. Parameters of functional response of Gα₁₆_fused receptors. Operational efficacy τ, agonist equilibrium dissociation constant K_A, and agonist relative intrinsic activity R_Ai were calculated according to Equations (6)–(8), respectively, from parameters of functional response EC₅₀ and E´_MAX (Supplementary Material, Table S3) obtained by fitting Equation (5) to data from measurement of the accumulation of inositol phosphates. Values of system E_MAX are (27.1 ± 0.5 for M₁_Gα₁₆; 30.7 ± 0.603 for M₂_Gα₁₆; 27.1 ± 0.6 for M₃_Gα₁₆; 27.1 ± 1.2 for M₄_Gα₁₆; and 28.9 ± 0.4 for M₅_Gα₁₆). K_A is expressed as negative logarithms. Values are means ± SD from three independent experiments performed in triplicate.

		Arecoline			Carbachol			Furmethide			Iperoxo			McN-A343			NDMC			Oxotremorine			Pilocarpine			Xanomeline			JR6	JR7
M1_G16	τ	0.594	±	0.057	0.887	±	0.017	0.655	±	0.005	1.577	±	0.055	0.472	±	0.02	0.5	±	0.009	0.869	±	0.027	0.668	±	0.04	0.795	±	0.007	0	0
	pKA	6.68	±	0.01	6.8	±	0.06	6.3	±	0.03	8.24	±	0.03	6.53	±	0.1	7.43	±	0.05	7.82	±	0.05	6.29	±	0.02	8.25	±	0.02	n.c.	n.c.
	RAi	0.45	±	0.025	1	±	0.01	0.234	±	0.001	45.9	±	0.9	0.248	±	0.006	2.4	±	0.02 *	10.3	±	0.2	0.234	±	0.008	25	±	0.1	0	0
M2_G16	τ	1.566	±	0.038	1.41	±	0.028	1.669	±	0.034	9.61	±	0.227	1.033	±	0.021	0.951	±	0.016	2.799	±	0.081	0.946	±	0.02	1.669	±	0.034	0	0
	pKA	6.73	±	0.06	6.6	±	0.06	6.18	±	0.03	7.62	±	0.08	6.68	±	0.04	6.7	±	0.02	7.64	±	0.08	6.39	±	0.02	8.15	±	0.03	n.c.	n.c.
	RAi	1.47	±	0.02 *	1	±	0.01	0.446	±	0.005 *	71.3	±	1	0.875	±	0.010 *	0.846	±	0.008	21.4	±	0.4 *	0.407	±	0.005 *	41.9	±	0.5 *	0	0
M3_G16	τ	0.541	±	0.004	0.918	±	0.02	0.656	±	0.009	2.926	±	0.876	0.307	±	0.046	0.497	±	0.003	0.834	±	0.027	0.697	±	0.039	0.777	±	0.022	0	0
	pKA	7.1	±	0.01	6.8	±	0.08	5.95	±	0.31	8.56	±	0.1	5.81	±	0.13	7.43	±	0.06	7.84	±	0.05	6.31	±	0.01	8.25	±	0.03	n.c.	n.c.
	RAi	1.05	±	0	1	±	0.01	0.102	±	0.001	176	±	30 *	0.028	±	0.002	2.35	±	0.01	10	±	0.2	0.247	±	0.008	23.8	±	0.4	0	0
M4_G16	τ	0.804	±	0.071	0.897	±	0.04	0.865	±	0.084	1.273	±	0.055	0.704	±	0.07	0.623	±	0.05	0.994	±	0.049	0.557	±	0.054	0.866	±	0.084	0	0
	pKA	7.19	±	0.04	7.1	±	0.01	6.64	±	0.01	7.87	±	0.03	6.93	±	0.02	6.8	±	0.02	7.95	±	0.01	6.47	±	0.01	8.59	±	0.01	n.c.	n.c.
	RAi	1.11	±	0.06	1	±	0.03	0.33	±	0.018	8.26	±	0.21	0.523	±	0.03	0.346	±	0.016	7.76	±	0.22	0.144	±	0.008	29.6	±	1.7	0	0
M5_G16	τ	0.51	±	0.005	1.126	±	0.015	1.072	±	0.01	0.825	±	0.031	0.358	±	0.009	0.709	±	0.009	1.555	±	0.016	0.8	±	0.023	1.172	±	0.015	0	0
	pKA	7.02	±	0.14	6.7	±	0.02	6.25	±	0.03	8.46	±	0.14	6.96	±	0.11	7.07	±	0.03	7.72	±	0.03	6.34	±	0.01	8.2	±	0.01	n.c.	n.c.
	RAi	0.76	±	0.005	1	±	0.01	0.336	±	0.002	35	±	1	0.452	±	0.006	1.47	±	0.01	14.5	±	0.1	0.309	±	0.005	33	±	0.2	0	0

n.c., not calculated; *, greater than at other subtypes (p < 0.05, according to ANOVA and Tukey-HSD post-test).

.

G_i/o-biased muscarinic partial agonists JR6 and JR7 did not stimulate the accumulation of IP_X at any fused protein. Although fusion with Gα₁₆ led to an increase in the affinity of JR6 to all fusion proteins, JR6 and JR7 induced conformation incompatible with activation of the Gα₁₆ signaling pathway. Except for JR6 and JR7, all tested agonists stimulated accumulation of IPx at all Gα₁₆_fused receptors.

Quantification of Agonist Bias towards Individual Gα₁₆ Fused Receptors.

To compare agonist specific activation of G₁₆ mediated pathway among individual muscarinic subtypes and to quantify agonist bias towards individual Gα₁₆_fused receptors, intrinsic activities relative to carbachol (RA_i) were calculated according to Equation (8) from the E´_MAX and EC₅₀ values of accumulation of inositol phosphates (Supplementary Materials Table S3). Values of RA_i are summarized in Table 2 and plotted in Figure 3. Interestingly, M₂ super-agonist iperoxo [32,33] displayed a strong bias to M₃_Gα₁₆ over the rest of the subtypes. Iperoxo RA_i values for other Gα₁₆_fused receptors were two (M₂) to 20-fold (M₄) lower. On the other hand, N-desmethylclozapine, considered as M₁ preferring agonist [34] displayed bias to M₁_Gα₁₆ and M₃_Gα₁₆ over the rest of the subtypes. The most pronounced bias was found in the case of McN-A-343. The RA_i for M₂_Gα₁₆ was more than 30-times higher than RA_i for M₃_Gα₁₆. On the other hand, signaling profiles to individual Gα₁₆_fused receptors of ligands like xanomeline, oxotremorine, pilocarpine were almost balanced. The majority of agonists (arecoline, furmethide, McN-A-343, pilocarpine, xanomeline, and oxotremorine) displayed bias to M₂_Gα₁₆. RA_i of arecoline decreases in order M₂ > M₄ ≈ M₃ > M₅ > M₁, RA_i of furmethide in order M₂ > M₅ ≈ M₄ > M₁ > M₃, McN-A-343 M₂ > M₄ > M₅ > M₁ > M₃, pilocarpine M₂ > M₅ > M₃ ≈ M₁ > M₄, xanomeline M₂ > M₅ > M₄ > M₁ > M₃, and oxotremorine M₂ > M₅ > M₁ ≈ M₃ > M₄. The same results were obtained using quantification of signaling bias by calculation of bias factor10^{∆∆log(τ/KA)} introduced by Kenakin et al., 2012 [35] (Supplementary Material Table S4, Figure S1). The variability in bias among agonists eliminates the possibility that protein fusion introduced a bias towards some of the receptors.

Functional Response of Gα₁₆_Fused, Gα₁₆ co-Transfected Receptors, and wt Receptors to Selected Agonists.

We analyzed activation of IP_X pathway by agonist carbachol, oxotremorine, and iperoxo at Gα₁₆_fused receptors, receptors co-transfected with Gα₁₆-subunit and wt M₂ and M₅ receptor (Figure 4,

Table 3. Comparison of parameters of functional response of variants of M₂ and M₅ receptor. Parameters of agonist-induced functional response EC₅₀ and E´_MAX were obtained by fitting Equation (6) to data from measurement of the accumulation of inositol phosphates. Operational efficacy τ, agonist equilibrium dissociation constant K_A and agonist relative intrinsic activity RA_i were calculated according to Equations (6)–(8), respectively. EC₅₀ and K_A are expressed as negative logarithms. Values of system E_MAX are (30.7 ± 0.603 for M₂_Gα₁₆; 29 ± 3 for M₅_Gα₁₆; 5.8 ± 0.4 for M₂ + Gα₁₆; 21 ± for M₅ + Gα₁₆; 5.5 ± 0.4 for wt M₂; 22 ± 2 for wt M₅). Values are means ± SD from 3 independent experiments performed in triplicate.

		pEC50			E′MAX			τ			pKA			RAi
M2+Gα16	carbachol	5.59	±	0.12	4.62	±	0.37	0.88	±	0.17	5.52	±	0.17 †	1	±	0.03
	oxotremorine	6.53	±	0.13	4.46	±	0.33	0.85	±	0.15	6.26	±	0.10 †	5.41	±	0.05 †
M2_Gα16	carbachol	6.99	±	0.06	18	±	0.4	1.41	±	0.03	6.6	±	0.06	1	±	0.01
	oxotremorine	8.22	±	0.08	22.4	±	0.6 *	2.8	±	0.08 *	7.64	±	0.08	21.4	±	0.4
M2 wt	carbachol	6.01	±	0.04	1.91	±	0.07	0.2	±	0.1	5.9	±	0.1	1	±	0.01
	oxotremorine	6.68	±	0.05	1.60	±	0.05	0.2	±	0.1	6.6	±	0.1	3.08	±	0.28
M5+Gα16	carbachol	6.61	±	0.08	11.7	±	0.4	0.814	±	0.03	6.35	±	0.08 †	1	±	0.02
	iperoxo	8.95	±	0.14	11.4	±	0.8	0.785	±	0.057	8.7	±	0.14 †	213	±	9 †
M5_Gα16	carbachol	7.03	±	0.02	15.3	±	0.2	1.126	±	0.015	6.7	±	0.02	1	±	0.01
	iperoxo	8.72	±	0.14	11.3	±	0.4 *	0.825	±	0.031 *	8.46	±	0.14	35	±	1
M5 wt	carbachol	6.09	±	0.16	10.1	±	1.1	0.68	±	0.077	5.86	±	0.16	1	±	0.06
	iperoxo	8.93	±	0.16	13	±	1.1 *	1.08	±	0.09 *	8.61	±	0.16	912	±	45 †

*, different from carbachol (p < 0.05), ^† different from fusion protein (p < 0.05), according to ANOVA and Tukey HSD post-test.

).

Agonist induced coupling with Gα₁₆: Data show better coupling of Gα₁₆ fused M₂ receptor in comparison to the co-transfected system for carbachol and oxotremorine. At IP_X pathway, the value of equilibrium dissociation constant expressed as the negative logarithm (pK_A) for reference agonist carbachol as well as tested agonist oxotremorine, was higher at Gα₁₆_fused receptors than at M₂ receptors co-transfected with Gα₁₆ (Table 3), indicating a better coupling in the case of the fusion protein. The better coupling to G₁₆ at fused receptors M₁_Gα₁₆ through M₅_Gα₁₆ than at co-transfected variants is obvious also from comparison of (Table 2 with our previous data Randakova et al. [28]). The pK_A value for reference agonist carbachol and oxotremorine, as well as pilocarpine or xanomeline, was higher at Gα₁₆_fused receptors than at corresponding wt receptors co-transfected with Gα_16. The increase in pK_A ranged from 3-fold for xanomeline at M₁ to 63-fold for oxotremorine at M₃.

In contrast, pK_A of iperoxo at the fused M_{5_}Gα₁₆ was lower than at co-transfected M₅+Gα₁₆, indicating worse coupling of the fusion protein (Table 3). The high variability in the observed shift in pK_A excludes a possibility of the systemic artifact caused by protein fusion.

Comparison of operational efficacies of selected agonists: In comparison to the co-transfected system M₂+Gα₁₆, the increase in operational efficacy τ of both reference agonist carbachol as well as tested agonist oxotremorine to stimulate the non-canonical accumulation of IP_X at M₂_G_α16 (Table 3) indicates the higher sensitivity of measurement of functional response at Gα_fused receptors. Oxotremorine had higher operational efficacy than carbachol at M₂_Gα₁₆ and M₅_Gα₁₆ (Table 2). At the rest of the Gα₁₆_fused receptors, the operational efficacies of oxotremorine and carbachol were the same. In contrast, oxotremorine stimulated accumulation of IP_X at M₂ + Gα₁₆ and M₂ wt with efficacy comparable (Table 3) or lower [19] to carbachol. Operational efficacies τ of functional responses of carbachol and oxotremorine at M₂_Gα₁₆ and M₂ + Gα₁₆ (Figure 4) are summarized in Table 3. In other words, at M₂_G_α16 fusion protein (where M₂ receptor signals only via G_α16) oxotremorine had higher efficacy than in co-transfected system (where binding of other Gα subunits to M₂ may take place) which indicates bias of oxotremorine towards Gα₁₆ mediated pathway at M₂ receptor.

Interestingly, agonist iperoxo had higher operational efficacy τ than carbachol at all Gα₁₆_fused receptors, except M₅_Gα₁₆ (Table 2). At fused M₅_Gα₁₆, τ value of iperoxo was almost 30% lower than τ values of carbachol. At the rest of Gα₁₆_fused receptors, τ values of iperoxo were greater than τ for carbachol, least by 40 % (M₄) and most nearly 7-fold (at M₂). In contrast to M₅_Gα₁₆, iperoxo stimulated accumulation of IP_X at M₅-wt with higher operational efficacy than carbachol. At co-transfected system M₅ + Gα₁₆, iperoxo and carbachol stimulated IP_X accumulation with comparable efficacy (Figure 4, Table 3) which indicates impairment of Gα₁₆ signaling of super-agonist iperoxo at fused M₅ receptor.

3. Discussion

In this study, we show that fusion proteins of receptor and α-subunit of G-protein are a suitable tool for studying agonist bias. We demonstrate it on the example of muscarinic receptors fused with Gα₁₆ subunit and 11 muscarinic agonists whose signaling profile (bias) varies among receptor subtypes.

Analysis of signaling bias of muscarinic receptors, concerning G-protein mediated signaling, has several pitfalls. Coupling promiscuity of muscarinic receptors leads to molecular crosstalk in downstream signaling. For example, calcium ions released upon activation of G_q/11 IP_X pathway modulate some adenylate cyclases and thus cAMP signaling. In turn, βγ-dimers released from G_i/o G-proteins modulate some calcium channels and thus calcium signaling [36,37]. Moreover, signals of non-preferential pathways are usually weak, thus, highly sensitive methods are needed. The main obstacle, in the study of the non-preferential G-protein pathways, is the competition of different (mainly preferential) Gα-subunits for the binding site at a given receptor. Activation of a non-preferential G-protein pathway may play important roles in processes characterized by fluctuation in an expression of individual G-proteins or GPCRs, e.g., immune cell maturation [28], progression of cancer [38], or Parkinson´s disease [39].

Several tools including G-protein-specific pharmacological inhibitors or toxins [40], C-terminus mimicking peptides [41], small interfering RNA [42,43], using artificial systems with limited endogenous G-proteins [44,45,46] or reconstitution of purified receptors and G-proteins in the artificial membrane [47,48] limit the signal mediated by certain G-proteins. Techniques like the immunoprecipitation with specific Gα antibodies [2,49], resonance energy transfer techniques, where bioluminescent (BRET) or fluorescent (FRET) donors and acceptors are fused on the C-terminus of the GPCR and in one of the subunits of the G-protein [50,51] were used to study specific GPCR-G-protein interactions. Although these methods diminish or eliminate signaling crosstalk, they are not aimed at high sensitivity.

Receptor_Gα fusion proteins are well described to study the activation of individual G-protein mediated signaling pathways at many GPCR [21,22,23,24,25,26]. We demonstrate their use to study agonist bias at non-canonical G₁₆ pathway among individual subtypes of muscarinic receptors. Gα₁₆₍₁₅₎ is expressed only in highly specific cell types such as hematopoietic and epithelial cells [27], which are characterized by a high rate of cell turnover. Muscarinic receptors expressed in these cells appear to be involved in the regulation of diverse cellular activities including immune response [28], cell proliferation, or cell differentiation [52,53].

The engineering of receptor-transducer fusion proteins seems to be an effective strategy to target cellular effectors more efficiently and specifically [21]. Fusion proteins enable the study of signaling mediated by G-proteins up to the level of individual G-proteins isoforms. Moreover, receptor-G-protein fusion forces a 1:1 stoichiometry and ensures efficient coupling of the given receptor to an attached Gα subunit. Receptor-G-protein stoichiometry is a relevant aspect of signaling bias and should be taken into account in the screening of biased agonists [54].

We have created fusion proteins of individual muscarinic receptors (M₁–M₅) and non-canonical promiscuous Gα₁₆ subunit and performed detailed binding and functional analysis of these constructs using 11 structurally different muscarinic agonists to evaluate the suitability of such fusion proteins to study agonist bias. Structurally different agonists vary in interactions in the orthosteric binding site of the muscarinic receptor [55]. The portfolio of used agonists included reference balanced full agonist carbachol, classic muscarinic agonists arecoline, furmethide, pilocarpine, oxotremorine, super-agonist iperoxo [32,33], bitopic agonists xanomeline [56], and McN-A343 [57], and Gi/o-biased agonists JR6 and JR7 [19] (Supplementary Materials Figure S2).

The use of Gα_fused receptors for analysis of signaling bias is conditioned by the full preservation of binding and functional properties of both receptor and Gα subunit. In the preparation of the construct, palmitoylation sites, at the C-terminus of the receptors [58], and the N-terminus of the Gα subunit [59], that mediate interaction with the membrane, were maintained. That is essential for keeping the native conformation of a receptor as well as G-protein. Comparison of homology models of prepared constructs M₁_Gα₁₆ and M₂_Gα₁₆ with cryo-EM structures of receptor-G-protein complexes M₁ + Gα₁₁ and M₂ + Gα_o [30] confirmed the natural arrangement of the receptor and Gα in fusion proteins (Figure 1). At the M₁ receptor, insertion of the C-terminus of related Gα₁₆ and Gα₁₁ subunits into the G-protein binding site of the receptor is practically identical. On the other hand, at the M₂ receptor, evolutionarily more distant Gα₁₆ and Gα_o differ in the angle at which they are inserted into the G-protein binding site. Gα-specific insertion of C-terminus into the intracellular cavity of cognate GPCR was observed in 3D structures of GPCR-G-protein complexes [30,60,61,62,63] and demonstrated using molecular dynamics (MD) as well [64]. Furthermore, the fusion of muscarinic receptors with Gα₁₆ subunit did not affect the binding affinity of the labeled antagonist [³H]N-methylscopolamine at any fusion protein (Supplementary Materials Table S1), indicating that fusion did not markedly influence receptor conformation.

The signaling of interest can be selectively analyzed when the binding of other competing G-proteins to the receptor is excluded. We hypothesized that tight fusion of the receptor with a particular Gα subunit prevents the binding of other G-proteins. The M₂ and M₄ receptors preferentially inhibit cAMP synthesis via Gα_i/o G-proteins and can also couple with non-preferential Gα_s to activate cAMP synthesis [65,66]. In contrast to the wt M₂ and M₄ receptors (Figure 2), carbachol did not induce changes in cAMP level at fused M₂_Gα₁₆ and M₄_Gα₁₆ receptors, indicating no coupling to endogenous G_i/o or G_s G-proteins. It suggests that, unlike some fusion constructs [67], our directly Gα₁₆_fused constructs indeed prevent the access of competitive Gα subunits to the receptor.

The binding analysis has shown that in contrast to wild-type (wt) receptors, at Gα₁₆_fused constructs, almost all tested agonists displayed only low-affinity binding. G-protein binding to a receptor might, in turn, allosterically influence ligand binding [68,69]. The absence of high-affinity binding of most agonists to Gα₁₆_fused receptors may be either due to lack of pre-coupling of Gα₁₆ to the receptor or receptor is pre-coupled Gα₁₆ that binds GDP [43]. Since the decrease in the value of equilibrium dissociation constant (K_A) of agonists at Gα_{16_}fused receptors in comparison to wt receptor co-expressed with Gα₁₆ (Table 3, Table 2 versus our previous data [28]) indicates pre-coupling, the absence of high-affinity binding indicates pre-coupling Gα₁₆ that binds GDP [43].

G₁₆ G-protein is efficiently capable to couple all muscarinic subtypes (M₁–M₅) via phospholipase C activation (IP_X accumulation). Thus, it may be possible to analyze the activation of all muscarinic subtypes using one assay (measurement of the accumulation of inositol phosphates, IP_X) and demonstrate agonist-specific activation of this pathway. Signaling bias among individual Gα₁₆_fused receptors was calculated from relative intrinsic activities RA_i to reference agonist carbachol [70] (Table 2). RAi values can be easily calculated for several pathways and many ligands and quickly compared. In principle, for a single signaling pathway and two or more receptors, a ligand that has greater RAi at one receptor than at other(s) is biased to a given pathway at that receptor. Additionally, we analyzed our data also by conventionally used bias factor [35]. Data are summarized in (Supplementary Materials Table S4) and plotted (Supplementary Materials Figure S1). Quantification of agonist bias obtained by both ways was the same, showing that a quick comparison of RAi factors is sufficient and that analysis was conducted correctly. Presented data demonstrate differences in the pattern of the Gα₁₆ pathway activation at five subtypes of Gα₁₆_fused muscarinic receptors after stimulation by structurally different agonists. It points to variations in the compatibility of agonist-specific conformations with Gα₁₆ coupling and activation. While some agonists have quite balanced Gα₁₆ pathway activation patterns—such as pilocarpine, oxotremorine, or xanomeline—profound bias towards individual Gα₁₆_fused muscarinic receptors was observed for agonists McN-A-343 (towards M₂_Gα₁₆) and iperoxo (towards M₃_Gα₁₆). McN-A343 is a bitopic agonist, capable of stimulating the G_q pathway while incapable of stimulating G_s at M₁ expressed in CHO cells [64]. We have shown that McN-A343 successfully activates G₁₆ pathway at all muscarinic subtypes with a bias towards M₂. Interestingly, M₂ super-agonist iperoxo displayed bias towards M₃_Gα₁₆ over other Gα₁₆_fused receptors. It was demonstrated that iperoxo-based dualsteric compounds exert bias G_i/o over G_s pathway at M₂ [71] but exert bias to G_q/11 over G_i/o signaling at the M₁ receptor [72]. On the other hand, M₁-preferring agonist N-desmethylclozapine displayed bias to M₁_Gα₁₆ and M₃_Gα₁₆ over the rest of the subtypes. It points to huge variability in signaling depending on the combination of a ligand–receptor-pathway system, promising a chance to find agonists with a bias to the desired pathway at the desired receptor subtype.

Comparison of parameters of functional response of selected agonists at Gα₁₆_fused and Gα₁₆ cotransfected wt receptors suggest better coupling of fused Gα subunit. The better coupling of Gα₁₆ in fusion proteins was demonstrated by a decrease in the value of equilibrium dissociation constant (K_A) of agonists at Gα_{16_}fused receptors (Table 2 vs. our previous data Randakova et al. [19], Table 3), except iperoxo at M₅_Gα₁₆ (Table 3, discussed below). The elimination of interaction with other competitive Gα subunits as well as fusion alone could lead to better coupling of fused Gα subunits. The operational equilibrium dissociation constant K_A quantifies an affinity of agonist to the conformation that initiates a given signaling pathway. Thus, it can be considered as one of the coupling parameters.

Furthermore, the operational efficacy τ to stimulate the non-canonical accumulation of IP_X induced both by reference agonist carbachol and tested agonist oxotremorine is higher at fusion protein M₂_Gα₁₆ than at co-transfected system M₂ + G_α16 (Table 3). The better coupling (both the decrease in K_A and increase in τ) indicates that the fusion protein strategy is highly sensitive and thus suitable for detection and analysis of low-efficacy pathways.

Despite the high sensitivity, we did not detect accumulation of IPx induced by G_i/o biased agonist JR6 and JR7 (Table 2) at any fused protein. These data further support the true G_i/o bias of these novel agonists and also support the suitability of these fusion systems in the analysis of signaling bias.

Our data demonstrate that oxotremorine stimulates accumulation of IPx at M₂_Gα₁₆ more efficiently in comparison with co-transfected system M₂+Gα₁₆, where the competition of endogenous G_i/o and G_q/11 occurs and more efficiently than at wt M₂ via endogenous G_q/11 (Table 3). In our previous study of Randáková et al. [19], oxotremorine displayed lower RA_i to stimulate the accumulation of IP_X in the co-expressed system M₂ + Gα₁₆ than in the presented study. This discrepancy can be explained by different levels of expression of Gα₁₆ in co-expressed systems and points to the advantage of using fusion proteins with 1:1 stoichiometry for easier spotting of agonist bias. In comparison with our previous data [19], oxotremorine exerts bias towards IPx accumulation (via M₂_Gα₁₆) over cAMP inhibition via G_i/o at wt M₂. Signaling bias of agonist oxotremorine to G₁₆ over G_i/o and G_q/11 pathway at M₂ receptor would be hard to reveal and quantify without fusion proteins due to signaling crosstalk or could be overlooked due to competition with other G-proteins. We show that using fusion proteins for this analysis can be very practical.

Furthermore, we demonstrate impairment of Gα₁₆ signaling of super-agonist iperoxo at the M₅ receptor. Besides worse coupling (lower pK_A) of iperoxo to fused M₅_Gα₁₆ (Table 3), unlike other Gα₁₆_fused receptors, super-agonist iperoxo stimulated accumulation of IP_X at M₅_Gα₁₆ with lower operational efficacy than reference agonist carbachol. In contrast at wt M₅ receptors expressed in CHO cells, iperoxo stimulated accumulation of IP_X through cognate Gα_q/11 with higher operational efficacy than carbachol. In CHO cells expressing wt M₅ co-transfected with Gα₁₆, operational efficacy for carbachol and iperoxo was the same (Figure 4, Table 3), which could be explained by competition of Gα₁₆ with endogenous preferential G_q/11. Combined data thus indicate incompatibility of active M₅ receptor conformation specific to iperoxo with Gα₁₆ coupling and activation.

4. Materials and Methods

4.1. Construct Preparation

Constructs containing sequences of human variants of muscarinic acetylcholine receptors M₁–M₅ fused with the human variant of Gα₁₅ subunit (also known as Gα₁₆ [73]) were prepared, and new stable cell lines of Chinese hamster ovary (CHO) expressing these fusion proteins were generated. Plasmids pcDNA3.1 coding human receptors M₁–M₅ and Gα₁₆ subunit were obtained from Missouri S&T cDNA Resource Center (Rolla, MO, USA). Plasmid pCMV6-A-Hygro containing hygromycin as a mammalian selection marker was purchased from Origene (Rockville, MD, USA). The coding sequence for Gα₁₆ subunit and subsequently sequences for M₁–M₅ receptors and were subcloned into the pCMV6-A-Hygro vector using restriction endonucleases. To this end, restriction site AflII at the N-terminus of the Gα₁₆ subunit and AgeI at the C-terminus of receptor sequences were created. Both parts were connected via short GATRARS linker, corresponding to the C-terminal amino acids in the M₂ sequence and N-terminal amino acid of the Gα₁₆ subunit. Cysteines needed for palmitoylation of receptors (C⁴³⁵ at M₁, C⁴⁵⁷ at M₂, C⁵⁶¹ at M₃, C⁴⁷⁰ at M₄, and C⁵¹² at M₅) were preserved. Sequences of all fusion proteins are in the Supplementary Material.

4.2. Homology Modeling

Homology models of fusion proteins were constructed as hybrid models using YASARA software, Biosciences (Vienna, Austria) [74]. For M₁_Gα₁₆ fusion protein structures PDB ID: 6WJC, 5CXV, 3SN6, 6OIJ, and 6PT0 were selected by the program as templates. For M₂_Gα₁₆ fusion protein structures PDB ID: 5ZK3, 6OIK, 3SN6, 6OIJ, and 6PT0 were selected by the program as templates. Modeling parameters were set as follows:

Modeling speed: Slow.

Number of PSI-BLAST iterations in template search: 4.

Maximum allowed (PSI-)BLAST E-value to consider template: 0.5.

Maximum number of templates to be used: 5.

Maximum number of templates with the same sequence: 1.

Maximum oligomerization state: 4 (tetrameric).

Maximum number of alignment variations per template: 5.

Maximum number of conformations tried per loop: 50.

Maximum number of residues added to the termini: 10.

4.3. Molecular Dynamics

The homology model of fusion proteins and structure of M₁ receptor in complex with G₁₁ G-protein (6OIJ) were aligned on the receptor part using MUSTANG [75]. The βγ-dimer from the 6OIJ structure was added to the homology model. To evaluate the stability of homology models, conventional molecular dynamics (MD) was simulated using Desmond/GPU ver. 6.1, D. E. Shaw Research (New York, NY, USA). The simulated system consisted of a receptor–G-protein complex in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane set to receptor helices in water and 0.15 M NaCl. The system was first relaxed by the standard Desmond protocol for membrane proteins. Then 120 ns of NPγT (Nosé–Hoover chain thermostat at 300 K, Martyna–Tobias–Klein barostat at 1.01325 bar, isotropic coupling, Coulombic cut-off at 0.9 nm) molecular dynamics without restrains was simulated. The quality of molecular dynamics simulation was assessed by Simulation Quality Analysis tools of Maestro.

4.4. Cell Culture and Membrane Preparation

CHO-K₁ cells, ATCC (Manassas, VA, USA) were transfected with the desired plasmids using Lipofectamine 3000, Invitrogen (Carlsbad, CA, USA). Subconfluent cells were washed with phosphate-buffered saline and then Opti-MEM, Life Technologies (Carlsbad, CA, USA) containing Lipofectamine at a final concentration of 5 μL/mL and plasmid DNA at a final concentration of 1 μg/mL was applied. After 48 h cells were diluted 1000-times by subculturing and hygromycin-B, Toku-E (Bellingham, WA, USA) was added at a final concentration of 200 µg/mL for selection of transfected clones. Selected clones of each construct were used up to passage 10. The expression level of fused muscarinic receptors was confirmed in radioligand binding experiments using ³H-N-methylscopolamine ([³H]NMS),ARC (ST.Louis, MO, USA). Additionally, CHO-K1 cells were also transiently co-transfected with plasmids coding muscarinic receptors and plasmid coding Gα₁₆ subunit. For transient transfection, linear polyethyleneimine PEI 25K, Polysciences, (Hirschberg, Germany) was used. Subconfluent cells were incubated 24 h in the growth medium containing PEI at a final concentration of 2.4 μg/mL and plasmid DNA at a final concentration of 0.8 μg/mL. After 24 h, fresh medium was added, and cells were harvested 48 h after transfection.

CHO cells expressing individual Gα₁₆_fused muscarinic receptors were grown to confluence in 75 cm² flasks in Dulbecco’s modified EaglE′s medium supplemented with 10% fetal bovine serum, Life Technologies (Carlsbad, CA, USA). One million cells were subcultured in 100 mm Petri dishes. Cells were washed with phosphate-buffered saline and harvested by mild trypsinization for functional experiments or manually for binding experiments on day five after subculture. After harvesting cells were centrifuged for 3 min at 250× g.

Membranes from CHO cells were prepared for binding experiments. The pellets of harvested cells were suspended in the ice-cold homogenization medium (100 mM NaCl, 20 mM Na-HEPES, 10 mM EDTA, pH = 7.4) and homogenized on ice by two 30 sec strokes using a Polytron homogenizer Ultra-Turrax; Janke & Kunkel GmbH & Co. KG, IKA-Labortechnik, (Staufen, Germany) with a 30-sec pause between strokes. Cell homogenates were centrifuged for 5 min at 1000× g. The supernatant was collected and centrifuged for 30 min at 30,000× g. Pellets were suspended in the washing medium (100 mM NaCl, 10 mM MgCl2, 20 mM Na-HEPES, pH = 7.4), left for 30 min at 4 °C, and then centrifuged again for 30 min at 30,000× g. The resulting membrane pellets were kept at −80 °C until assayed.

4.5. Radioligand Binding Experiments

All radioligand binding experiments were optimized and carried out as described by El-Fakahany and Jakubik [76]. Briefly, membranes (approximately 10 μg of membrane proteins per sample) were incubated in 96-well plates for 3 h at 25 °C in the incubation medium (100 mM NaCl, 20 mM Na-HEPES,10 mM MgCl₂, pH = 7.4). In the case of the M₅ receptor, which has very slow kinetics of binding, the incubation time was extended to 5 h. Incubation volume for competition and saturation experiments with [³H]NMS was 400 μL or 800 μL, respectively.

In saturation experiments of binding of [³H]NMS six concentrations of the radioligand (ranging from 63 to 2000 pM) were used. Agonist binding was determined in competition experiments with 1 nM [³H]NMS. Nonspecific binding was determined in the presence of 10 μM unlabeled atropine. Incubation was terminated by filtration through Whatman GF/C glass fiber filters, Whatman (Maidstone, GB using a Brandel harvester, Brandel (Gaithersburg, MD, USA). Filters were dried in a microwave oven (3 min, 800 W), and then solid scintillator Meltilex A was melted on filters (105 °C, 90 s) using a hot plate. The filters were cooled and counted in a Microbeta scintillation counter, PerkinElmer Waltham, MA, USA).

4.6. Measurement of Production of cAMP

Agonist-induced changes in the cAMP level were analyzed at Gα₁₆_fused M₂ and M₄ receptors and M₂ and M₄ wild types. The level of cAMP was determined in radio-chromatographical separation of [³H]-cAMP as described previously [4]. To determine levels of cAMP, cells in suspension were pre-incubated for 1 h with 0.4 μM [³H]adenine, ARC (St.Louis, MO, USA), washed, and incubated for 10 min in the presence of 1 mM isobutyl methylxanthine and 10 μM forskolin. Then about 200,000 cells per 0.8 mL of sample were incubated for 1 h with tested agonists. Incubation was ended by the addition of 0.2 mL of 2.5 M HCl to the samples. Samples were applied to alumina columns (1.5 g of alumina per column, Sigma, USA), washed with 2 mL of ammonium acetate (1 M, pH = 7.0), and eluted from columns with 4 mL of ammonium acetate and measured by liquid scintillation spectrometry. Level of cAMP was expressed in dpm (decay per minute). Data are expressed as fold over basal level (after subtraction of blank value), the bottom (basal) is equal to 1.

4.7. Accumulation of Inositol Phosphates

The functional response of Gα₁₆_fused muscarinic receptors was measured as an agonist-stimulated accumulation of inositol phosphates (IPX) using radiochemical chromatography as described previously [4]. The assay was performed in cells in suspension. IPX was determined after separation on ion-exchange columns Dowex 1 × 8-200, Sigma (St.Louis, MO, USA). Harvested cells were resuspended in Krebs-HEPES buffer (KHB; 138 mM NaCl; 4mM KCl; 1.3 mM CaCl₂; 1mM MgCl₂; 1.2 mM NaH₂PO₄; 20 mM HEPES; 10 mM glucose; pH adjusted to 7.4) and centrifuged 250 g for 3 min. Cells were resuspended in KHB supplemented with 500 nM [³H]myo-inositol, ARC (St.Lous, MO) and incubated at 37 °C for 1 h. Then they were washed once with an excess of KHB, resuspended in KHB containing 10 mM LiCl, and incubated for 1 h at 37 °C in the presence of indicated concentrations of agonists. The total reaction volume was 800 µL. Incubation was terminated by the addition of 0.5 mL of stopping solution (chloroform: methanol: 35% HCl; 2: 1: 0.1) and placed in 4 °C for 1 h. An aliquot (0.6 mL) of the upper (aqueous) phase was taken and loaded onto ion-exchange columns. Columns were washed with 10 mL of deionized water and 20 mL of 60 mM ammonium formate/5 mM sodium borate solution. IPX were collectively eluted from columns by 4 mL of 1 M ammonium formate-0.1 M/formic acid buffer. Level of IPx is expressed in dpm (decay per minute). Data are expressed as fold over basal level (after subtraction of blank value), the bottom (basal) is equal to 1.

4.8. Used Agonists

Muscarinic agonists arecoline, carbachol furmethide, iperoxo, McN-A343, N-desmethylclozapine, oxotremorine, pilocarpine (Sigma, St.Louis, MO, USA), xanomeline (Tocris Bioscience, Bristol, UK), JR-6, and JR-7 (synthesized at Barry University, Miami Shores, FL, USA [19]) were used in this study. Structures of all used agonists are in the Supplementary Material (Figure S2).

4.9. Data and Analysis

Experiments were independent, using different seedings of CHO cells. Binding experiments were carried out in three experiments with samples in quadruplicates and functional assays were carried out at least in three experiments with samples in triplicate. Experimenters were blind to tested agonists.

After subtraction of non-specific binding (binding experiments) or background/blank values (functional experiments) data were normalized to control values determined in each experiment. IC₅₀ and EC₅₀ values and parameters derived from them (Ki and K_A) were treated as logarithms. All data were included in the analysis, no outliers were excluded. In statistical analysis value of p < 0.05 was taken as significant for all data. In multiple comparison tests ANOVA with p < 0.05 was followed by Tukey HSD post-test (p < 0.05). Data were processed in Microsoft office, analyzed, and plotted using the program Grace. The statistic was calculated using R (www.r-project.org, accessed on 13 September 2021).

4.9.1. [³H]NMS Saturation Binding

The equilibrium dissociation constant (K_D) and maximum binding capacity (B_MAX) were determined in the saturation experiments. Non-specific binding in the presence of 10 µM atropine was subtracted to determine specific binding. Free concentration of [³H]NMS was calculated by subtraction of values of specific binding from the final concentration of [³H]NMS calculated from measurements of added radioactivity. Equation (1) was fitted to the data.

$$\mathrm{y}=\frac{{\mathrm{B}}_{\mathrm{MAX}}\ast \mathrm{x}}{{\mathrm{K}}_{\mathrm{D}}+\mathrm{x}}$$

(1)

where y is specific binding at free concentration x. K_D values are expressed as negative logarithms and B_MAX values as pmol of binding sites per mg of membrane protein.

4.9.2. Competition Binding

The binding of tested agonists was determined in competition experiments with 1 nM [³H]NMS fitting of Equation (2) for one-site competition or Equation (3) for two-site competition

$$\mathrm{y}=100-\frac{100*\mathrm{x}}{{\mathrm{x}+\mathrm{IC}}_{50}}$$

(2)

$$\mathrm{y}=100-\frac{(100-\mathrm{flow})*\mathrm{x}}{{\mathrm{x}+\mathrm{IC}}_{50\mathrm{high}}}-\frac{\mathrm{flow}*\mathrm{x}}{{\mathrm{x}+\mathrm{IC}}_{50\mathrm{low}}}$$

(3)

where y is specific radioligand biding at concentration x of competitor expressed as a percent of binding in the absence of a competitor, IC₅₀ is concentration causing 50% inhibition of radioligand binding, flow is the fraction of low-affinity binding sites expressed in percents.

Inhibition constants K_I for analyzed agonists were calculated as

$${\mathrm{K}}_{\mathrm{I}}=\frac{{\mathrm{IC}}_{50}}{1+\frac{\left[\mathrm{D}\right]}{{\mathrm{K}}_{\mathrm{D}}}}$$

(4)

where IC₅₀ is concentration causing 50 % inhibition of [³H]NMS binding calculated according to Equation (2) or (3) from competition binding data, [D] is the concentration of [³H]NMS used, and K_D is its equilibrium dissociation constant calculated according to Equation (1) from saturation binding data. Inhibition constants K_I are expressed as negative logarithms.

4.9.3. Functional Response

The potency of analyzed agonists (EC₅₀) to induce maximal response (E′_MAX) were obtained by fitting Equation (5) to the data from measurement of the accumulation of inositol phosphates,

$$\mathrm{y}=1+\frac{\left({{\mathrm{E}}^{\prime }}_{\mathrm{MAX}}-1\right){*\mathrm{x}}^{\mathrm{nH}}}{{\mathrm{EC}}_{50}^{\mathrm{nH}}{+\mathrm{x}}^{\mathrm{nH}}}$$

(5)

where y is a functional response at a concentration of tested compound x, E′_MAX is the apparent maximal response to the tested compound, EC₅₀ is concentration causing half-maximal effect and ^nH is slope factor (Hill coefficient). EC₅₀ values are expressed as negative logarithms and E_MAX values as folds over basal.

4.9.4. Operational Model of Functional Agonism

The operational efficacy coefficient τ [77] was determined by fitting Equation (6) to data from the functional assay.

$$\mathrm{y}=\frac{{\mathrm{E}}_{\mathrm{MAX}}*\mathsf{\tau }*\mathrm{x}}{{\mathrm{K}}_{\mathrm{A}}+\left(\mathsf{\tau }+1\right)\ast \mathrm{x}}$$

(6)

where y is a functional response at a concentration of tested compound x, E_MAX is the maximal response of the system, K_A is the equilibrium dissociation constant. Equation (6) was fitted to data from functional experiments. Equation (6) was fitted to data by the two-step procedure described earlier [31]. In the first step, system E_MAX was determined using carbachol, oxotremorine, and pilocarpine as internal standards by global fit to all data for a given receptor subtype and signaling pathway. In the second step, Equation (6) with E_MAX fixed to the value determined in the first step was fitted to individual experimental data sets.

4.9.5. Relative Intrinsic Activity

For comparison of effects of agonists at different receptors fused with alpha Gα₁₆ to IPX signaling pathways, relative intrinsic activity (R_Ai) was calculated according to Griffin et al. [70].

$${\mathrm{RA}}_{\mathrm{i}}=\frac{{\mathsf{\tau }}_{\mathrm{carbachol}}{*\mathrm{K}}_{\mathrm{Aa}}}{{\mathsf{\tau }}_{\mathrm{a}}{*\mathrm{K}}_{\mathrm{Acarbachol}}}$$

(7)

where τa and K_Aa are half-effective concentration and apparent maximal response to the tested compound, respectively. As Hill coefficients were equal to one, R_Ai values were calculated according to Equation (8).

$${\mathrm{RA}}_{\mathrm{i}}=\frac{{{\mathrm{E}}^{\prime }}_{\mathrm{MAXcarbachol}}{*\mathrm{EC}}_{50\mathrm{a}}}{{{\mathrm{E}}^{\prime }}_{\mathrm{MAXa}}{*\mathrm{EC}}_{50\mathrm{carbachol}}}$$

(8)

where EC_50a and E′_MAXa are half-effective concentration and apparent maximal response to the tested compound, respectively.

4.9.6. Signaling Bias

For receptors activating two or more signaling pathways, a ligand that has greater R_Ai value for one pathway than for other(s) is biased to that pathway. Analogically, for a single signaling pathway and two or more receptors, a ligand that has greater R_Ai at one receptor than at other(s) is biased to a given pathway at that receptor.

Analysis of signaling bias via bias factor 10^{∆∆log(τ/KA)} introduced by Kenakin et al., 2012 [35] is summarized in Supplementary Material (Table S4; Figure S1).

5. Conclusions

The analysis of agonist bias at individual G-protein mediated pathways including non-preferential ones, plays a relevant role in the agonist screening and the development of drugs with reduced side effects that temper their clinical use. Our data showed that fusion proteins of muscarinic receptors and Gα subunits can serve as a suitable approach to analyze agonist bias and to serve as a convenient screening tool. Fusion proteins provide 1:1 receptor Gα stoichiometry, which makes quantification of agonist bias easier. We demonstrate that fusion of muscarinic receptors with Gα₁₆ limits access of other competitive Gα subunits to the receptor. That, in turn, makes it easier to quantify signaling via the non-canonical Gα₁₆. We demonstrated agonist-specific activation of G₁₆ mediated pathway among individual subtypes of muscarinic receptors. We have confirmed functional selectivity of novel muscarinic agonists JR6 and JR7 for G_i/o signaling pathway [19]. Furthermore, our data revealed signaling bias of oxotremorine towards non-canonical G₁₆ at M₂ and impairment of iperoxo mediated signaling through G₁₆, regarding G_i/o and G_q/11 for M₂ and G_q/11 for M₅ G-proteins.