Cleavage-resistant fusion proteins of the M2 muscarinic receptor and Gαi1. Homotropic and heterotropic effects in the binding of ligands

Biochimica et Biophysica Acta 1810 (2011) 592–602

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

Cleavage-resistant fusion proteins of the M2 muscarinic receptor and Gαi1. Homotropic and heterotropic effects in the binding of ligands Amy W.-S. Ma, John Y. Dong, Dengbo Ma, James W. Wells ⁎ Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario, Canada M5S 3M2

a r t i c l e

i n f o

Article history: Received 27 August 2010 Received in revised form 28 January 2011 Accepted 2 March 2011 Available online 11 March 2011 Keywords: Cooperativity Fusion protein G protein-coupled receptor Gα-subunit Oligomers Radioligand binding studies

a b s t r a c t Background: G protein-coupled receptors fused to a Gα-subunit are functionally similar to their unfused counterparts. They offer an intriguing view into the nature of the receptor–G protein complex, but their usefulness depends upon the stability of the fusion. Methods: Fusion proteins of the M2 muscarinic receptor and the α-subunit of Gi1 were expressed in CHO and Sf9 cells, extracted in digitonin–cholate, and examined for their binding properties and their electrophoretic mobility on western blots. Results: Receptor fused to native αi1 underwent proteolysis near the point of fusion to release a fragment with the mobility of αi1. The cleavage was prevented by truncation of the α-subunit at position 18. Binding of the agonist oxotremorine-M to the stable fusion protein from Sf9 cells was biphasic, and guanylylimidodiphosphate promoted an apparent interconversion of sites from higher to lower affinity. With receptor from CHO cells, the apparent capacity for N-[3H]methylscopolamine was 60% of that for [3H]quinuclidinylbenzilate; binding at saturating concentrations of the latter was inhibited in a noncompetitive manner at low concentrations of unlabeled N-methylscopolamine. Conclusions: A stable fusion protein of the M2 receptor and truncated αi1 resembles the native receptor–G protein complex with respect to the guanyl nucleotide-sensitive binding of agonists and the noncompetitive binding of antagonists. General significance: Release of the α-subunit is likely to occur with other such fusion proteins, rendering the data ambiguous or misleading. The properties of a chemically stable fusion protein support the notion that signaling proceeds via a stable multimeric complex of receptor and G protein. © 2011 Elsevier B.V. All rights reserved.

1. Introduction A widely held view of G protein-mediated signaling is based on the notion of a ligand-regulated, transient complex between one holo-G protein and one receptor (e.g. [1,2]). According to that view, the receptor interconverts spontaneously between G protein-coupled and uncoupled states that correspond to sites of high and low affinity, respectively, seen in the binding of agonists [3]; thus, the heterogeneity revealed by agonists is thought to be induced by G proteins in an otherwise homogeneous population of mutually independent sites. Such schemes can account qualitatively for many of the biochemical and pharmacological properties of G protein-coupled receptors, but

Abbreviations: CHO, Chinese hamster ovary; DEAE, diethylaminoethyl; DTT, dithiothreitol; GMP-PNP, guanylylimidodiphosphate; GPCR, G protein-coupled receptor; HA, influenza hemagglutinin; IB, immunoblot; IP, immunoprecipitate; MOI, multiplicity of infection; NMS, N-methylscopolamine; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonylfluoride; QNB, (−)-quinuclidinylbenzilate; Sf9, Spodoptera frugiperda ⁎ Corresponding author. Fax: +1 416 978 8511. E-mail address: [email protected] (J.W. Wells). 0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2011.03.003

they become problematic when applied in a quantitative and mechanistically consistent manner ([4] and references therein). An alternative view emerges from the realization that receptors and G proteins can exist as oligomers (e.g. [5–8]), in which heterogeneity can be attributed to cooperativity and perhaps asymmetry among interacting sites [7,9,10]. Oligomers of G protein-coupled receptors (GPCRs)1 as well as complexes of GPCRs, G proteins, and effectors have been shown to form early on during biosynthesis and to be trafficked intact to the plasma membrane (e.g. [11]), where they appear in some studies to remain intact throughout the signaling process [12,13]. The properties of such aggregates, including their size, their composition, and the degree to which their existence is transient, have implications for the mechanism of GPCR-initiated signaling [10]. An intriguing development related to these questions has been the advent of fusion proteins in which a GPCR is linked via its C-terminus to the N-terminus of a Gα-subunit. The permanence of the association between receptor and α-subunit is thereby assured, and the prototype in which the β–adrenergic receptor was fused to αs [14] has been followed by various combinations of other GPCRs and other α-subunits (reviewed in [15]). Most such combinations are functional, as indicated by the guanyl nucleotide-sensitive binding of agonists (e.g.

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[16]), agonist-induced GTPase activity [17], and the activation of effectors [14,18,19]. In some cases, the efficiency of coupling and activation was greater with the fusion protein than with the unfused receptor and G protein [19,20]. Receptors in fusion proteins also mimic their unfused counterparts in their formation of homo- and heterooligomers that display cooperativity in the binding of ligands [21]. Although not physiological, fusion proteins have contributed to studies into various aspects of ligand-regulated signaling (reviewed in [22]). They have been used to probe the effect of modifications in the receptor or the G protein on signal transduction (e.g. [23]) and in the study of internalization and recycling following agonist stimulation [24]. In several cases, they have been used to demonstrate transactivation of the α-subunit in one fusion protein by the receptor in another (e.g. [17,25–28]) and the activation of a fused α-subunit by a wild-type receptor (e.g. [26,29]). Such effects imply the existence of complexes that contain multiple equivalents of both receptor and G protein. Conclusions drawn from studies on fusion proteins presuppose that the protein remains intact throughout the experiment. During the present investigation, however, we identified an apparent cleavage within fusion proteins of the M2 muscarinic receptor and Gαi1 following expression in Sf9 and CHO cells. The cleavage occurs within the N-terminus of the α-subunit, and it is prevented upon deletion of the first 17 amino acids from that region. The stable adduct binds the agonist oxotremorine-M in a guanyl nucleotide-sensitive, biphasic manner indicative of a functional interaction between the receptor and the α-subunit. It also exhibits a difference in the apparent capacity for two antagonists, [3H]quinuclidinylbenzilate and N-[3H]methylscopolamine, and related noncompetitive effects that previously have been rationalized in terms of cooperativity among interacting sites. Sequential homology between the N-terminus of αi1 and that of other α-subunits raises the possibility that a cleavage similar to that described here may occur in other such fusion proteins. 2. Materials and methods 2.1. Ligands, antibodies and other materials (−)-[3H]Quinuclidinylbenzilate was purchased from PerkinElmer Life Sciences (lot 3467877, 42.0 Ci/mmol; lot 3595138, 50.5 Ci/mmol) or from Amersham Biosciences (lot B-49, 49 Ci/mmol; lot B-50, 48 Ci/mmol). N-[3H]Methylscopolamine was from Amersham Biosciences (lot B-37, 80 Ci/mmol). Unlabeled N-methylscopolamine hydrobromide, carbamoycholine chloride (carbachol), and oxotremorine-M were purchased from Sigma-Aldrich. Digitonin used for solubilization and purification of the receptor was purchased from Wako Bioproducts at a purity near 100%. Digitonin for the buffers used to pre-equilibrate and to elute the columns of Sephadex G-50 in binding assays was from Calbiochem. Sephadex G-50 (fine) was purchased from Sigma-Aldrich, and all restriction enzymes and ligases were from New England BioLabs. Suppliers of other chemicals were as follows: ACP Chemicals (magnesium sulfate), BDH, Inc. (bromophenol blue and sodium chloride), Bioshop Canada (DTT and EDTA), Caledon Laboratories (glycerol), EM Science (glycine, potassium phosphate, and SDS), EMD Chemicals, Inc. (methanol), MBI Fermentas (DNA Ladder), Roche Diagnostics (HEPES), and Sigma-Aldrich (sodium cholate, Trizma base, Tween-20, PMSF, and other protease inhibitors). Receptor was concentrated using Centricon and Centriprep concentrators (Amicon) purchased from Millipore. Protein concentration was estimated by means of bicinchoninic acid using the BCA Protein Assay Kit and bovine serum albumin, taken as the standard, purchased from Pierce. Ascites fluid (mouse) containing a monoclonal antibody directed against the M2 muscarinic receptor was purchased from Affinity Bioreagents, and a polyclonal antibody (rabbit) to a region of the third intracellular loop (i.e. position 225 to 356) was from Millipore. A

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monoclonal antibody (mouse) directed against Gαi1 was from Millipore, and a polyclonal antibody (rabbit) to the C-terminus of Gβ1–4 was from Santa Cruz Biotechnology. Anti-IgGs conjugated to horseradish peroxidase were purchased from Amersham Biosciences. 2.2. M2 receptor and M2 receptor–αi1 fusion proteins Baculoviruses coding for the HA-, FLAG-, and c-Myc-tagged forms of the human M2 muscarinic receptor have been described previously [30]. They were digested with KpnI and XbaI for subsequent cloning in pcDNA3.1 and expression in CHO cells. Baculoviruses coding for the β1- and γ2-subunits of G protein were obtained from Dr. Tohru Kozasa (University of Illinois at Chicago). Baculoviruses coding for two forms of the human M2 receptor fused at the C-terminus to the α-subunit of bovine Gi1 were obtained from Dr. Tatsuya Haga (Gakushuin University, Tokyo, Japan). One coded for the wild-type receptor (M2αi1); the other coded for a mutant lacking amino acids 233–380 from the third intracellular loop (M2LDαi1) and therefore resistant to proteolysis within that region [31]. The loop-deleted form of the fusion protein also was obtained in the pFASTBac1 vector, which was digested with BamHI or BglII at the 5′ end of the cDNA and with NotI at the 3′ end. The NotI and KpnI digestion sites were added at the 5′ and 3′ ends, respectively, for subcloning into the pBudCE4.1 vector; the KpnI and XbaI digestion sites likewise were added for subcloning into the pcDNA3.1 vector. The pcDNA3.1 vector yielded higher protein levels than the pBudCE4.1 vector when transfected into CHO cells, and subsequent constructs therefore were inserted into pcDNA3.1. All amino acid substitutions, truncations, and additions to M2LDαi1 were prepared using the gene from pcDNA3.1 (Fig. 1, line 1), taken as the template, and the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's directions. Mutated plasmids were transfected into XL1-Blue supercompetent cells, and transformed cells were identified by means of ampicillin selection. The fidelity of mutations was confirmed by sequencing, and the expressed proteins were identified by western blotting. To express the stable fusion protein designated M2(ΔR)His6αi1(NT) in CHO cells, the gene for the wild-type M2 receptor was isolated from pcDNA3.1, and the codon for the terminal arginine was removed by means of PCR. The gene for human Gαi1 within pcDNA3.1 (Missouri S&T cDNA Resource Center) was digested with KpnI and XhoI at the 5′ and 3′ ends, respectively, and the bases corresponding to the first 17 residues of the N-terminus were replaced in a second PCR by those coding for six histidyl residues. The mutated receptor and α-subunit then were ligated in a third PCR, and the KpnI and XbaI digestion sites were added at the 5′ and 3′ ends for reinsertion into pcDNA3.1. To express M2(ΔR)His6αi1(NT) in Sf9 cells, genes for the M2 receptor in the pBlueBac4.5 baculovirus transfer vector (Invitrogen) were digested with KpnI and BstEII at the N-terminus and position 105 of the gene, respectively. The gene for M2(ΔR)His6αi1(NT) in pcDNA3.1 also was digested with BstEII at position 105 and with XbaI at the C-terminus. The resulting fragments were fused at the BstEII site and reinserted into pBlueBac4.5 at the KpnI and XbaI sites. Other details have been described previously [30]. 2.3. Cell culture and extraction of receptor Sf9 cells were cultured at 27 °C in Ex-Cell 400 insect media (JRH Biosciences) containing 2% fetal bovine serum, 1% Fungizone, and 0.1% gentamycin (all from Gibco-BRL). Cells growing at a density of 2 × 106 per mL were infected with baculovirus and collected 48 h after infection. To obtain the wild-type M2 receptor or M2(ΔR)His6αi1(NT) alone, the virus was added at a multiplicity of infection (MOI) of 5; to obtain the receptor or fusion protein together with the β1- and γ2subunits of G protein, the three viruses were added in the ratio 1:1:1 at a total MOI of 5 and in the ratio 1:3:3 at a total MOI of 7.

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Fig. 1. Amino acid sequences of fusion proteins, or mutants thereof, containing the loop-deleted (LD) or full-length M2 receptor and the Gαi1-subunit. The point of fusion is indicated by the arrow. Deleted residues are marked by a hyphen; substitutions and insertions are set in bold.

CHO cells were cultured at 37 °C with 6% CO2 in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and supplemented with 100 μM nonessential amino acids (all from Gibco-BRL). Cells growing at a density of 5.6 × 106 per mL were plated on T75 flasks in 15 mL of media, cultured overnight, and transfected with 30 μg of total DNA using 0.5% Lipofectamine 2000 (Invitrogen) and 10% OPTI-MEM® (Gibco-BRL). The cells were collected 24 h after transfection. Both types of cell were harvested by centrifugation (1000g), resuspended in buffer A (20 mM KH2PO4, 20 mM NaCl, and NaOH to pH 7.40) supplemented with a mixture of protease inhibitors (Sigma) [9,30,32] or with Complete Protease Inhibitor Cocktail Tablets (1 tablet per 50–100 mL) (Roche Diagnostics) [10]. The resuspended cells were solubilized in digitonin–cholate (0.86% digitonin, 0.17% cholate), as described previously [30], and the solubilized material was divided into aliquots that were stored at − 75 °C. Solubilized receptor was monitored routinely for the specific binding of [3H] quinuclidinylbenzilate, measured as described below; the Hill coefficient was near or indistinguishable from 1 throughout, and the affinity varied by up to sixfold among different batches of receptor over the course of the investigation. 2.4. Immunoprecipitation, electrophoresis, and western blotting Most procedures were carried out essentially as described previously [10,30,33]. Samples for electrophoresis were prepared by heating for 5 min at 65 °C, which has been shown not to induce aggregation of the M2 receptor [33]. Gels were loaded with 0.5–7.0 ng of solubilized receptor, as estimated from the binding of 100 nM [3H]quinuclidinylbenzilate, or with whole cell lysates containing the equivalent of 0.1– 0.2 × 105 cells. The latter were incubated with DNaseI (Fermentas) (9 μL per 2 × 106 cells) for 1 h at room temperature. Samples were loaded and subjected to electrophoresis on precast polyacrylamide gels (Bio-Rad, Ready Gel Tris–HCl, 10%) in parallel with molecular weight standards (Bio-Rad or Amersham Biosciences). Coimmunoprecipitation of the wild-type M2 receptor or M2(ΔR) His6αi1(NT) with the β1-subunit was monitored by means of the ExactaCruz™ F kit from Santa Cruz Biotechnology. Polyclonal antibodies to the receptor (8 μg) or Gβ1–4 (2 μg) were mixed with the immunoprecipitation matrix (50 μL) in PBS (500 μL). The mixture was shaken for 1 h at 4 °C and added to an aliquot of extract from Sf9 cells expressing the wild-type M2 receptor (5.6 ng/500 μL) or M2(ΔR) His6αi1(NT) (9.9 ng/500 μL) alone or together with β1 and γ2. Baculoviruses to the three proteins were added at the same MOI. The mixture was shaken overnight at 4 °C, and immunoadsorbed material was collected by centrifugation for 40 s at 4 °C and 16,162g. Precipitated beads were washed four times by resuspension in PBS

(500 μL) and subsequent centrifugation, and the entire precipitate then was applied to a precast polyacrylamide gel (Bio-Rad, MiniProtean® TGX™, 10%). The amount of receptor taken for immunoprecipitation was tenfold greater than that taken from the same sample and applied directly to the same gel. Resolved proteins were transferred onto nitrocellulose membranes (Bio-Rad, 0.45 μm) in a Mini Trans-Blot Transfer Cell (Bio-Rad). The membranes were treated for 2 h with the primary antibody at a dilution of 1:1000 and, unless stated otherwise, for 1 h with the horseradish peroxidase-conjugated secondary antibody at a dilution of 1:3000. Proteins were visualized by chemiluminescence using reagents and film purchased from Amersham Biosciences (ECL™, Hyperfilm MP). 2.5. Binding assays Binding was measured essentially as described previously [9,10]. The radioligand and any unlabeled ligand were dissolved in buffer B (20 mM HEPES, 20 mM NaCl, 5 mM MgSO4, 1 mM EDTA, and NaOH to pH 7.40) supplemented with 0.1% digitonin, 0.02% cholate, and 0.1 mM PMSF. An aliquot of the ligand-containing solution (50 μL) was added to the wild-type receptor or fusion protein (3 μL) in polypropylene microcentrifuge tubes. Bound radioligand was separated by applying an aliquot of the sample (50 μL) to a column of Sephadex G-50 fine (0.8 × 6.5 cm) pre-equilibrated with buffer B supplemented with 0.017% digitonin. All of the eluant up to and including the void volume was collected (wild-type receptor, 1.60 mL; fusion protein, 1.40 mL) and assayed for radioactivity. 2.6. Analysis of data All data were analyzed with total binding taken as the dependent variable (Bobsd) and with the total concentrations of all ligands taken as the independent variables. Values listed for total receptor ([R]t), maximal specific binding (Bmax) and the concentrations of ligands ([P]t, [A]t) are the concentrations in the binding assays. Data acquired at graded concentrations of [3H]quinuclidinylbenzilate were analyzed empirically according to the Hill equation, formulated as shown in Eq. (1).

Bobsd = Bmax

ð½Pt −Bsp ÞnH + NSð½Pt −Bsp Þ K nH + ð½Pt −Bsp ÞnH

ð1Þ

The parameter Bmax represents the total concentration of receptor, and Bsp represents specific binding of the radioligand at the total concentration [P]t. The parameter K represents the concentration of

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unbound radioligand that corresponds to half-maximal specific binding, and nH is the Hill coefficient. The parameter NS represents the fraction of unbound radioligand that appears as nonspecific binding. Eq. (1) was solved numerically [34]. Data acquired at a single concentration of [3H]quinuclidinylbenzilate and graded concentrations of oxotremorine-M were analyzed empirically in terms of Eq. (2), in which the inhibitory effect of the agonist is described as a sum of n Hill terms. nHð jÞ Fj′ IC50ð jÞ nHð jÞ nHð jÞ j = 1 IC 50ð jÞ + ½At

  n Bobsd = B½A = 0 −B½A→∞ ∑

+ B½A→∞

ð2Þ

The variable [A]t is the total concentration of unlabeled ligand, and the parameter IC50( j) is the concentration that corresponds to halfmaximal inhibition at the fraction F′j of labeled sites (∑ nj = 1F′j = 1); nH(j) is the corresponding Hill coefficient. When n was taken as 1, the potency and Hill coefficient are designated IC50 and nH, respectively. The parameters B[A] = 0 and B[A] → ∞ represent the levels of binding when [A]t = 0 and as [A]t → ∞. Mechanistic analyses were performed according to Scheme 1, in which the radioligand (P) and an unlabeled ligand (A) compete for distinct and mutually independent sites (Rj, j = 1, 2, …, n). Sites of type j bind P and A with equilibrium dissociation constants KPj and KAj, respectively, and constitute the fraction Fj of all sites (i.e. Fj = [Rj]t / [R]t, where [Rj]t = [Rj] + [ARj] + [PRj], and [R]t = ∑ nj = 1[Rj]t). Estimates of total binding were analyzed in terms of Scheme 1 by fitting Eq. (3), in which the quantities Bobsd, Bsp, and NS are as described above.   Bobsd = Bsp + NS ½Pt −Bsp

ð3Þ

Total specific binding of the probe was calculated as Bsp =∑ nj = 1 [PRj], in which the value of [PRj] for each j was computed numerically from the total concentrations of Rj, A, and P [34].

Scheme 1.

Parametric values were estimated throughout by nonlinear regression. When two or more sets of data were analyzed in concert, the errors associated with the fitted parametric values were calculated from the diagonal elements of the covariance matrix at the minimum in the weighted sum of squares. The results from simultaneous analyses have been presented with reference to a single fitted curve. To obtain the values plotted on the y-axis, estimates of Bobsd from individual experiments were adjusted according to the
equation B′obsd = Bobsd f xi ; a = f ðxi ; aÞ[7]. Further details regarding the analyses and related procedures have been described elsewhere [7,34].

3. Results 3.1. Proteolytic cleavage of M2 receptor–αi1 fusion proteins Fusion proteins comprising the full-length or loop-deleted M2 muscarinic receptor and the α-subunit of Gi1—designated here as M2αi1 and M2LDαi1, respectively—were expressed in Sf9 cells and solubilized in digitonin–cholate. Both products yielded bands with the mobility expected of a monomer when examined on western blots with antibodies to αi1 (M2αi1, Mr = 102.8 ± 1.2, N = 31; M2LDαi1, Mr = 68.6 ± 1.7, N = 6) (Fig. 2A, long arrows). Some preparations of

Fig. 2. Proteolysis of fusion proteins comprising the M2 muscarinic receptor and the α-subunit of Gi1. (A and B) Samples containing M2αi1 or M2LDαi1 expressed in Sf9 cells were lysed and probed on western blots with the antibody to αi1 (A) or solubilized in digitonin–cholate and probed with the antibody to the M2 receptor (B). (C) M2 LDαi1 was expressed in CHO cells, and the lysate was probed with the antibody to αi1. The long arrows indicate full-length M2αi1 or M2LDαi1; the short arrows indicate αi1 -subunit that presumably was cleaved from the full-length fusion protein. Controls were prepared in parallel from uninfected Sf9 cells (A, B) or from CHO cells that were transfected with an empty vector (C) (lane 1).

each product also revealed bands corresponding to dimers and larger oligomers. All preparations of M2αi1 and M2LDαi1 yielded a band with the mobility expected of the native α-subunit (M2αi1, Mr = 40.9 ± 0.2, N = 35; M2LDαi1, Mr = 40.7 ± 0.6, N = 8) (Fig. 2A, short arrow). Such a band was absent from uninfected Sf9 cells (Fig. 2A, lane 1), and it therefore appears to result from cleavage of the fusion protein during growth or subsequent processing of the cells. Although the degree of cleavage varied from batch to batch of material, a substantial fraction of the parent fusion protein was affected in most preparations; in many cases, the intensity of the signal from free αi1 exceeded that from M2αi1 or M2LDαi1. Since the molecular weight of the cleaved product closely matches that of native αi1 (Mr = 40.4), the cleavage occurs at or near the junction of the receptor and the α-subunit. The band corresponding to free αi1 was not eliminated when the cells were treated with the protease inhibitor Pefabloc SC (4 mM) (Roche Diagnostics) during growth or with Complete Protease Inhibitor Cocktail or Aprotinin (20 μg/mL) (Roche Diagnostics) during processing of the membrane. In the case of M2αi1, the anti-αi1 antibody also identified a band migrating between αi1 and the holo-fusion protein (Fig. 2A, lane 3) (Mr = 64.1 ± 1.2, N = 20). The M2 receptor has been shown to undergo proteolytic cleavage within the third intracellular loop [10,33], and a corresponding band was absent from samples of M2LDαi1. The intermediate band obtained with M2αi1 therefore may represent the last two transmembrane regions of the receptor fused to αi1. Appearance of the 64-kDa band generally was avoided by including a sufficient amount of Complete Protease Inhibitor Cocktail in the buffer solution during harvesting and subsequent processing of the cells. M2αi1 migrated as a singlet or a doublet when probed with an antibody that recognizes the third intracellular loop of the M2 receptor (Fig. 2B) (singlet, M r = 105.4 ± 2.2, N = 13; doublet, M r = 89.5 ± 1.2 and 98.9 ± 1.3, N = 17). There was no band corresponding to a monomer of the M2 receptor alone, despite the evidence for cleavage of the αi1-subunit. The absence of a monomeric band may result from further cleavage of the receptor within the third intracellular loop, although there was no evidence of the 40-kDa band that is expected from such a cleavage [10,33]. If the cleaved receptor were to migrate as a dimer (Mr = 104), however, it would be difficult to distinguish from the parent fusion protein. Since the wild-type M2 receptor extracted from Sf9 cells migrates primarily as a monomer

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[10,33], such an explanation implies that a dimer of receptors is stabilized by the cleaved αi1-subunit. The anti-M2 antibody also detected bands corresponding to oligomers of M2αi1 on blots of some preparations. There was no signal with M2LDαi1, which lacks the epitope recognized by the antibody. M2LDαi1 also was expressed in CHO cells, and the pattern of migration was similar to that obtained with the product from Sf9 cells: the anti-αi1 antibody identified bands corresponding to the cleaved αi1-subunit (Mr = 40.6 ± 0.3, N = 32), the full-length fusion protein (Mr = 62.8 ± 1.3, N = 22), and apparent oligomers of the latter (Fig. 2C). The intensity of the signal from free αi1 often exceeded that from intact M2LDαi1. There was no signal with samples from untransfected CHO cells (Fig. 2C, lane 1). The fusion protein therefore undergoes cleavage when expressed in either Sf9 or CHO cells. 3.2. Cleavage-resistant M2 receptor–αi1 fusion proteins Since M2αi1 and M2LDαi1 both appeared to undergo cleavage at or near the point of fusion, that region was modified in an attempt to identify the location and to prevent the reaction. The M2 receptor contains a number of arginyl and lysyl residues among the last 20 amino acids, including arginine at the C-terminus (Fig. 1). Among those GPCRs that have been fused to an α-subunit, only the 5-HT1A receptor and the H2 histamine receptor terminate in arginine or lysine, creating a potential substrate for tryptic-like proteolysis. To probe for cleavage within the C-terminus of the receptor, several mutants of M 2 LDα i1 were constructed and examined after expression in CHO cells (Fig. 1). Some included the insertion of six histidyl residues between the loop-deleted receptor and αi1 (e.g. M2LDHis6αi1), a modification that itself did not prevent the appearance of a band with the electrophoretic mobility of the free α-subunit (Fig. 3B, lane 2). Little or no protection was afforded by replacement of the terminal arginine with alanine in M2LDαi1 [Fig. 1, M2LD(R N A)αi1; Fig. 3A, lane 8]. There also was no effect when four contiguous segments, each comprising 5 amino acids, were removed sequentially from the C-terminus of the loop-deleted receptor [Fig. 1, M2LD(CTn)αi1; Fig. 3A, lanes 3–6] or when the last 20 amino acids were removed

from the receptor and replaced by six histidyl residues [Fig. 1, M2LD(CT)His6αi1; Fig. 3A, lane 7]. Since the molecular weight of the cleaved αi1-subunit closely matches that of purified αi1 (Fig. 3A, lane 9), the point of cleavage is unlikely to be more than about 20 residues (i.e. 2.4 kDa) from the C-terminus of the receptor. Gα-subunits have been shown previously to undergo proteolysis at their N-termini [35], and several arginyl and lysyl residues are located within the first 17 amino acids of αi1 (Fig. 1). An M2 receptor truncated 20 amino acids from the C-terminus therefore was linked via a hexahistidyl segment to an αi1-subunit truncated at position 18 [Fig. 1, M2LD(CT)His6αi1(NT)]. That deletion eliminated most of the signal corresponding to free αi1 on western blots (Fig. 3B, lane 4), and similar or better protection was achieved when the modification to the receptor was restricted to removal of the terminal arginyl residue [Fig. 1, M2LD(ΔR)His6αi1(NT); Fig. 3B, lane 3]. The segment corresponding to the loop-deleted receptor in M2LD(ΔR)His6αi1(NT) (Fig. 3B, lane 3) was replaced by the fulllength receptor minus the terminal arginine to obtain the construct designated M2(ΔR)His6αi1(NT) (Fig. 1). Expressed in CHO cells, the latter gave little or no signal at the position corresponding to free αi1 on western blots probed with the anti-αi1 antibody (Fig. 4A, lane 3). The intact fusion protein appeared to migrate primarily as a dimer (Mr = 180 ± 6, N = 6) or larger oligomer, with a weak signal at the position corresponding to a monomer (Fig. 4A, arrow). Blots probed with the anti-M2 antibody revealed a mixture of apparent monomers (Mr = 93.1 ± 2.2, N = 10), dimers (Mr = 191 ± 2, N = 4) and larger oligomers (Fig. 4B, lane 2). There similarly was no evidence for cleaved αi1 when M2(ΔR)His6αi1(NT) was expressed in Sf9 cells (Fig. 4C, lane 3). The intact protein migrated mostly as a monomer when probed with the antibody to either αi1 (Mr = 92.4 ± 0.9, N = 6) (Fig. 4C) or the M2 receptor (Mr = 94.7 ± 0.7, N = 6) (Fig. 4D). The foregoing observations indicate that fusion proteins of the M2 receptor and full-length αi1 undergo proteolysis within the N-terminal region of the latter, as might be expected on the basis of previous reports that αi, αo, and αt are susceptible to tryptic hydrolysis at lysyl residues near position 20 [e.g. 36]. The cleavage occurs irrespective of the status of the third intracellular loop of the receptor and is precluded by truncation of αi1. Since the reaction was not prevented by various

Fig. 3. Release of αi1 from variants of M2LDαi1. Fusion proteins containing the loop-deleted form of the receptor were expressed in CHO cells, and lysates were probed on western blots with the antibody to αi1. The sequences around the point of fusion are shown in Fig. 1, and the deletions for the mutants in lanes 3–7 of panel A are as follows: CT1, Δ462–466; CT2, Δ457–461; CT3, Δ452–456; CT4, Δ447–451; CT, Δ447–466. The arrows indicate the cleaved product, which migrated at the same position as purified αi1 (A, lane 9; B, lane 5). Controls were prepared as described in the legend to Fig. 2. Panels A and B are from different gels.

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Fig. 4. Stability of M2(ΔR)His6αi1(NT). A fusion protein in which the terminal arginine of the full-length M2 receptor and the first 17 amino acids of αi1 were replaced by six histidyl residues (Fig. 1) was expressed in CHO (A and B) and Sf9 (C and D) cells, and the cell lysates (A) or digitonin-solubilized extracts (B–D) were probed on western blots with the antibody to αi1 (A and C) or the M2 receptor (B and D). The arrows indicate the full-length monomeric fusion protein. Controls were prepared as described in the legend to Fig. 2.

protease inhibitors, at least in Sf9 cells, it is unclear whether or not trypsin or a trypsin-like protease is responsible. 3.3. Interaction of a stable M2 receptor–αi1 fusion protein with Gβ1 A fusion protein of the α2A–adrenergic receptor and αi1 expressed in COS-7 cells has been found to exhibit agonist-stimulated GTPase activity, the magnitude of which was about twofold greater upon coexpression with Gβ1 and Gγ2 [37]. Extracts from Sf9 cells therefore were examined on western blots for interactions between endogenous β-subunits or exogenous β1 and either the M2 receptor or the fusion protein. The results are illustrated in Fig. 5, where the samples in panels A and B were blotted with a non-specific anti-β antibody and the anti-M2 antibody, respectively; the immunoprecipitates in both panels were obtained with an immobilized anti-M2 antibody. The antibody to β1–4 revealed faint bands indicative of a β-subunit in extracts from cells expressing the wild-type receptor or M2(ΔR) His6αi1(NT) alone (Fig. 5A, lanes 2 and 4), but there was no detectable signal from the immunoprecipitate in either case (Fig. 5A, lanes 3 and 5). In contrast, a strong signal was obtained from blots of the extract (Fig. 5A, lanes 6 and 8) and the immunoprecipitate (Fig. 5A, lanes 7 and 9) when the fusion protein or the receptor was coexpressed with β1 and γ2. Sf9 cells therefore contain one or more β-subunits that are recognized by the antibody, but any complex formed with M2(ΔR) His6αi1(NT) or the wild-type receptor was unstable or below the level

597

Fig. 5. Interaction of Gβ with M2(ΔR)His6αi1(NT) and wild-type M2 receptor. The fusion protein or the M2 receptor was expressed in Sf9 cells either alone or together with Gβ1 and Gγ2 at a relative MOI of 1:1:1. The cells were solubilized in digitonin–cholate, and an aliquot of each preparation was applied directly to the gel (even-numbered lanes); a second aliquot was incubated with an immobilized anti-M2 antibody, and the precipitate was applied to the gel (odd-numbered lanes). For the control (lane 1), uninfected Sf9 cells were solubilized in digitonin–cholate; an aliquot (60 μL) was incubated with the immobilized anti-M2 antibody, and the precipitate was applied to the gel. The transferred material was blotted with the anti-β1–4 antibody (A) or the anti-M2 antibody (B), and the bands were detected using the Western Blotting Detection Reagent (Santa Cruz, ExactaCruz™ F) at a dilution of 1:1000 in place of a secondary antibody.

of detection. Both forms of the receptor formed a stable complex with coexpressed β1. The anti-M2 antibody revealed bands with the mobility expected of M2(ΔR)His6αi1(NT) (Fig. 5B, lanes 2, 3, 6, and 7) and the wild-type receptor (Fig. 5B, lanes 4, 5, 8, and 9) in the extract (lanes 2, 4, 6, and 8) and in the precipitate (lanes 3, 5, 7, and 9). The receptor was quantified by means of [3H]quinuclidinylbenzilate, and the same amount was applied to each of the even-numbered lanes depicted in both panels of Fig. 5; the amount mixed with the immobilized anti-M2 antibody to obtain the precipitate subsequently applied to the oddnumbered lanes was tenfold greater than the amount applied directly to the gel. A comparison of the intensities of the bands in Fig. 5B suggests that the efficiency of immunoprecipitation was similar for both forms of the receptor and unaffected by coexpression with β1 and γ2. It follows that there is comparatively little endogenous βsubunit in Sf9 cells and that the absence of a signal in lanes 3 and 5 of Fig. 5A was due to the absence of receptor-bound β-subunit rather than a failure of the anti-M2 antibody to pull down the receptor.

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Table 1 Hill coefficients for the binding of oxotremorine-M and [3H]quinuclidinylbenzilate to wild-type M2 receptor and M2(ΔR)His6αi1(NT). [3H]Quinuclidinylbenzilate

Oxotremorine-M No GMP-PNP

M2 receptor (4, 3) M2 receptor + β1γ2 (3, 2) M2(ΔR)His6αi1(NT) (7, 7) M2(ΔR)His6αi1(NT) + β1γ2 (3, 2)

0.1 mM GMP-PNP

No GMP-PNP

0.1 mM GMP-PNP

nH

p

nH

p

nH

p

nH

p

1.16 ± 0.06 1.34 ± 0.01 0.59 ± 0.03 0.78 ± 0.02

0.57 0.00002 b 0.00001 0.0046

1.14 ± 0.02 1.34 ± 0.02 1.02 ± 0.05 1.21 ± 0.04

0.40 b 0.00001 0.99 0.0014

0.87 ± 0.09 0.83 ± 0.08 1.06 ± 0.03 0.85 ± 0.03

0.067 0.033 0.66 0.043

0.99 ± 0.04 0.79 ± 0.01 1.06 ± 0.03 0.86 ± 0.02

0.82 0.010 0.69 0.016

The data from each experiment represented in Figs. 6 and 7 and from other assays at graded concentrations of [3H]QNB were analyzed independently in terms of Eq. (1) or (2) (n = 1), and the individual values of nH were averaged to obtain the means (± S.E.M.) listed in the table. The corresponding values of p indicate the level of significance for the increase in the global sum of squares when the same data were analyzed with the value of nH set at 1. The number of independent experiments is shown in parentheses (oxotremorine-M, QNB). Assays with and without GMP-PNP were conducted in parallel.

3.4. Functionality of a stable M2 receptor–αi1 fusion protein A functional interaction between the M2 receptor and the fused αi1-subunit was demonstrated by the allosteric effect of GMP-PNP on the binding of the muscarinic agonist oxotremorine-M. The effect was examined in extracts from Sf9 cells in order to avoid potentially misleading contributions from endogenous G proteins. Although CHO cells appear not to contain appreciable amounts of αi1 (Fig. 4A), they do contain αi2 and αi3 [38], which also can be activated by the M2 muscarinic receptor. Wild-type M2 receptor bound [3H]quinuclidinylbenzilate and the agonist oxotremorine-M with Hill coefficients near 1 (Table 1). The data can be described in terms of Scheme 1 with a single class of sites to obtain the fitted curves shown in Fig. 6 (panels A and B) and the parametric values listed in Table 2. The sum of squares for either ligand is essentially unchanged with two classes of sites rather than one

(p N 0.5). There also was little or no effect of GMP-PNP on the binding of either ligand (Tables 1 and 2), consistent with reports that the M2 receptor does not interact with endogenous G proteins in Sf9 cells [39]. Fusion of truncated αi1 to the receptor markedly decreased the Hill coefficient for oxotremorine-M in the absence of guanyl nucleotide, from 1.16 in the wild-type receptor to 0.59 in M2(ΔR)His6αi1(NT) (Table 1). The effect was reversed by GMP-PNP to yield a Hill coefficient of 1.02 (Table 1, Fig. 6D). M2(ΔR)His6αi1(NT) bound [3H]quinuclidinylbenzilate with a Hill coefficient indistinguishable from 1 either with or without the nucleotide (Table 1, Fig. 6C). Two classes of sites are required to describe the binding of oxotremorine-M in terms of Scheme 1, whereas one class is sufficient for [3H]quinuclidinylbenzilate; the fitted curves are illustrated in Fig. 6 (panels C and D), and the parametric values are listed in Table 2. There is no appreciable effect on the sum of squares if the values of KAj for oxotremorine-M are assumed to be unaffected by GMP-PNP (p = 0.30), which therefore appears to

Fig. 6. Binding of oxotremorine-M and [3H]quinuclidinylbenzilate to wild-type M2 receptor (A and B) and M2(ΔR)His6αi1(NT) (C and D) extracted from Sf9 cells in digitonin–cholate. (A and C) Total binding was measured at graded concentrations of [3H]QNB in the absence of guanyl nucleotide (closed symbols) and in the presence of 0.1 mM GMP-PNP (open symbols), either alone (upper curves) or in the presence of 1 mM unlabeled NMS (baseline). Assays with and without the nucleotide were performed in parallel, and different symbols denote data from different experiments (A, ▲, Δ; ▼,∇; ◆, ◇; C, ■,□; ▲, Δ;▼,∇; ◆, ◇; ●,○;×,+; ⁎, ‫)׀‬. (B and D) Total binding was measured at half-saturating concentrations of [3H]QNB (B, 3.52 ± 0.25 nM and 3.59 ± 0.25 nM, N = 4; D, 1.94 ± 0.17 nM, N = 7) and graded concentrations of oxotremorine-M. Binding in the absence of guanyl nucleotide (●) and in the presence of 0.1 mM GMP-PNP (○) was measured in parallel in each experiment. The lines represent the best fits of Eq. (3) to the pooled data from the seven experiments represented in panels A and B (n = 1) and, in a separate analysis, to the pooled data from the 14 experiments represented in panels C and D (n = 2). The value of [R]t was estimated separately for each set of data. For data acquired in the absence of GMP-PNP, the mean values used to obtain the adjusted values of Bobsd plotted on the y-axis are as follows: A and B, 283 ± 11 pM (N = 7); C and D, 292 ± 36 pM (N = 14). The corresponding values of [R]t for data acquired in the presence GMP-PNP were calculated from the mean ratios of the individual values measured in the absence (−) and presence (+) of GMP-PNP in each experiment; that is, [R]t(+)/[R]t(−). The values of the mean ratio are as follows: A and B, 1.04 ± 0.13 (N = 3); C and D, 1.01 ± 0.02 (N = 7). Points shown at the lower and upper ends of the x-axis in panels B and D indicate binding in the absence or presence of oxotremorine-M at concentrations less than 100 nM and in the presence of 1 mM NMS, respectively. Other parametric values and further details regarding the analyses are given in Table 2.

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599

Table 2 Parametric values for the binding of quinuclidinylbenzilate and oxotremorine-M to wild-type M2 receptor and M2(ΔR)His6αi1(NT) extracted from Sf9 cells, estimated empirically in terms of Scheme 1.

[[3H]QNB] Preparation

GMP-PNP

M2 receptor

0 0.1 mM

M2(ΔR)His6αi1(NT)

0 0.1 mM

n

(nM)

Oxotremorine-M

Quinuclidinylbenzilate log KP1

1

3.52 ± 0.25 3.59 ± 0.25

–9.34 ± 0.03 –9.13 ± 0.03

2

1.94 ± 0.17

–9.54 ± 0.02 –9.44 ± 0.02

log KP2

log KA1

log KA2

F2

–3.83 ± 0.04 –3.69 ± 0.04

α α

–5.86 ± 0.11

–4.21 ± 0.04

0.44 ± 0.03 1.00b

The data illustrated in the paired left- and right-hand panels of Fig. 6 were pooled (panels A and B, panels C and D) and analyzed according to Eq. (3) to obtain the parametric values listed in the table. The parameters KAj and KPj represent the affinities of oxotremorine-M and [3H]QNB, respectively. A single class of sites was sufficient for the wild-type M2 receptor (n = 1), and separate values of KPj and KAj were assigned to data acquired with and without GMP-PNP. Two classes of sites were required for M2(ΔR)His6αi1(NT) (n = 2), with separate values of F2 assigned to data acquired with and without GMP-PNP. The heterogeneity was transparent to [3H]QNB, and the values of KPj were constrained accordingly. Separate values of KP were assigned to data acquired with and without GMP-PNP, whereas single values of KA(j) were common to all of the data. Values of [R]t were assigned separately to data acquired with and without the nucleotide to accommodate small differences in the apparent capacity in some experiments. a The data were analyzed with a single value of KP for both classes of sites (i.e. KP1 = KP2). b The fitted value of F2 in the presence of GMP-PNP was 1.003 ± 0.004.

promote a net interconversion of sites from the state of higher affinity for the agonist (KA1) to that of lower affinity (KA2). Coexpression of the wild-type M2 receptor with β1 and γ2 was accompanied by an increase from about 1.15 to 1.34 in the Hill coefficient for oxotremorine-M (Table 1). GMP-PNP was without effect on either the apparent affinity or the Hill coefficient of the agonist (Table 1, Fig. 7A). Coexpression of M2(ΔR)His6αi1(NT) with β1 and γ2 was accompanied by an increase from 0.59 to 0.78 in the Hill coefficient for oxotremorine-M in the absence of GMP-PNP (Table 1). The data nevertheless require two classes of sites (p = 0.0046), and the nucleotide effected an apparent interconversion from higher to lower affinity (Fig. 7B). In the presence of GMP-PNP, the Hill coefficient increased from 1.02 to 1.21. GMP-PNP was without effect on the apparent affinity or the Hill coefficient of [3H]quinuclidinylbenzilate at either form of the receptor irrespective of β1γ2. Upon extraction from CHO cells, M2(ΔR)His6αi1(NT) displayed an apparent difference in the capacity for N-[3H]methylscopolamine and [3H]quinuclidinylbenzilate (Fig. 8A). When data from parallel assays with both radioligands were analyzed in terms of the Hill equation, the capacity for N-[3H]methylscopolamine was only about 60% of that for [3H]quinuclidinylbenzilate ([3H]NMS, log K = −8.47 ± 0.24, nH = 1.11 ± 0.20, N = 2; [3H]QNB, log K = −8.83 ± 0.06, nH = 1.35 ±0.18, N = 3; [R] t,NMS / [R] t,QNB = 0.61 ± 0.04, N = 3). The capacity for N-[ 3 H] methylscopolamine was the same after incubation of the reaction mixture for 30, 45, and 75 min, indicating that the shortfall was not due to a time-dependent loss of receptor under the conditions of the assays.

Specific binding at near-saturating concentrations of [3H]quinuclidinylbenzilate was inhibited by unlabeled N-methylscopolamine in a biphasic manner that was complete at comparatively low concentrations of the latter (Fig. 8B). The data represented in both panels of Fig. 8 were analyzed simultaneously in terms of Scheme 1, taken initially with two classes of sites. The fitted curves are shown in the figure, and the parametric values are listed in Table 3. The model predicts that the sites are noninterconverting and that binding is strictly competitive. It follows that all of the data are expected to be accommodated by a single value of F2; similarly, the values of KLj are expected to be the same for N-[3H]methylscopolamine (panel A) and for unlabeled N-methylscopolamine at both concentrations of [3H]quinuclidinylbenzilate (panel B). The value of F2 therefore was constrained accordingly, and the values of KLj were estimated separately for radiolabeled and unlabeled N-methylscopolamine in order to test for consistency as required by the model. An alternative approach would be to constrain the values of KLj and to test for consistency between separate estimates of F2. About 50% of the sites were of anomalously weak affinity for N[3H]methylscopolamine (log KP2 = −5.77, Table 3), whereas the affinity of unlabeled N-methylscopolamine for the same sites was about 170-fold higher (log KA2 = −8.00, Table 3). Similarly, there was a 40-fold difference in the affinity of radiolabeled and unlabeled N-methylscopolamine for the sites of higher affinity (cf. log KP1 = −8.65 and log KA1 = −10.29, Table 3). These discrepancies are confirmed by an increase of more than 1.8-fold in the global sum of

Fig. 7. Binding of oxotremorine-M to wild-type M2 receptor (A) and M2(ΔR)His6αi1(NT) (B) coexpressed with Gβ1 and Gγ2 in Sf9 cells and extracted in digitonin–cholate. Total binding of [3H]QNB in the absence of guanyl nucleotide (●) and in the presence of 0.1 mM GMP-PNP (○) was measured at a half-saturating concentration of the radioligand (●, 2.96 ± 0.02 nM, N = 3; ○, 2.92 ± 0.01 nM, N = 3) and graded concentrations of oxotremorine-M. Binding with and without GMP-PNP was measured in parallel in each experiment. The lines represent the best fits of Eq. (2) to the pooled data represented in panel A and, in a separate analysis, to those represented in panel B. Single values of log IC50(j), nH(j), and F′j were common to all data acquired under the same conditions with respect to GMP-PNP. Separate values of B[A]=0 and B[A]→∞ were assigned to each set of data, and individual estimates of Bobsd were normalized to the mean value of B[A]=0 to obtain the adjusted values plotted on the y-axis. The mean values of B[A]=0 in the absence and presence of GMP-PNP are as follows (N = 3): A, 308 ± 8 pM and 302 ± 4 pM; B, 292 ± 3 pM and 307 ± 6 pM. Points shown at the lower and upper ends of the x-axis indicate binding in the absence of oxotremorine-M and in the presence of 1 mM NMS, respectively. The fitted parametric values are as follows: A, (●, n = 1) log IC50 = − 3.12 ± 0.02, nH = 1.34 ± 0.01, (○, n = 1) log IC50 = − 3.10 ± 0.02, nH = 1.33 ± 0.01; B, (●, n = 2) log IC50(1) = − 5.04 ± 0.21, log IC50(2) = − 3.20 ± 0.04, F′2 = 0.80 ± 0.03, (○, n = 1) log IC50 = − 3.21 ± 0.02, nH = 1.20 ± 0.05. The value of nH(j) was taken as 1 for both terms of Eq. (2) when n = 2.

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Fig. 8. Binding of quinuclidinylbenzilate and N-methylscopolamine to M2(ΔR)His6αi1(NT) extracted from CHO cells in digitonin–cholate. (A) Total binding was measured at graded concentrations of [3H]QNB (▼,●, ◆) and [3H]NMS (▽, ○), either alone (upper curves) or in the presence of 1 mM unlabeled NMS (baseline). Each experiment included assays with both radioligands taken in parallel, and different symbols denote data from different experiments (▼,▽; ●, ○; ◆). (B) Total binding was measured at near-saturating (▲) and subsaturating (■) concentrations of [3H]QNB [22 ± 2 nM (N = 6) and 2.18 ± 0.16 nM (N = 4), respectively] and graded concentrations of unlabeled NMS. The lines represent the best fit of Eq. (3) (n = 2) to the pooled data from the experiments represented in panels A and B taken together. The mean value of [R]t used to obtain the adjusted values of Bobsd plotted on the y-axis is 380 ± 68 pM (N = 13). Points shown at the lower end of the x-axis in panel B indicate binding in the absence of NMS; values obtained at concentrations greater than 10 μM were indistinguishable from zero and are not shown. Other parametric values and further details regarding the analyses are given in Table 3.

squares when the data represented in the two panels are reanalyzed with the parameters assigned in a mechanistically consistent manner (p b 0.00001): that is, with a single value of KLj rather than two for N-methylscopolamine at each class of sites. The sum of squares from such an analysis is not reduced appreciably with three or four classes of sites rather than two (p N 0.10), and the data therefore are inconsistent with Scheme 1 at any degree of heterogeneity. Such discrepancies in the affinity of N-methylscopolamine point to noncompetitive effects in the binding of antagonists. 4. Discussion Fusion proteins comprising the M2 muscarinic receptor and fulllength Gαi1 were found to undergo cleavage within the N-terminal region of the α-subunit. The consequent release of free αi1 defeats the purpose of the fusion, which is at least partly to create a stable “complex” of fixed composition, and effects of the tethered α-subunit become difficult to distinguish from those of subunits released through proteolysis. That in turn is likely to cloud the interpretation of data, particularly when the conclusions relate to the composition of the receptor–G protein complex or to its nature as transient or otherwise. The cleavage was avoided by fusing the M2 receptor with N-truncated αi1 in two adducts designated as M2LD(ΔR)His6αi1(NT) and M2(ΔR)His6αi1(NT) (Fig. 1). Sequential homology among different α-subunits suggests that other GPCR–Gα fusion proteins may undergo proteolysis similar to that described here (Fig. 9). A fusion protein of the β2–adrenergic receptor and Gαs was reported to release the α-subunit [14], although a variant in which the receptor and αs were linked by six histidyl residues or a thrombin-specific site appears to have been stable [20,40]. No similar stabilization was achieved by the inclusion of a

hexahistidyl segment between the receptor and the α-subunit in the adduct M2LDHis6αi1 (Fig. 1). In most cases, however, the stability of GPCR–Gα fusion proteins is unclear owing to their expression in cells containing endogenous G proteins of the same subtype (e.g. [41]) or to published western blots that omit the region corresponding to the free α-subunit (e.g. [16]). Digitonin-solubilized M2(ΔR)His6αi1(NT) extracted from CHO cells revealed noncompetitive effects similar to those reported previously for the wild-type M2 receptor under somewhat different conditions: namely, M2 receptor extracted from porcine atria in cholate–NaCl [9], and M2 receptor purified from Sf9 membranes and reconstituted without G protein in phospholipid vesicles [10]. In each case, the apparent capacity for [3H]quinuclidinylbenzilate exceeded that for N[3H]methylscopolamine, yet binding of the former to those sites inaccessible to the latter was blocked by unlabeled N-methylscopolamine at concentrations that were anomalously low for competitive inhibition. N-Methylscopolamine therefore appears to inhibit at sites to which it cannot bind, a noncompetitive effect that has been rationalized quantitatively in terms of cooperativity among interacting sites [9,10]. In contrast, noncompetitive effects were not observed when the wild-type M2 receptor was investigated under conditions similar to those used in the present investigation. There was little or no difference in the capacity for N-[3H]methylscopolamine and [3H]quinuclidinylbenzilate when M2 receptor was extracted from Sf9 cells in digitonin–cholate [33]. There similarly was no difference when the muscarinic receptor was extracted from porcine sarcolemmal membranes, also in digitonin–cholate [9], despite the presence of comparable amounts of G proteins [32]. In each case, unlabeled N-methylscopolamine was found to inhibit the binding of [3H]quinuclidinylbenzilate in a competitive manner. These considerations suggest that a fused αi1-subunit can promote

Table 3 Parametric values for the binding of quinuclidinylbenzilate and N-methylscopolamine to M2(ΔR)His6αi1(NT) extracted from CHO cells, estimated empirically in terms of Scheme 1.

Preparation

L

M2(ΔR)His6αi1(NT)

P (2,3) A (6) A (4)

[[3H]QNB]

N-Methylscopolamine

(nM)

log KL1

log KL2

–8.65 ± 0.07

–5.77 ± 0.48

–10.29 ± 0.16

–8.00 ± 0.17

2.18 ± 0.16 22 ± 2

Quinuclidinylbenzilate log KP1

log KP2

F2

–8.70 ± 0.12

–8.66 ± 0.15

0.47 ± 0.03

The data illustrated in Fig. 8 were pooled and analyzed according to Eq. (3) to obtain the parametric values listed in the table. QNB was present only as the radioligand (L ≡ P), while NMS was present either as the radioligand (L ≡ P, panel A of Fig. 8) or as the unlabeled analogue (L ≡ A, panel B of Fig. 8). The number of experiments is shown in parentheses [(NMS, QNB), (NMS)]. Two classes of sites were required, and a single value of F2 was common to all of the data in both panels of the figure. In the case of [3H]QNB, single values of KPj were common to the data in both panels. In the case of NMS, single values of KPj were assigned to the data acquired at graded concentrations of [3H]NMS (panel A), and single values of KAj were assigned to the data acquired with unlabeled NMS at both concentrations of [3H]QNB (panel B). There was no appreciable increase in the sum of squares with single rather than separate values of KAj for the data at the two concentrations of [3H]QNB (p N 0.05).

A.W.-S. Ma et al. / Biochimica et Biophysica Acta 1810 (2011) 592–602

Fig. 9. Comparison of the N-termini of Gα-subunits. Potential targets of trypsin-like proteolysis are shown in bold (i.e. lysine and arginine).

interactions among orthosteric sites of the M2 receptor, presumably within an oligomer, thereby giving rise to noncompetitive effects that are not observed with the wild-type receptor under similar conditions. The fused subunit may act by favoring the retention of cooperativity within the oligomer or of the oligomer itself during solubilization. The fused α-subunit in M2(ΔR)His6αi1(NT) interacts with the receptor in a manner that permits the characteristic effect of guanyl nucleotides on the binding of agonists. Oxotremorine-M revealed two classes of sites, and those of high affinity were converted to low affinity in the presence of GMP-PNP. The heterogeneity cannot be attributed to endogenous β-subunits. The latter appear to be scarce in Sf9 cells, and they do not interact with the fusion protein in a manner that could be detected by coimmunoprecipitation. Furthermore, β1-subunits that were coexpressed with γ2 and M2(ΔR)His6αi1(NT) formed a complex with the fusion protein without increasing the relative number of highaffinity sites detected by oxotremorine-M. It follows that the sites of high affinity do not arise from an interaction between a subpopulation of fusion proteins and a limited quantity of endogenous β-subunits or βγ heterodimers. A similar result has been described previously for a stable fusion protein of the β2–adrenergic receptor and αs expressed in Sf9 cells, where the fraction of sites exhibiting higher affinity for isoproterenol was not increased upon coexpression with β1 and γ2 [20]. The nucleotide-sensitive binding of oxotremorine-M to M2(ΔR) His6αi1(NT) suggests that the interaction between the M2 receptor and αi1 in the fusion protein resembles that in preparations such as myocardial membranes, where the magnitude of the effect of guanyl nucleotides is a measure of efficacy [42]. Since the α-subunit in M2 (ΔR)His6αi1(NT) is linked covalently to the receptor, the similarity suggests that an α-subunit in natural tissues remains associated with its attendant receptor throughout the signaling process. The present results resemble those reported for other receptor–Gα fusion proteins (e.g. [16,20,40,43]), but the involvement of cleaved or endogenous α-subunit in earlier studies often cannot be ruled out. Also, a liberated α-subunit may remain associated with the receptor in those cases when cleavage occurs [40]. Tethering of the α-subunit to the receptor ensures that the association is immutable and that the ratio of components is 1:1. A monomeric fusion protein therefore is expected to yield a Hill coefficient of 1 for ligands to either component in a system at equilibrium [4]. It follows that the heterogeneity revealed in the binding of oxotremorine-M is induced in a population of sites that otherwise would appear homogeneous. A plausible explanation for such an induced effect attributes the heterogeneity to asymmetry or cooperativity among interacting sites within an oligomer [10,44]. Although the balance of those contributions is unclear, the cooperative potential of the system is illustrated by the noncompetitive binding of quinuclidinylbenzilate and N-methylscopolamine. Cooperativity also can be inferred from the Hill coefficient of 1.34 obtained for oxotremorine-M at the wild-type receptor in the presence of β1γ2. The tendency of β1γ2 to increase the value of nH may arise from an effect on the degree of cooperativity between successive equivalents of agonist binding to linked sites. Several lines of evidence suggest that the M2 receptor exists as a tetramer [10,44,45]; in the case of the fusion protein, bands corresponding to dimers and larger oligomers have been observed routinely on western blots (Fig. 4).

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These considerations suggest that the nucleotide-sensitive dispersion of affinities revealed by agonists is a property of the oligomeric state of the receptor. Within such a complex, an α-subunit might interact with its fused counterpart or with the receptor of a contiguous fusion protein. The feasibility of an intramolecular interaction has been demonstrated by studies in which free G proteins were mixed with monomers of GPCRs embedded in nanodiscs to yield a functional complex of G protein and receptor [46–49]. Evidence for intermolecular effects has emerged from studies on receptor–Gα fusion proteins and their unfused counterparts [17,25–27,29]. With the D2 dopamine receptor fused to αqi5, a chimera that is resistant to pertussis toxin and mediates the activation of phospholipase C by the wild-type receptor [29], there was no signaling unless the receptor and the α-subunit were separated by an eight-amino acid linker or the D2-αqi5 fusion protein was coexpressed with the wild-type D2 receptor. The fused α-subunit therefore appears to have been activated by a neighboring receptor, presumably within an oligomer. The N-terminus of Gα-subunits has been implicated in a variety of functions, including post-translational modifications [50], attachment to the membrane [50], interaction with the βγ heterodimer [36], and coupling of the G protein to the receptor [50]. Truncation of αi1 at the N-terminus eliminated the site of palmitoylation, but the role of the latter in membrane targeting may be irrelevant when the α-subunit is fused to a GPCR. Truncation also had no apparent effect on the proper coupling of fused αi1 to the M2 receptor, as indicated by the GMPPNP-sensitive binding of oxotremorine-M, nor did it prevent binding of the β1-subunit to the fusion protein, as indicated by coimmunoprecipitation. The link between the C-terminus of the receptor and the N-terminus of the α-subunit therefore appears to be long enough for the fused components to interact in the proper manner. Although indicative of coupling between the receptor and αi1, the GMP-PNP-sensitive binding of oxotremorine-M differs in at least one respect from that reported for agonists at the muscarinic receptor in myocardial membranes and in liposomal preparations reconstituted with purified M2 receptor and G proteins [[10] and references therein]. In those cases, three classes of sites were required to describe the binding of agonists either alone or in the presence of GMP-PNP. In contrast, only two classes of sites are required to account for the binding of oxotremorine-M to M2(ΔR)His6αi1(NT). G proteins reconstituted with the purified M2 receptor were a mixture of Go, Gi1, Gi2, and Gi3 [10], and the same G proteins are associated with muscarinic receptors in the heart [32]. It may be that the relative simplicity of the present data is due to the presence of only one subtype of α-subunit. The covalently determined ratio of 1 receptor per α-subunit in M2 (ΔR)His6αi1(NT) is in agreement with reports that the number of receptors equals the number of G proteins in purified complexes containing the cardiac muscarinic receptor [32] and the μ-opioid receptor from rat brain [51]. In contrast, modeling and biochemical studies have suggested in some cases that the ratio of α-subunits to receptors is 1:2 (e.g. [52,53]). Differences in the composition reported for purified complexes might be reconciled if the receptor or complex of receptors bound one G protein more tightly than another, perhaps because the second G protein is linked to the receptor via the first and prone to dissociation under some conditions. A similar rationale is not readily applied to the fusion protein, particularly in extracts from Sf9 cells. If the overall complement is indeed two receptors per G protein in vivo, one half of the fused α-subunits presumably are redundant in the oligomer formed by M2(ΔR)His6αi1(NT). Acknowledgments This work was supported by the Heart and Stroke Foundation of Ontario (grant T6280) and the Canadian Institutes of Health Research (grants MOP 43990 and MOP 97978). The authors are grateful to Dr. Tatsuya Haga of the Institute for Biomolecular Science, Gakushuin University, for kindly providing the plasmids and baculoviruses

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