W276 mutation in the endothelin receptor subtype B impairs Gq coupling but not Gi or Go coupling

The mutation of W276 to cysteine within the human endothelin receptor subtype B (ET(B)R) is associated with Hirschsprung's disease, a congenital intestinal disease. The sequence surrounding W276 is highly conserved between the endothelin receptor subtypes A and B. We have introduced sets of mutations into W275 and W276 of the ET(B)R gene, and the corresponding W257 and W258 of the ET(A)R gene, and studied their coupling properties with G(i), G(o), and G(q) in reconstituted phospholipid vesicles. The prepared mutants all showed a similar affinity for endothelin-1. The W276C/ET(B)R and W276A/ET(B)R mutants had reduced activities in G(q) coupling but not in G(i)/G(o) coupling, while the W275A/ET(B)R displayed reduced activities in G(i)/G(q) coupling, with normal G(o) coupling. On the other hand, W257A/ET(A)R and W258A/ET(A)R exhibited wild-type activities in all examined G protein couplings. These results suggest that the defects in the G(q) signaling pathway by the ET(B)R are connected with Hirschsprung's disease and that the two conserved tryptophans play distinct roles in signal transduction by the two receptor subtypes. In addition, W275 and W276, which are thought to be located near the extracellular side of the transmembrane helix 5, play important roles in forming the active structure of ET(B)R.     

Mechanisms of muscarinic receptor action on Go in reconstituted phospholipid vesicles

Purified muscarinic receptors (0.5-10 nmol of L-[3H]quinuclidinyl benzilate-binding sites/mg of protein) from bovine brain and the GTP-dependent regulatory protein, Go, were reconstituted with a lipid mixture of phosphatidylcholine and cholesterol. Essentially all of the receptors could interact with Go as evinced by increases in affinity for agonist as large as 800-fold. Both the alpha and beta gamma subunits of Go were required for this effect. Similarly, both subunits were required for the stimulation of guanine nucleotide exchange by agonists. This latter action of the receptor on Go was catalytic and potentiated markedly by prior treatment with dithiothreitol. Initially, agonist stimulation of association of GTP and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to Go was small and variable due to high basal rates. Prior addition of excess GDP inhibited the basal rate of exchange but allowed stimulation by agonists. Under these conditions, oxotremorine stimulated the rates of association of GTP gamma S up to 10-fold. This selective effect was not mimicked by GTP which inhibited both the basal and hormone-dependent rates. Direct examination of the association of GTP and GDP to Go demonstrated that agonist caused either stimulation or marked inhibition, respectively. These results indicate that receptors stimulate guanine nucleotide exchange on G proteins by both increasing the rates of dissociation of nucleotides and altering their relative affinities such that binding of GTP becomes highly favored over GDP. This would ensure the activation of G proteins by receptors in the presence of both nucleotides.     

Functional interactions of recombinant alpha 2 adrenergic receptor subtypes and G proteins in reconstituted phospholipid vesicles.

The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 adrenergic receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.     

Activation of the inhibitory GTP-binding protein of adenylate cyclase, Gi, by beta-adrenergic receptors in reconstituted phospholipid vesicles

beta-Adrenergic receptors and the inhibitory GTP-binding protein, Gi of the adenylate cyclase system were reconstituted into phospholipid vesicles by the method described previously for reconstituting receptors and the stimulatory GTP-binding protein, Gs (Brandt, D. R., Asano, T., Pedersen, S. E., and Ross, E. M. (1983) Biochemistry 22, 4357-4362). In the receptor-Gi vesicles, beta-adrenergic agonists stimulated both the high-affinity binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to Gi and GTPase activity to an extent similar to that observed in vesicles containing beta-adrenergic receptors and Gs. Stimulation required receptors and displayed appropriate beta-adrenergic specificity. The prior treatment of receptor-Gi vesicles with islet-activating protein (pertussis toxin) plus NAD markedly inhibited both the isoproterenol-stimulated binding of GTP gamma S and the isoproterenol-stimulated GTPase activity. No contamination of Gi by Gs was apparent. These data suggest that receptors that typically stimulate adenylate cyclase activity may also activate the inhibitory system, perhaps as one mechanism of desensitization.     

Selectivity of the beta-adrenergic receptor among Gs, Gi's, and Go: assay using recombinant alpha subunits in reconstituted phospholipid vesicles.

The selective regulation of Gs (long and short forms), Gi's (1, 2, and 3), and Go by the beta-adrenergic receptor was assessed quantitatively after coreconstitution of purified receptor, purified G-protein beta gamma subunits, and individual recombinant G-protein alpha subunits that were expressed in and purified from Escherichia coli. Receptor and beta gamma subunits were incorporated into phospholipid vesicles, and the alpha subunits bound to the vesicles stoichiometrically with respect to beta gamma. Efficient regulation of alpha subunit by receptor required the presence of beta gamma. Regulation of G proteins was measured according to the stimulation of the initial rate of GTP gamma S binding, steady-state GTPase activity, and equilibrium GDP/GDP exchange. The assays yielded qualitatively similar results. GDP/GDP exchange was a first-order reaction for each subunit. The rate constant increased linearly with the concentration of agonist-liganded receptor, and the dependence of the rate constant on receptor concentration was a reproducible measurement of the efficiency with which receptor regulated each G protein. Reconstituted alpha s (long or short form) was stimulated by receptor to approximately the extent described previously for natural Gs. Both alpha i,1 and alpha i,3 were regulated with 25-33% of that efficiency. Stimulation of alpha o and alpha i,2 was weak, and stimulation of alpha o was barely detectable over its high basal exchange rate. Reduction of the receptor with dithiothreitol increased the exchange rates for all G proteins but did not alter the relative selectivity of the receptor.     

The purified alpha subunits of Go and Gi from bovine brain require beta gamma for association with phospholipid vesicles

The purified G-proteins from bovine brain were examined for potential solubility in the absence of detergent. The isolated alpha o and alpha i subunits migrated through sucrose with rates consistent with the existence of monomeric species either in the presence or the absence of cholate. The beta gamma subunits or holo-G-proteins aggregated extensively if cholate was absent. Al3+, Mg2+, and F- prevented the aggregation of alpha o and alpha i caused by the addition of beta gamma and could also prevent the aggregation of alpha s when Gs was examined at higher temperature. The association of subunits with phospholipid vesicles was examined. Whereas beta gamma associated totally with phospholipid vesicles, purified alpha o showed little interaction. alpha o did bind to vesicles containing beta gamma (beta gamma vesicles) in a saturable fashion that indicated a stoichiometric association between the subunits. Treatment with guanosine 5'-(3-O-thio)triphosphate could partially dissociate alpha o that was bound to beta gamma vesicles. These data suggest that beta gamma may be an anchor for association of alpha subunits with membranes and that regulation by these proteins may not be limited to the plasma membrane. This possibility and its implications are discussed. The reversible association of alpha o to beta gamma vesicles may provide a very sensitive system for the study of the interactions between these subunits.     

Electron microscopy of phospholipid vesicles reconstituted with purified renal Na,K-ATPase

Purified Na,K-ATPase after reconstitution into phospholipid vesicles catalyzed an active coupled transport with a ratio close to 3Na/2K. A uniform population of closed vesicles with average diameters close to 900 A are observed after freeze-fracture and thin sectioning. After freeze-fracture intramembranous particles with diameters of 80-100 A are observed. The data suggest that these particles correspond to Na,K-ATPase molecules.     

Incorporation of bovine enterokinase in reconstituted soybean phospholipid vesicles

Bovine enterokinase was incorporated into vesicles reconstituted from a soybean phospholipid mixture. A thin film hydration procedure (MacDonald, R. I., and MacDonald, R. C. (1975) J. Biol. Chem. 250, 9206-9214) produced vesicles with 40% of the enterokinase activity bound in the membrane. The highest incorporation was observed when cholesterol or dimyristoylphosphatidylethanolamine was added to the soybean phospholipids. Crude and highly purified enterokinase preparations were incorporated to the same extent suggesting that other membrane components were not required for a successful reconstitution. The properties of enterokinase in phospholipid vesicles were compared with those of alkaline phosphatase, which was also added to the reconstitution system, and with the enzyme activities present in vesicles prepared from brush-border membranes. The enzyme activities were not released by solutions of high ionic strength and remained associated with the phospholipid vesicles on gel filtration, ultracentrifugation, and sucrose density centrifugation. Enterokinase and alkaline phosphatase had their active sites exposed to substrate in the brush-border membrane vesicles. In soybean phospholipid vesicles half of the active sites of both enzymes were on the outside, since release of the enzyme with Triton X-100 almost doubled the units of enzyme present. Incubation of the soybean phospholipid and brush-border membrane vesicles with papain released the exposed molecules of enterokinase. The released enzyme molecules were fully active but could not be reincorporated into phospholipid vesicles. This suggests that the structure imbedded in the lipid bilayer was essential for a successful reconstitution. We conclude that the reconstituted soybean phospholipid vesicles are a suitable membrane system for the further study of membrane-bound enterokinase.     

Fusion of phospholipid vesicles reconstituted with cytochromec oxidase and mitochondrial hydrophobic protein

Summary Reconstituted cytochrome oxidase liposomes were fused with liposomes reconstituted with mitochondrial hydrophobic protein, which acts as a membrane-bound uncoupler of cytochrome oxidase. Fusion was assayed by the loss of respiratory control of cytochrome oxidase as measured by the increased rate of ascorbate oxidation induced by hydrophobic protein when both proteins shared the same vesicles. Fusion was dependent on the presence of phosphatidylserine in the liposomes and Ca⁺⁺ in the aqueous medium. Phosphatidylcholine-phosphatidylserine liposomes required higher concentrations of phosphatidylserine and Ca⁺⁺ than did phosphatidylethanolamine-phosphatidylserine liposomes. Cytochrome oxidase vesicles containing high concentrations of phosphatidylserine showed little or no respiratory control, while those with lower concentrations showed high respiratory control; respiratory control could be induced by fusing cytochrome oxidase vesicles containing high phosphatidylserine with protein-free liposomes containing low phosphatidylserine concentration. If cytochrome oxidase vesicles and hydrophobic protein vesicles were prefused separately for 15 min, they lost the ability to fuse upon being subsequently mixed together. The reconstituted vesicles had diameters of about 200 Å; fusion yielded vesicles with diameters in excess of 1000 Å.     

Magnetic resonance study of 13C-enriched glycophorin A reconstituted into phospholipid vesicles

$\text{[S-[}{}^{13}\text{C}\text{]}\text{methylmethionine-8 and -81]glycophorin}$ A was reconstituted into l-α-phosphatidyl choline vesicles. Results indicate that the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethyionine}\text{-81}$ residue in the phospholipid bilayer has limited mobility and is not susceptible to dealkylation, whereas the opposite effects are indicated for the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethionine}\text{-8}$ residue.     

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.     

Specificity of receptor-G protein interactions. Discrimination of Gi subtypes by the D2 dopamine receptor in a reconstituted system

The selectivity of D2 dopamine receptor-guanine nucleotide-binding protein (G protein) coupling was studied by reconstitution techniques utilizing purified D2 dopamine receptors from bovine anterior pituitary and resolved G proteins from bovine brain, bovine pituitary, and human erythrocyte. Titration of a fixed receptor concentration with varying G protein concentrations revealed two aspects of receptor-G protein coupling. First, Gi2 appeared to couple selectively with the D2 receptor with approximately 10-fold higher affinity than any other tested Gi subtype. Second, the G proteins differed in the maximal receptor-mediated agonist stimulation of the intrinsic GTPase activity. Gi2 appeared to be maximally stimulated by agonist-receptor complex with turnover numbers of approximately 2 min-1. The other Gi subtypes, Gi1 and Gi3, could be only partially activated, resulting in maximal rates of GTPase of approximately 1 min-1. Agonist-stimulated GTPase activity was not detected in preparations containing Go from bovine brain. The differences in maximal agonist-stimulated GTPase rates observed among the Gi subtypes could be explained by differences in agonist-promoted guanyl nucleotide exchange. Both guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding and GDP release parameters were enhanced 2-fold for the Gi2 subtype over the other Gi subtypes. These results suggest that even though several types of pertussis toxin substrate may exist in most tissues, a receptor may interact discretely with G proteins, thereby dictating signal transduction mechanisms.     

Interactions of polymerized phospholipid vesicles with cells. Uptake, processing and toxicity in macrophages

We have studied the uptake of photopolymerized multilamellar vesicles composed of bis(1,2(methacryloyloxy)dodecanoyl)-L-alpha-phosphatidylchol ine (DPL) by mouse peritoneal macrophages in vitro. Vesicles composed of polymerized DPL are taken up more rapidly and extensively than vesicles composed of conventional phosphatidylcholine. The uptake of radioactive DPL vesicles was not blocked by incubation with unlabelled phosphatidylcholine vesicles in either the fluid or gel state. Likewise, fluid-phase negatively charged vesicles failed to block uptake of DPL vesicles, whereas solid-phase negatively charged vesicles did have a blocking effect. A radioactive lipophilic marker (dipalmitoylphosphatidyl[N-methyl-3H]choline) incorporated into DPL vesicles was metabolized at essentially the same rate whether the vesicles were polymerized or not. Nonpolymerized DPL vesicles were quite toxic to macrophages, whereas polymerized DPL vesicles or vesicles composed of conventional phosphatidylcholines were not toxic.     

Fusion of phospholipid vesicles reconstituted with cytochrome c oxidase and mitochondrial hydrophobic protein.

Reconstituted cytochrome oxidase liposomes were fused with liposomes reconstituted with mitochondrial hydrophobic protein, which acts as a membrane-bound uncoupler of cytochrome oxidase. Fusion was assayed by the loss of respiratory control of cytochrome oxidase as measured by the increased rate of ascorbate oxidation induced by hydrophobic protein when both proteins shared the same vesicles. Fusion was dependent on the presence of phosphatidylserine in the liposomes Ca++ in the aqueous medium. Phosphatidylcholine-phosphatidylserine liposomes required higher concentrations of phosphatidylserine and Ca++ than did phosphatidylethanolamine-phosphatidylserine liposomes. Cytochrome oxidase vesicles containing high concentrations of phosphatidylserine showed little or no respiratory control, while those with lower concentrations showed high respiratory control; respiratory control could be induced by fusing cytochrome oxidase vesicles containing high phosphatidylserine with protein-free liposomes containing low phosphatidylserine concentration. If cytochrome oxidase vesicles and hydrophobic protein vesicles were prefused separately for 15 min, they lost the ability to fuse upon being subsequently mixed together. The reconstituted vesicles had diameters of about 200 A; fusion yielded vesicles with diameters in excess of 1000 A.     

Conformational transitions in fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles

Fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles has been used to study conformational transitions. Addition of K+ or Na+ to the vesicle medium induces fluorescence changes characteristic of the E2(K) or E1Na states of fluorescein-labeled (Na,K)ATPase (Karlish, S.J.D. (1980) J. Bioenerg. Biomembr. 12, 111-136). The cation effects are exerted from the cytoplasmic surface of inside-out-oriented pumps. Equilibrium cation titrations and measurements of rates of conformational transitions have led to the following observations. 1) The rate of E2(K)----E1Na or E2(T1)----E1Na is 4-6-fold faster and E1K----E2(K) is about 2-fold slower in vesicles compared to enzyme. In equilibrium titrations the K0.5 for K+ is higher and that for Na+ is lower for vesicles compared to enzyme. The conformational equilibrium E(1)2K----E2(2K) is apparently shifted toward E(1)2K in vesicles compared to enzyme. 2) Diffusion potentials, positive-outside, induced with valinomycin or Li+ ionophore AS701, do not affect the rates of E2(T1)----E1Na or E1K----E2(K), or equilibrium cation titrations. This demonstrates that the conformational transitions E(1)2K----E2(2K) are voltage-insensitive steps, confirming a prediction based on transport experiments. 3) In vesicles containing choline, K+, Na+, or Li+, the rate of E2(T1)----E1Na increases in the order given. Vesicles with reconstituted fluorescein-labeled (Na,K)ATPase provide a convenient system for correlating directly properties of conformational transitions with cation transport.     

Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers

Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Although both receptor subtypes are co-expressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ET(B)-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ET(A) receptor and CFP (ET(A)myc.CFP) and a fusion protein consisting of an ET(B) receptor and YFP (ET(B).YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ET(B) and myc-tagged ET(A) receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ET(A)myc.CFP and ET(B).YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ET(B) receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A.dynamin I or incubation with 450 mm sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ET(A)myc.CFP/ET(B)flag.YFP receptors revealed a slower sequestration of the ET(B)flag.YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ET(B)flag.YFP receptors alone. The data suggest that ET(A) and ET(B) receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ET(B)-selective stimulation.     

Interactions of phospholipid vesicles with rat hepatocytes in vitro influence of vesicle-incorporated glycolipids

We examined the interaction of glycolipid-containing phospholipid vesicles with rat hepatocytes in vitro. Incorporation of either $\text{N-}\text{lignoceroyldihydrolactocerebroside}$ or the monosialoganglioside, G_M1, enhanced liposomal lipid uptake 4–5-fold as judged by the uptake of radioactive phosphatidylcholine as a vesicle marker. Cerebroside enhanced phospholipid uptake only when incorporated into dimyristoyl, but not into egg phosphatidylcholine vesicles. The lack of cerebroside effect in egg phosphatidylcholine-containing vesicles appeared to be due to a limited exposure of the carbohydrate part of the glycolipid as suggested by the reduced agglutinability of those vesicles by Ricinus communis agglutinin.In contrast to the results with radioactive phosphatidylcholine, we observed only a 20% increase in vesicle-cell association as a result of glycolipid incorporation, when a trace amount of [¹⁴C]cholesteryloleate served as a marker of the liposomal lipids or when using the fluorescent dye, carboxyfluorescein, as a marker of the aqueous space of the vesicles. By the same token, intracellular delivery of vesicle-contents was only slightly enhanced (approx. 10%).The discrepancy between the association with the cells of phosphatidylcholine on the one hand and cholesteryoleate or entrapped marker on the other suggests different mechanisms of uptake for these markers. Our results are compatible with the notion that the main effect of incorporation of glycolipids into the vesicles is the enhancement of exchange or transfer of phospholipid molecules between vesicles and cells. Incubation of the cells with galactose or lactose, prior to addition of vesicles, suggests that this enhanced phospholipid exchange or transfer involves specific recognition of the terminal galactose residues of the glycolipid vesicles by a receptor present on the plasma membranes of hepatocytes.     

Recovery of oligomers and cooperativity when monomers of the M2 muscarinic cholinergic receptor are reconstituted into phospholipid vesicles

FLAG- and HA-tagged M2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[3H]methylscopolamine (NMS) was only 60% of that for [3H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [3H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [3H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations.