Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system.

Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.

Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors.

Measurements of affinity and efficacy are fundamental for work on agonists both in drug discovery and in basic studies on receptors. In this review I wish to consider methods for measuring affinity and efficacy at G protein coupled receptors (GPCRs). Agonist affinity may be estimated in terms of the dissociation constant for agonist binding to a receptor using ligand binding or functional assays. It has, however, been suggested that measurements of affinity are always contaminated by efficacy so that it is impossible to separate the two parameters. Here I show that for many GPCRs, if receptor/G protein coupling is suppressed, experimental measurements of agonist affinity using ligand binding (K(obs)) provide quite accurate measures of the agonist microscopic dissociation constant (KA). Also in pharmacological functional studies, good estimates of agonist dissociation constants are possible. Efficacy can be quantitated in several ways based on functional data (maximal effect of the agonist (E(max)), ratio of agonist dissociation constant to concentration of agonist giving half maximal effect in functional assay (K(obs)/EC50), a combined parameter E(max)K(obs)/EC50). Here I show that E(max)K(obs)/EC50 provides the best assessment of efficacy for a range of agonists across the full range of efficacy for full to partial agonists. Considerable evidence now suggests that ligand efficacy may be dependent on the pathway used to assess it. The efficacy of a ligand may, therefore, be multidimensional. It is still, however, necessary to have accurate measures of efficacy in different pathways.

Mechanisms underlying agonist efficacy.

Agonist efficacy is a measure of how well an agonist can stimulate a response system linked to a receptor. Efficacy can be assessed in functional assays and various parameters (E(max), K(A)/EC(50), E(max).K(A)/EC(50)) determined. The E(max).K(A)/EC(50) parameter provides a good estimate of efficacy across the full range of efficacy. A convenient assay for the efficacy of agonists for some receptors is provided by the [(35)S]GTP[S] (guanosine 5'-[gamma-[(35)S]thio]triphosphate)-binding assay. In this assay, the normal GTP-binding event in GPCR (G-protein-coupled receptor) activation is replaced by the binding of the non-hydrolysable analogue [(35)S]GTP[S]. This assay may be used to profile ligands for their efficacy, and an example here is the D(2) dopamine receptor where an efficacy scale has been set up using this assay. The mechanisms underlying the assay have been probed. The time course of [(35)S]GTP[S] binding follows a pseudo-first-order reaction with [(35)S]GTP[S] binding reaching equilibrium after approx. 3 h. The [(35)S]GTP[S]-binding event is the rate-determining step in the assay. Agonists regulate the maximal level of [(35)S]GTP[S] bound, rather than the rate constant for binding. The [(35)S]GTP[S]-binding assay therefore determines agonist efficacy on the basis of the amount of [(35)S]GTP[S] bound rather than the rate of binding.

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor CCR5.

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca(2+) mobilisation in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1alpha (D26A) (MIP-1alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5), MIP-1beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin-stimulated cAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCL5, CCL8 and CCL13 were able to stimulate Ca(2+) mobilisation through CCR5, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca(2+) responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca(2+) mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca(2+) mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCR5.

Mechanisms of G protein activation via the D2 dopamine receptor: evidence for persistent receptor/G protein interaction after agonist stimulation.

BACKGROUND AND PURPOSE: The aim of this report is to study mechanisms of G protein activation by agonists. EXPERIMENTAL APPROACH: The association and dissociation of guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding at G proteins in membranes of CHO cells stably transfected with the human dopamine D(2short) receptor was studied in the presence of a range of agonists. KEY RESULTS: Binding of [(35)S]GTPgammaS was dissociable in the absence of agonist and dissociation was accelerated both in rate and extent by dopamine, an effect which was blocked by the dopamine D(2) receptor antagonist raclopride and by suramin, which inhibits receptor/G protein interaction. A range of agonists of varying efficacy increased the rate of dissociation of [(35)S]GTPgammaS binding, with the more efficacious agonists resulting in faster dissociation. Agonists were able to dissociate about 70% of the pre-bound [(35)S]GTPgammaS, leaving a component which may not be accessible to the agonist-bound receptor. The dissociable component of the [(35)S]GTPgammaS binding was reduced with longer association times and increased [(35)S]GTPgammaS concentrations. CONCLUSIONS AND IMPLICATIONS: These data are consistent with [(35)S]GTPgammaS binding being initially to receptor-linked G proteins and then to G proteins which have separated from the agonist bound receptor. Under the conditions used typically for [(35)S]GTPgammaS binding assays, therefore, much of the agonist-receptor complex remains in proximity to G proteins after they have been activated by agonist.

Assays for enhanced activity of low efficacy partial agonists at the D(2) dopamine receptor.

BACKGROUND AND PURPOSE: Low efficacy partial agonists at the D2 dopamine receptor may be useful for treating schizophrenia. In this report we describe a method for assessing the efficacy of these compounds based on stimulation of [35S]GTPgammaS binding. EXPERIMENTAL APPROACH: Agonist efficacy was assessed from [(35)S]GTPgammaS binding to membranes of CHO cells expressing D2 dopamine receptors in buffers with and without Na+. Effects of Na+ on receptor/G protein coupling were assessed using agonist/[3H]spiperone competition binding assays. KEY RESULTS: When [35S]GTPgammaS binding assays were performed in buffers containing Na+, some agonists (aripiprazole, AJ-76, UH-232) exhibited very low efficacy whereas other agonists exhibited measurable efficacy. When Na+ was substituted by N-methyl D-glucamine, the efficacy of all agonists increased (relative to that of dopamine) but particularly for aripiprazole, aplindore, AJ-76, (-)-3-PPP and UH-232. In ligand binding assays, substitution of Na+ by N-methyl D-glucamine increased receptor/G protein coupling for some agonists -. aplindore, dopamine and (-)-3-PPP - but for aripiprazole, AJ-76 and UH-232 there was little effect on receptor/G protein coupling. CONCLUSIONS AND IMPLICATIONS: Substitution of Na+ by NMDG increases sensitivity in [(35)S]GTPgammaS binding assays so that very low efficacy agonists were detected clearly. For some agonists the effect seems to be mediated via enhanced receptor/G protein coupling whereas for others the effect is mediated at another point in the G protein activation cycle. AJ-76, aripiprazole and UH-232 seem particularly sensitive to this change in assay conditions. This work provides a new method to discover these very low efficacy agonists.

Agonist regulation of D(2) dopamine receptor/G protein interaction. Evidence for agonist selection of G protein subtype.

The D(2) dopamine receptor has been expressed in Sf21 insect cells together with the G proteins G(o) and G(i2), using the baculovirus system. Expression levels of receptor and G protein (alpha, beta, and gamma subunits) in the two preparations were similar as shown by binding of [(3)H]spiperone and quantitative Western blot, respectively. For several agonists, binding data were fitted best by a two-binding site model in either preparation, showing interaction of expressed receptor and G protein. For some agonists, binding to the higher affinity site was of higher affinity in D(2)/G(o) than in the D(2)/G(i2) preparation. Some agonists exhibited binding data that were best fitted by a two-binding site model in D(2)/G(o) and a one-binding site model in D(2)/G(i2). Therefore, receptor/G protein interaction seemed to be stronger in the D(2)/G(o) preparation. Agonist stimulation of [(35)S]GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) binding in the two preparations also gave evidence for higher affinity D(2)/G(o) interaction. In the D(2)/G(o) preparation, agonist stimulation of [(35)S]GTP gamma S binding occurred at higher potency for several agonists, and a higher stimulation (relative to dopamine) was achieved in D(2)/G(o) compared with D(2)/G(i2). Some agonists were able to stimulate [(35)S]GTP gamma S binding in the D(2)/G(o) preparation but not in D(2)/G(i2). The extent of D(2) receptor selectivity for G(o) over G(i2) is therefore dependent on the agonist used, and thus agonists may stabilize different conformations of the receptor with different abilities to couple to and activate G proteins.

Mechanisms of inverse agonism of antipsychotic drugs at the D(2) dopamine receptor: use of a mutant D(2) dopamine receptor that adopts the activated conformation.

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.

Dopamine D2 receptor dimer formation: evidence from ligand binding.

We have examined the binding of two radioligands ([(3)H]spiperone and [(3)H]raclopride) to D(2) dopamine receptors expressed in Chinese hamster ovary cells. In saturation binding experiments in the presence of sodium ions, both radioligands labeled a similar number of sites, whereas in the absence of sodium ions [(3)H]raclopride labeled about half the number of sites labeled by [(3)H]spiperone. In competition experiments in the absence of sodium ions, however, raclopride was able to inhibit [(3)H]spiperone binding fully. In saturation analyses with [(3)H]spiperone in the absence of sodium ions raclopride exerted noncompetitive effects, decreasing the number of sites labeled by the radioligand. These data are interpreted in terms of a model where the receptor exists as a dimer, and in the absence of sodium ions, raclopride exerts negative cooperativity across the dimer both for its own binding and the binding of spiperone. A model of the receptor has been produced that provides a good description of the experimental phenomena described here.

Antipsychotic drugs: importance of dopamine receptors for mechanisms of therapeutic actions and side effects.

Interaction of the antipsychotic drugs with dopamine receptors of the D2, D3, or D4 subclasses is thought to be important for their mechanisms of action. Consideration of carefully defined affinities of the drugs for these three receptors suggests that occupancy of the D4 subclass is not mandatory for achieving antipsychotic effects, but actions at D2 or D3 receptors may be important. A major difference between typical and atypical antipsychotic drugs is in the production of extrapyramidal side effects by the typical drugs. Production of extrapyramidal side effects by typical drugs seems to be due to the use of the drugs at doses where striatal D2 receptor occupancy exceeds approximately 80%. Use of these drugs at doses that do not produce this level of receptor blockade enables them to be used therapeutically without producing these side effects. The antipsychotic drugs have been shown to act as inverse agonists at D2 and D3 dopamine receptors, and this property may be important for the antipsychotic effects of the drugs. It is suggested that the property of inverse agonism leads to a receptor up-regulation upon prolonged treatment, and this alters the properties of dopamine synapses. Several variants of the dopamine receptors exist with different DNA sequences and in some cases different amino acid sequences. These variants may have different properties that alter the effects of dopamine and the antipsychotic drugs. The determination of such variants in patients may help in the prediction of drug responsiveness.

Site-directed mutations in the third intracellular loop of the serotonin 5-HT(1A) receptor alter G protein coupling from G(i) to G(s) in a ligand-dependent manner.

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.

Regulation of human D(1), d(2(long)), d(2(short)), D(3) and D(4) dopamine receptors by amiloride and amiloride analogues.

1. The modulatory effects of the allosteric effectors methylisobutylamiloride (MIA), benzamil and amiloride have been examined at human D(1), D(2), D(3) and D(4) dopamine receptors. The subtype selectivity and the mechanism of action of this allosteric regulation was examined. 2. In radioligand dissociation experiments each modulator accelerated dissociation from all four receptor subtypes indicating allosteric regulation. MIA displayed selectivity for the D(3) subtype for acceleration of radioligand dissociation. 3. In equilibrium binding (pseudo-competition) experiments the three compounds inhibited radioligand binding at the four receptor subtypes. Inhibition curves for D(1), D(2(short)), D(2(long)) and D(3) receptors were described by Hill coefficients exceeding unity and data were fitted best by a model that assumes binding of modulator to both the primary and allosteric binding sites of the receptor (the allosteric/competitive model). 4. At the D(4) subtype, Hill coefficients of unity described the binding data for amiloride and benzamil, consistent with competitive inhibition. The Hill coefficient for MIA at the D(4) subtype was less than unity and data could be fitted well by the allosteric/competitive model, but it was not possible to define unambiguously the modulatory mechanism. For this effect a better definition of the mechanism could be obtained by simultaneous analysis of data obtained in the presence of a range of concentrations of a purely competitive ligand. 5. MIA reduced the potency with which dopamine stimulated [(35)S]-GTPgammaS binding at the D(2) receptor. The effects of MIA could be described by the allosteric/competitive model with effects of MIA to inhibit the binding of dopamine but not its ability to induce a response.

Mechanisms of agonism and inverse agonism at serotonin 5-HT1A receptors.

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.

Effect of multiple serine/alanine mutations in the transmembrane spanning region V of the D2 dopamine receptor on ligand binding.

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.

G-protein coupled receptors: conformations and states.

Activation of G-protein coupled receptors by agonists is thought to involve the stabilisation of a ternary complex of agonist/receptor/G-protein, leading to effector activation, but this mechanism may be an oversimplification, as follows: (a) Agonist binding to the free receptor (uncoupled from G-proteins) is not a neutral event, but includes a component of the activation process and may be described in terms of the stabilisation of a partly activated form of the receptor (R*) that is able to couple to the G-protein. Stabilisation of R*, therefore, may contribute to agonist efficacy. Also, determinations of agonist affinity even in the absence of G-protein coupling do not necessarily describe the affinities of agonists for the ground state of the receptor. (b) R* is a partly activated intermediate between the ground state of the receptor (R) and the activated form coupled to G-protein (R*G). There is some indication that different agonists may stabilise different conformational states of the receptor, i.e. different R* species. (c) Agonists also stabilise the activated, coupled form of the receptor (AR*G), and for some agonists acting at a single receptor, the activated states may be similar, although there is evidence for other agonists that different activated states with different activities may be stabilised. (d) Two or more efficacy-generating steps are involved in the activation of G-protein coupled receptors by agonists: the stabilisation of R*, the stabilisation of R*G, and possibly the modulation of the activity of the activated state (AR*G). (e) The experimentally observed excess of G-proteins over receptors in membranes is inconsistent with data obtained from ligand-binding assays on these receptors. Receptors and G-proteins, therefore, may exist in some form of higher order array with cooperative interactions.

Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells.

In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.

Agonism and inverse agonism at dopamine D2-like receptors.

1. The processes that follow the binding of ligands to receptors are critical for their physiological functions. In the present paper I intend to review our own work and the work of other laboratories attempting to understand these processes for the dopamine D2-like receptors (D2, D3, D4) and how they contribute to the mechanisms of drug action. It is thought that the key event in agonist action for these receptors is the stabilization, by the agonist, of the agonist-receptor-G-protein ternary complex. The majority of the work I shall describe has been performed using recombinant receptors expressed in cell lines and the mechanisms of receptor action have been probed using ligand binding (competition vs [3H]-spiperone), the stimulation of [35S]-GTP gamma S binding and inhibition of adenylyl cyclase. 2. Measures of the ability of agonists to stabilize the agonist-receptor-G-protein ternary complex may be obtained in ligand-binding studies using the ratio of dissociation constants for the higher and lower affinity states (KI/KH ratio). The stimulation of [35S]-GTP gamma S binding provides a very convenient assay for agonist action and allows the determination of agonist potency and maximal response. Estimates of these quantities may also be obtained from the inhibition of adenylyl cyclase. For a range of agonists at the D2 receptor, there is a tendency for high values of KI/KH to predict high maximal activity and vice versa, but there is no general correlation. This suggests that the simple scheme of agonist action depending on the stabilization of the ternary complex is an over-simplification and further efficacy determining steps need to be included. For a number of receptors, including the D2 and D3 receptors, it has now been shown that there is activity in the absence of agonist (so-called constitutive activity). This agonist-independent activity can be inhibited by compounds previously considered to be antagonists (e.g. the antipsychotic drugs). Therefore, these compounds are inverse agonists rather than antagonists. The mechanism of this inverse agonist effect is unclear and we are examining this using a variety of biochemical approaches, including the use of constitutively active mutants. 3. The mechanisms of agonism and inverse agonism may be probed using biochemical assays and these studies are of great relevance to the understanding of drug action.

Comparison of the ability of dopamine receptor agonists to inhibit forskolin-stimulated adenosine 3'5'-cyclic monophosphate (cAMP) accumulation via D2L (long isoform) and D3 receptors expressed in Chinese hamster ovary (CHO) cells.

The pharmacological properties of the human D2L (long isoform) and rat D3 dopamine receptors in functional assays were examined. A range of dopamine agonists were assessed for their ability to inhibit adenosine 3'5'-cyclic monophosphate (cAMP) accumulation via the two receptors expressed stably in Chinese hamster ovary cells. Dopamine caused a significantly greater maximal inhibition (P < 0.05) of cAMP accumulation via the D2L receptor (approximately 70%) as compared to the D3 receptor (approximately 50%). The pattern of agonist effects was different at the two receptors. The absolute and relative potencies for inhibition of cAMP accumulation were different for a range of agonists acting at the two receptors. Similarly, the maximal inhibitions achieved by a range of agonists were different for the two receptors.

Role of conserved serine residues in the interaction of agonists with D3 dopamine receptors.

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.