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J. Neurochem. (2010) 112, 1442–1453. Abstract There is much evidence to suggest that G protein coupled receptors exist as oligomers but the relevance to their function is unclear. We have, therefore, examined the binding of the radiolabelled agonist [3H]NPA to membranes of CHO cells expressing the D2 dopamine receptor in dissociation rate experiments. When [3H]NPA dissociation was started by dilution, the dissociation rate in the absence of sodium ions was unaffected by addition of the antagonist/inverse agonist (+)-butaclamol, but was accelerated by addition of agonists e.g. dopamine, suggesting that the receptor was not behaving as a monomer with a single binding site. The very low efficacy partial agonist, aripiprazole provided an intermediate level of acceleration of dissociation. [3H]NPA dissociation experiments started by addition of ligands without dilution gave a similar pattern. [3H]NPA dissociation could also be accelerated by GTP. Dissociation of [3H]NPA in the presence of GTP and dopamine provided a greater acceleration than for either modulator alone, suggesting synergistic effects related to receptor/G protein interaction. When [3H]NPA dissociation experiments were performed in the presence of sodium ions, dissociation was faster than in their absence but the rate still depended on the ligand present in the assay. Overall the data cannot be explained by a ternary complex model and are consistent with an oligomeric receptor in which binding of [3H]NPA, as an example of an agonist ligand, can be modulated co-operatively by ligands binding elsewhere in the oligomer. Interactions with G proteins also occurs providing further modulation of [3H]NPA binding. Both agonists and G proteins are proposed to modulate the oligomer by switching high affinity agonist binding sites to low affinity sites.

A histidine residue (His(394)) that is likely to be located in the ligand-binding region of the D-2 dopamine receptor has been mutated to a leucine (Leu(394)), and the properties of the mutant receptor have been determined. For a range of antagonists the mutation has only a minor effect on the affinity of the receptor for the antagonist. The mutation does, however, elicit a structurally specific effect on the affinity with which certain members of the substituted benzamide class of antagonist bind to the receptor. Some of these drugs, e.g., sulpiride, sultopride, and tiapride, bind with reduced affinity to the mutated receptor, whereas others, e.g., clebopride and metoclopramide, bind with increased affinity. However, the Na+/H+ sensitivity of the binding of sulpiride to the receptor is not reduced by the mutation. These findings have been interpreted in terms of the productive or unfavourable interaction of the His(394) residue with these compounds.

Mechanisms of ligand binding and receptor activation for the human D2(short) dopamine receptor have been probed using two homologous series of monohydroxylated and dihydroxylated agonists (phenylethylamines and 2-dipropylaminotetralins). In ligand binding studies, the majority of compounds exhibited competition curves versus [3H]spiperone that were best fitted using a two site binding model. The compounds had different abilities (potencies and maximal effects) to stimulate [35S]GTPgammaS binding and to inhibit forskolin-stimulated cAMP accumulation. From the data it can be concluded that: (i) the ability of an agonist to stabilize receptor/G protein coupling can be used to predict agonist efficacy for some groups of compounds (2-dipropylaminotetralins) but not for others (phenylethylamines); (ii) the receptor may be activated by unhydroxylated compounds; (iii) single hydroxyl groups or pairs of hydroxyl groups on the agonist may contribute to binding affinity, potency and efficacy; and (iv) for the 2-dipropylaminotetralin series two modes of agonist/receptor interaction have been identified associated with different relative efficacy.

The effect of mutations (V344E and T343A/V344E) in the third intracellular loop of the serotonin 5-HT(1A) receptor expressed transiently in human embryonic kidney 293 cells have been examined in terms of receptor/G protein interaction and signaling. Serotonin, (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT], and buspirone inhibited cyclic AMP production in cells expressing native and mutant 5-HT(1A) receptors. Serotonin, however, produced inverse bell-shaped cyclic AMP concentration-response curves at native and mutant 5-HT(1A) receptors, indicating coupling not only to G(i)/G(o), but also to G(s). (R)-8-OH-DPAT, however, induced stimulation of cyclic AMP production only after inactivation of G(i)/G(o) proteins by pertussis toxin and only at the mutant receptors. The partial agonist buspirone was unable to induce coupling to G(s) at any of the receptors, even after pertussis toxin treatment. The basal activities of native and mutant 5-HT(1A) receptors in suppressing cyclic AMP levels were not found to be significantly different. The receptor binding characteristics of the native and mutant receptors were investigated using the novel 5-HT(1A) receptor antagonist [(3)H]NAD-299. For other receptors, analogous mutations have produced constitutive activation. This does not occur for the 5-HT(1A) receptor, and for this receptor the mutations seem to alter receptor/G protein coupling, allowing ligand-dependent coupling of receptor to G(s) in addition to G(i)/G(o) proteins.

[3H]Nemonapride and [3H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [3H]nemonapride and [3H]spiperone give markedly different Bmax values for preparations of D2 dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D2(short), D2(long), and D3] in different buffers. Bmax values derived using either radioligand differ by an average of < 20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [3H]spiperone and [3H]nemonapride do not differentiate between different forms or populations of D2-like receptors.

Three serine residues (Ser193, Ser194, Ser197) in the fifth transmembrane-spanning region of the D2 dopamine receptor have been mutated separately to alanine and the effects of the mutations determined in ligand-binding experiments with [3H] spiperone. For many antagonists the mutations had little effect, showing that the overall conformation of the mutant receptors was similar to that of the native, although there were effects on the binding of certain antagonists. The effect of the mutations on agonist binding to the free receptor (uncoupled from G proteins) was determined in the presence of GTP (100 microM). This showed that there was no single mode of binding of catecholamine agonists to the receptor and that all three serine residues can participate in the binding of some agonists, possibly through hydrogen bonds to the catechol hydroxyl groups. Coupling of the mutant receptors to G proteins was assessed from agonist-binding curves in the absence of GTP, when higher and lower affinity agonist-binding sites were seen. Receptor/G protein coupling was generally unaffected by the Ala193 and Ala194 mutations, but the Ala197 mutation eliminated receptor/G protein coupling for some agonists. These data show that the interactions of agonists with the free and coupled forms of the receptor are different.

Mechanisms of agonist action at the G protein-coupled D-2(short) dopamine receptor expressed in Chinese hamster ovary cells have been investigated. Agonist binding was assayed in the presence and absence of GTP (100 mu M). Data in the absence of GTP were fitted best by a two-site model (apomorphine, dopamine, 10,11-dihydroxy-N-n-propylnorapomorphine hydrochloride, and quinpirole) or a one-site model [bromocriptine, dihydroergocristine, and (-)-3-(3-hydroxyphenyl)-N-propylpiper idine hydrochloride], whereas in the presence of GTP a one-site model was the best fit for all compounds. Agonist binding parameters were used to provide a measure of the ability of the agonist to stabilise the ternary complex of agonist/receptor/G protein. Agonist stimulation of [S-35]guanosine 5'-O-(3-thiotriphosphate) ([S-35]GTP gamma S) binding for a range of agonist concentrations was measured and the EC50 and maximal effects determined. The initial rates of [S-35]GTP gamma S binding induced by maximally stimulating agonist concentrations were also recorded. Simultaneous inhibition of agonist-stimulated [S-35]GTP gamma S binding and receptor occupancy by spiperone was determined. Agonist inhibition of forskolin-stimulated cyclic AMP accumulation was determined for a range of agonist concentrations and the EC50 and maximal inhibition recorded. The data on the maximal agonist responses showed that it was possible to detect a spectrum of agonist efficacy (partial and full agonism) in both functional assays. The data on the apparent potencies of agonists to elicit the functional responses showed that different extents of amplification of response were seen for different agonists in both assays, The maximal activity data have been compared with the stabilisation of the agonist/receptor/G protein ternary complex as measured in binding assays.

To understand the role of conserved serine residues in the fifth transmembrane domain (Ser192, Ser193, and Ser196) of the D3 dopamine receptor, these have been mutated individually to alanine, and the ligand binding properties of the mutant receptors have been evaluated. The mutations had little or no effect on the binding of the antagonist spiperone and the agonist quinpirole, indicating that the overall conformation of the receptor was unaffected. The binding of dopamine and 7-hydroxydipropylaminotetralin, agonists containing hydroxyl groups, was, however, of lower affinity for the Ser192 mutation but unaffected by the other mutations (Ser193 and Ser196). Therefore, for the agonists tested, the hydroxyl groups interact exclusively with Ser192.

Mechanisms of agonist and inverse agonist action at the serotonin 5-HT1A receptor have been studied using the modulation of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding in membranes of Chinese hamster ovary (CHO) cells expressing the receptor (CHO-5-HTA1A cells). A range of agonists increased [35S]GTPgammaS binding with different potencies and to different maximal extents, whereas two compounds, methiothepin and spiperone, inhibited both agonist-stimulated and basal [5S]GTPgammaS binding, thus exhibiting inverse agonism. Potencies of agonists to stimulate [35S]GTPgammaS binding in membranes from CHO-5-HT1A cells were reduced by adding increasing concentrations of GDP to assays, whereas changes in sodium ion concentration did not affect agonist potency. The maximal effect of the agonists was increased by increasing sodium ion concentrations. The affinities of agonists in ligand binding assays were unaffected by changes in sodium ion concentration. Increasing GDP in the assays of the inverse agonists increased potency for spiperone to inhibit [35S]GTPgammaS binding and had no effect for methiothepin, in agreement with the sensitivity of these compounds to guanine nucleotides in ligand binding assays. Potencies for these inverse agonists were unaffected by changes in sodium ion concentration. These data were simulated using the extended ternary complex model. These simulations showed that the data obtained with agonists were consistent with these compounds achieving agonism by stabilising the ternary complex. For inverse agonists, the simulations showed that the mechanism for spiperone may be to stabilise forms of the receptor uncoupled from G proteins. Methiothepin, however, probably does not alter the equilibrium distribution of different receptor species; rather, this inverse agonist may stabilise an inactive form of the receptor that can still couple to G protein.

The antipsychotic drugs have been shown to be inverse agonists at the D(2) dopamine receptor. We have examined the mechanism of this inverse agonism by making mutations in residue T343 in the base of the sixth transmembrane spanning region of the receptor. T343R, T343S and T343K mutant D(2) dopamine receptors were made and the T343R mutant characterized in detail. The T343R mutant D(2) dopamine receptor exhibits properties of a receptor that resides more in the activated state, namely increased agonist binding affinity (independent of G-protein coupling and dependent on agonist efficacy), increased agonist potency in functional tests (adenylyl cyclase inhibition) and increased inverse agonist effects. The binding of agonists to the mutant receptor also shows sensitivity to sodium ions, unlike the native receptor, so that isomerization of the receptor to its inactive state may be driven by sodium ions. The binding of inverse agonists to the receptor is, however, unaffected by the mutation. We conclude that inverse agonism at this receptor is not achieved by the inverse agonist binding preferentially to the non-activated state of the receptor over the activated state. Rather the inverse agonist appears to bind to all forms of the receptor but then renders the receptor inactive.

Mechanisms of ligand binding and receptor activation for the human D2(short) dopamine receptor have been probed using two homologous series of monohydroxylated and dihydroxylated agonists (phenylethylamines and 2-dipropylaminotetralins). In ligand binding studies, the majority of compounds exhibited competition curves versus [3H]spiperone that were best fitted using a two site binding model. The compounds had different abilities (potencies and maximal effects) to stimulate [35S]GTPgammaS binding and to inhibit forskolin-stimulated cAMP accumulation. From the data it can be concluded that: (i) the ability of an agonist to stabilize receptor/G protein coupling can be used to predict agonist efficacy for some groups of compounds (2-dipropylaminotetralins) but not for others (phenylethylamines); (ii) the receptor may be activated by unhydroxylated compounds; (iii) single hydroxyl groups or pairs of hydroxyl groups on the agonist may contribute to binding affinity, potency and efficacy; and (iv) for the 2-dipropylaminotetralin series two modes of agonist/receptor interaction have been identified associated with different relative efficacy.

The antipsychotic drugs have been shown to be inverse agonists at the D(2) dopamine receptor. We have examined the mechanism of this inverse agonism by making mutations in residue T343 in the base of the sixth transmembrane spanning region of the receptor. T343R, T343S and T343K mutant D(2) dopamine receptors were made and the T343R mutant characterized in detail. The T343R mutant D(2) dopamine receptor exhibits properties of a receptor that resides more in the activated state, namely increased agonist binding affinity (independent of G-protein coupling and dependent on agonist efficacy), increased agonist potency in functional tests (adenylyl cyclase inhibition) and increased inverse agonist effects. The binding of agonists to the mutant receptor also shows sensitivity to sodium ions, unlike the native receptor, so that isomerization of the receptor to its inactive state may be driven by sodium ions. The binding of inverse agonists to the receptor is, however, unaffected by the mutation. We conclude that inverse agonism at this receptor is not achieved by the inverse agonist binding preferentially to the non-activated state of the receptor over the activated state. Rather the inverse agonist appears to bind to all forms of the receptor but then renders the receptor inactive.

Three conserved serine residues (Ser193, Ser194, and Ser197) in transmembrane spanning region (TM) V of the D2 dopamine receptor have been mutated to alanine, individually and in combination, to explore their role in ligand binding and G protein coupling. The multiple Ser -->Ala mutations had no effect on the binding of most antagonists tested, including [3H]spiperone, suggesting that the multiple mutations did not affect the overall conformation of the receptor protein. Double or triple mutants containing an Ala197 mutation showed a decrease in affinity for domperidone, whereas Ala193 mutants showed an increased affinity for a substituted benzamide, remoxipride. However, dopamine showed large decreases in affinity (>20-fold) for each multiple mutant receptor containing the Ser193Ala mutation, and the high-affinity (coupled) state of the receptor (in the absence of GTP) could not be detected for any of the multiple mutants. A series of monohydroxylated phenylethylamines and aminotetralins was tested for their binding to the native and multiple mutant D2 dopamine receptors. The results obtained suggest that Ser193 interacts with the hydroxyl of S-5-hydroxy-2-dipropylaminotetralin (OH-DPAT) and Ser197 with the hydroxyl of R-5-OH-DPAT. We predict that Ser193 interacts with the hydroxyl of R-7-OH-DPAT and the 3-hydroxyl (m-hydroxyl) of dopamine. Therefore, the conserved serine residues in TMV of the D2 dopamine receptor are involved in hydrogen bonding interactions with selected antagonists and most agonists tested and also enable agonists to stabilise receptor-G protein coupling.

Serotonin 5-HT 1A receptors expressed stably in recombinant Chinese hamster ovary cells have been studied using radioligand binding with the radiolabelled agonist [ 3 H]lisuride. Competition studies with a range of antagonists versus [ 3 H]lisuride confirmed that all of the specific [ 3 H]lisuride binding was to 5-HT 1A receptors on the cells. Competition studies with the antagonist spiperone and several agonists gave data that fitted best to two-binding-site models. The affinities of these competing ligands at the two classes of sites were generally in agreement with their corresponding affinities determined in previous work with either 8-[ 3 H]hydroxydipropylaminotetralin ([ 3 H]8-OH-DPAT ; labels receptor coupled to G protein) or [ 3 H]spiperone (labels free receptor). Saturation analyses with [ 3 H]lisuride showed that this radioligand labels a single class of binding sites, but the level of radioligand binding was approximately twice that seen when either [ 3 H]8-OH-DPAT or [ 3 H]spiperone was used. [ 3 H] Lisuride binding was partially inhibited by addition of guanine nucleotides, and the extent of inhibition decreased as the [ 3 H]lisuride concentration was increased. This inhibition was due to the effect of guanine nucleotide to decrease slightly the affinity of [ 3 H]lisuride for binding to the 5-HT 1A receptors on the cells. It is concluded that [ 3 H]lisuride can label both the free receptor and the receptor coupled to G proteins but with slightly different affinities and that these two states of the receptor exist in roughly equal amounts in the cells. Agonists generally have a higher affinity for the receptor coupled to G protein, whereas antagonists, with the exception of spiperone (which has a higher affinity for the free receptor), have roughly equal affinities for the free receptor and the receptor coupled to G proteins.

The short and long forms of the D2 dopamine receptor have been expressed at similar levels in fibroblast Ltk− cells, and the long form of the receptor has also been expressed in Chinese hamster ovary cells. The ligand binding properties of the two forms of receptor expressed in different systems have been compared in saturation analyses with [3H]spiperone and in competition studies with a range of antagonists. The long form of the receptor expressed in two different cell hosts exhibits essentially identical properties. However, whereas the long and short forms of the receptor show very similar affinities for several compounds representative of different chemical classes, the short form shows a two- to fivefold higher affinity for several substituted benzamide drugs. The conformation of the receptor binding site may therefore be different in the two receptor isoforms.

A series of group specific modifying reagents were tested for their effects on [3H]spiperone binding to brain D2 dopamine receptors to identify amino acid residues at the binding site of the D2 dopamine receptor that are critical for ligand binding. The dependence of ligand binding to the receptor on the pH of the incubation medium was also examined. N-Acetylimidazole, 5,5'-dithiobis(2-nitrobenzoic acid), 1,2-cyclohexanedione, and acetic anhydride had no specific effect on [3H]spiperone binding, indicating the lack of participation of tyrosine, free sulphydryl, arginine, or primary amino groups in ligand binding to the receptor. N,N'-Dicyclohexylcarbodiimide (DCCD) potently reduced the number of [3H]spiperone binding sites, indicating that a carboxyl group is involved in ligand binding to the receptor. The effects of DCCD could be prevented by prior incubation of the receptor with D2 dopamine receptor selective compounds. The pH-binding profile for [3H]spiperone binding indicated the importance of an ionising group of pKa 5.2 for ligand binding which may be the same carboxyl group. Diethyl pyrocarbonate, the histidine modifying reagent, also inhibited [3H]spiperone binding, reducing the affinity of the receptor for this ligand but the effects were not at the ligand binding site. From the effects of pH changes on ligand binding some evidence was obtained for a second ionising group (pKa 7.0) that specifically affects the binding of substituted benzamide drugs to the receptor. It is concluded that the D2 dopamine receptor binding site contains separate but over-lapping binding regions for antagonists such as spiperone and substituted benzamide drugs. The former region contains an important carboxyl group; the latter region contains another group that may be a second carboxyl group or a histidine.

[3H]Spiperone binding has been used to study neurotransmitter receptors in bovine caudate nucleus in displacement and saturation binding experiments. Displacement curves for several antagonists are biphasic and can be analysed into contributions from dopaminergic and serotonergic sites. Antagonist binding at each class of sites follows the simple mass action equations for binding at a homogeneous set of sites (slope factors close to unity). Agonist displacement curves also indicate complex behaviour, but agonist binding to the dopaminergic sites alone exhibits heterogeneous properties (slope factors less than unity). Saturation binding experiments have been conducted on each class of site, defining dopaminergic binding of [3H]spiperone as that binding displaced by 0.1 mM-dopamine and serotonergic binding as that displaced by 0.3 microM-mianserin. In each case, a single class of binding sites was detected: the binding parameters derived in this way have been used to calculate the proportions of the two classes of binding site observed in displacement experiments. Good agreement was obtained between calculated and observe values.

The effect of compounds that activate sodium channels on the number of muscarinic acetylcholine receptors in neuroblastoma NIE 115 cells has been investigated. The cells were used in electrically unexcitable (“control” cells) and excitable (“differentiated” cells) states. Although receptor assays using a single concentration of the radioligand [3H]scopolamine methyl chloride indicated a loss of receptors after a 6-h incubation of cells with veratrine, no true loss of receptors was seen with any of the compounds tested (veratridine, veratrine, aconitine) when full saturation analyses were performed in either control or differentiated cells. The apparent receptor loss seen with veratrine was due to a muscarinic receptor-active component of veratrine (not veratridine) occluded by the cells and released into the binding assays upon cell breakage. Veratridine and aconitine have a very low affinity for muscarinic acetylcholine receptors, and the binding of carbamoylcholine to the receptors is unaffected by tetrodotoxin, so that there is no evidence in this system for interaction between muscarinic receptors and sodium channels.

The lipophilic permeant cation [3H]triphenylmethylphosphonium (TPMP) was used to estimate membrane potential in neuroblastoma N1E 115 cells under carefully controlled conditions. The cation distributes into the cells only in the presence of a lipophilic anion, and tetraphenylboron and picrate have been used for this purpose. The potassium salt of tetraphenylboron is poorly soluble, so that studies in high [K+] media are difficult with this anion whereas picrate, at the concentrations required, hyperpolarises the cells. The effect of muscarinic receptor activation was investigated by treating cells with carbachol but no effect was seen either on [3H] TPMP distribution or electrophysiological parameters. The use of [3H]TPMP for qualitative and quantitative evaluation of membrane potential in these cells is discussed.

The use of cholate (in the presence of sodium chloride) is described for solubilising brain D2 dopamine receptors. The method described gives in high yield a preparation of solubilised D2 receptors which when assayed by [3H]spiperone binding show little interference from nonspecific and nonstereospecific binding sites. Characterisation by ultrafiltration, electron microscopy, gel filtration, and sucrose density gradient centrifugation has been achieved, showing the receptors to be truly solubilised. Pharmacological characterisation using [3H]spiperone binding suggested the presence of serotonergic S2 receptors in addition to D2 receptors. The pharmacological properties of the solubilised D2 receptors show a good correlation with those of membrane-bound receptors, although ligand binding affinities are lower in the solubilised preparation.

A series of detergents of varying chemical properties has been tested for solubilisation of bovine caudate nucleus D2 dopamine receptors using [3H]spiperone binding to assay the solubilised sites. The properties of the lysophosphatidylcholine (LPC)- and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulphonate (CHAPS)-solubilised preparations are described in detail. The preparations are truly solubilised, and sucrose density gradient and gel filtration data are reported. Specific [3H]spiperone binding in the LPC-solubilised preparation assayed at 4 degrees C is solely to D2 dopamine receptors. If the assay temperature is raised to 25 degrees C, the amount of specific [3H]spiperone binding is largely unchanged, but it forms a greater proportion of the total [3H]spiperone binding owing to a reduction in nonstereospecific (spirodecanone) [3H]spiperone binding at the higher temperature. The effect of raising the assay temperature is important as it enables more precise determinations of specific [3H]spiperone binding to be made. Part of the specific [3H]spiperone binding at 25 degrees C is to solubilised S2 serotonin receptors in addition to D2 dopamine receptors. Good correlations are observed between the affinities for binding of ligands to the solubilised D2 receptors and corresponding data obtained on membrane-bound receptors. Agonist binding in LPC-solubilised preparations is insensitive to guanine nucleotides. It is speculated that the spirodecanone sites represent, in part, proteolysed or damaged D2 dopamine, or S2 serotonin, receptors. In the CHAPS-solubilised preparation the pharmacological profile of [3H]spiperone binding is unclear when assayed at 4 degrees C, but in assays at 25 degrees C a clear serotonin S2 receptor component of specific [3H]spiperone binding can be discerned.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of guanine nucleotides on the binding properties of presynaptic muscarinic receptors has been studied in a membrane preparation from the electric organ of Torpedo marmorata by measuring the competitive displacement of the radiolabelled antagonist, [3H]quinuclidinyl benzilate, by nonradioactive muscarinic ligands. The binding of the antagonists, atropine, scopolamine and pirenzepine was to a single class of sites [slope factors (pseudo Hill coefficients) close to 1] and was unaffected by 0.1 mM GTP. The binding of the N-methylated antagonists, N-methylatropine and N-methyl-scopolamine was more complex (slope factors less than 1) but also insensitive (N-methylatropine) to 0.1 mM GTP. Agonist binding was complex and could be resolved into two binding sites with relatively high and low affinities. The proportion of high-affinity sites varied with the nature of the agonist (15-80%). Agonist binding was depressed by 0.1 mM GTP, and the order of sensitivity was oxotremorine-M greater than carbamoylcholine greater than muscarine greater than acetylcholine greater than arecoline greater than oxotremorine. The binding of pilocarpine, a partial agonist, was unaffected by GTP. With carbamoylcholine as a test ligand the GTP effect on agonist binding was half-maximal at 12 microM. GDP and guanylylimidodiphosphate produced comparable inhibition of carbamoylcholine binding, but GMP and cyclic GMP were ineffective, as were various adenine nucleotides. Analysis of agonist binding in terms of a two-site model indicates that the predominant effect of guanine nucleotides is to reduce the number of sites of higher affinity.

The distribution of 5'-nucleotidase activity, dopaminergic [3H]spiperone binding sites, and [3H]quinuclidinyl benzilate (QNB) binding sites in different subcellular fractions of bovine caudate nucleus has been studied. Each activity was enriched in a microsomal (P3) preparation from that tissue. The microsomal preparation was further fractionated by different techniques. First, the P3 fraction, or a sonicated P3 fraction, was fractionated on a discontinuous sucrose density gradient. Second, the P3 fraction, or a digitonin pretreated P3 fraction, was fractionated on a continuous sucrose density gradient. The results obtained demonstrate that 5'-nucleotidase activity does not cofractionate with radioligand binding activity, although no difference between the distributions of [3H]spiperone binding and [3H]QNB binding were seen. It is concluded that the two radioligand binding activities are located on nonglial membranes.