The influence of G protein subtype on agonist action at D-2 dopamine receptors

In previous studies, we have shown that agonists influence the ability of D-2 dopamine receptors to couple to G proteins and here we extend this work. The human D-2Short dopamine receptor and a natural polymorphism of this D-2Short(Ser(311)Cys), have been studied by co-expressing the receptors in insect cells with Gbeta(1)gamma(2) and either Galpha(o), Galpha(i1), Galpha(i2) or Galpha(i3) G protein subunits. These preparations have been used to study the G protein coupling profiles of the two receptors and the influence of agonists. Receptor/G protein coupling was analysed in dopamine/[H-3]spiperone competition binding experiments and through stimulation of [S-35]GTPgammaS binding. Although the Ser(311)Cys polymorphism itself had no appreciable effect on the G protein coupling specificity of the D-2 receptor, agonist stimulation of [S-35]GTPgammaS binding, revealed that both dopamine and (+)-3PPP showed a clear preference for Galpha(o) compared to the Galpha(i) subtypes, but quinpirole did not. These results indicate that agonists are able to stabilise different receptor conformations with different abilities to couple to G proteins. (C) 2004 Elsevier Ltd. All rights reserved.

Mechanisms of agonist action at D-2 dopamine receptors

In this study, we investigated the biochemical mechanisms of agonist action at the G protein-coupled D-2 dopamine receptor expressed in Chinese hamster ovary cells. Stimulation of guanosine 5'-O-(3-[S-35]thio) triphosphate ([S-35]GTPgammaS) binding by full and partial agonists was determined at different concentrations of [S-35]GTPgammaS (0.1 and 10 nM) and in the presence of different concentrations of GDP. At both concentrations of [S-35]GTPgammaS, increasing GDP decreased the [S-35]GTPgammaS binding observed with maximally stimulating concentrations of agonist, with partial agonists exhibiting greater sensitivity to the effects of GDP than full agonists. The relative efficacy of partial agonists was greater at the lower GDP concentrations. Concentration-response experiments were performed for a range of agonists at the two [S-35]GTPgammaS concentrations and with different concentrations of GDP. At 0.1 nM [S-35]GTPgammaS, the potency of both full and partial agonists was dependent on the GDP concentration in the assays. At 10 nM [S-35]GTPgammaS, the potency of full agonists exhibited a greater dependence on the GDP concentration, whereas the potency of partial agonists was virtually independent of GDP. We concluded that at the lower [S-35]GTPgammaS concentration, the rate-determining step in G protein activation is the binding of [S-35]GTPgammaS to the G protein. At the higher [S-35]GTPgammaS concentration, for full agonists, [S-35]GTPgammaS binding remains the slowest step, whereas for partial agonists, another (GDP-independent) step, probably ternary complex breakdown, becomes rate-determining.

Investigation of the mechanism of agonist and inverse agonist action at D-2 dopamine receptors

This study investigated, for the D-2 dopamine receptor, the relation between the ability of agonists and inverse agonists to stabilise different states of the receptor and their relative efficacies. K-i values for agonists were determined in competition, versus the binding of the antagonist [H-3]spiperone. Competition data were fitted best by a two-binding site model (with the exception of bromocriptine, for which a one-binding site model provided the best fit) and agonist affinities for the higher (K-h) (G protein-coupled) and lower affinity (K-l) (G protein-uncoupled) sites determined. Ki values for agonists were also determined in competition versus the binding of the agonist [H-3]N-propylnorapomorphine (NPA) to provide a second estimate of K-h,. Maximal agonist effects (E-max) and their potencies (EC50) were determined from concentration-response curves for agonist stimulation of guanosine-5'-O-(3-[S-32] thiotriphosphate) ([S-35]GTPgammaS) binding. The ability of agonists to stabilise the G protein-coupled state of the receptor (K-l/K-h, determined from ligand-binding assays) did not correlate with either of two measures of relative efficacy (relative E-max, Kl/EC50) of agonists determined in [S-35]GTPgammaS-binding assays, when the data for all of the compounds tested were analysed For a subset of compounds, however, there was a relation between K-l/K-h and E-max.. Competition-binding data versus [H-3]spiperone and [H-3]NPA for a range of inverse agonists were fitted best by a one-binding site model. K-i values for the inverse agonists tested were slightly lower in competition versus [H-3]NPA compared to [H-3]spiperone. These data do not provide support for the idea that inverse agonists act by binding preferentially to the ground state of the receptor. (C) 2004 Elsevier Inc. All rights reserved.

Functional coupling of the human dopamine D-2 receptor with G alpha(i1), G alpha(i2), G alpha(i3) and G alpha(o) G proteins: evidence for agonist regulation of G protein selectivity

1 The human dopamine D-2long (D-2L) receptor was expressed with four different G proteins in Sf9 cells using the baculovirus expression system. When co-expressed with G(i)/G(o) G proteins (G(i1)alpha, G(i2)alpha, G(i3)alpha, or G(o)alpha, plus Gbeta(1) and Ggamma(2)) the receptor displayed a high-affinity binding site for the agonists (dopamine and NPA), which was sensitive to GTP (100 mum), demonstrating interaction between the receptor and the different G proteins. 2 The receptor to G protein ratio (R: G ratio) was evaluated using [H-3]-spiperone saturation binding (R) and [S-35]-GTPgammaS saturation binding (G). R: G ratios of 1: 12, 1: 3, 1: 14 and 1: 5 were found for G(i1), G(i2), G(i3), and Go preparations, respectively. However, when R:G ratios of 1:2 and 1: 12 were compared for G(i2) and G(o), no difference was found for the stimulation of [S-35]-GTPgammaS binding. 3 Several agonists were tested for their ability to stimulate [S-35]-GTPgammaS binding to membranes co-expressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G(i3) and G(o). However, for G(i2) and G(i1) preparations, compounds such as S-(-)-3-PPP and p-tyramine were unable to stimulate [S-35]-GTPyS binding. 4 Most of the compounds showed higher relative efficacies (compared to dopamine) and higher potencies in the preparation expressing G(o). Comparison of the effects of different agonists in the different preparations showed that each agonist differentially activates the four G proteins. 5 We conclude that the degree of selectivity of G protein activation by the D-2L receptor can depend on the conformation of the receptor stabilised by an agonist.

High-affinity small molecule-phospholipid complex formation: Binding of siramesine to phosphatidic acid

Siramesine (SRM) is a sigma-2 receptor agonist which has been recently shown to inhibit growth of cancer cells. Fluorescence spectroscopy experiments revealed two distinct binding sites for this drug in phospholipid membranes. More specifically, acidic phospholipids retain siramesine on the bilayer surface due to a high-affinity interaction, reaching saturation at an apparent 1:1 drug-acidic phospholipid stoichiometry, where after the drug penetrates into the hydrocarbon core of the membrane. This behavior was confirmed using Langmuir films. Of the anionic phospholipids, the highest affinity, comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction Of X-PA = 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 +/- 80 x 10(6). An MD simulation on the siramesine:PA interaction was in agreement with the above data. Taking into account the key role of PA as a signaling molecule promoting cell growth our results suggest a new paradigm for the development of anticancer drugs, viz. design of small molecules specifically scavenging phospholipids involved in the signaling cascades controlling cell behavior.

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/EC50, E-max center dot K-A/EC50) determined. The E-max center dot K-A/EC50 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 [S-35]GTP[S] (guanosine 5'-[gamma-[S-35]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 [S-35]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 [S-35]GTP[S] binding follows a pseudo-first-order reaction with [S-35]GTP[S] binding reaching equilibrium after approx. 3 h. The [S-35]GTP[S]-binding event is the rate-deter mining step in the assay. Agonists regulate the maximal level of [S-35]GTP[S] bound, rather than the rate constant for binding. The [S-35]GTP[S]-binding assay therefore determines agonist efficacy on the basis of the amount of [S-35]GTP[S] bound rather than the rate of binding.

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.

Mechanisms of inverse agonist action at D-2 dopamine receptors

1 Mechanisms of inverse agonist action at the D-2(short) dopamine receptor have been examined. 2 Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [H-3]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. 3 Competition of inverse agonists versus [H-3] NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. K-i values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. K-coupled and K-uncoupled were statistically different for the set of compounds tested ( ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. 4 These observations were supported by simulations of these competition experiments according to the extended ternary complex model. 5 Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [S-35]GTPγ S binding to varying degrees in concentration-response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (-)-sulpiride was unaffected. 6 These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism.

Mechanisms of G protein activation via the D-2 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-[S-35] thio) triphosphate ([S-35] GTP gamma S) 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 [S-35] GTPgS 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 [S-35] GTPgS binding, with the more efficacious agonists resulting in faster dissociation. Agonists were able to dissociate about 70% of the pre-bound [S-35] GTPgS, leaving a component which may not be accessible to the agonist-bound receptor. The dissociable component of the [S-35] GTPgS binding was reduced with longer association times and increased [S-35] GTPgS concentrations. Conclusions and implications: These data are consistent with [S-35] GTPgS 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 [S-35] GTPgS binding assays, therefore, much of the agonist-receptor complex remains in proximity to G proteins after they have been activated by agonist.

Dual role of the beta(2)-adrenergic receptor C terminus for the binding of beta-arrestin and receptor internalization

Homologous desensitization of beta(2)-adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind beta-arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by beta-arrestins, we have investigated beta-arrestin interaction and internalization of a set of mutants of the human beta(2)-adrenergic receptor. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor-beta-arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of beta-arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and beta-arrestin2 translocation. A beta(2)-adrenergic receptor truncated distal to residue 381 interacted normally with beta-arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and beta-arrestin, but its last eight amino acids facilitate receptor internalization in concert with beta-arrestin2.

beta-arrestin binding to the beta(2)-adrenergic receptor requires both receptor phosphorylation and receptor activation

Homologous desensitization of beta(2)-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.

Agonist-dependent internalization of D2 receptors: Imaging quantification by confocal microscopy

In positron emission tomography and single photon emission computed tomography studies using D2 dopamine (DA) receptor radiotracers, a decrease in radiotracer binding potential (BP) is usually interpreted in terms of increased competition with synaptic DA. However, some data suggest that this signal may also reflect agonist (DA)-induced increases in D2 receptor (D2R) internalization, a process which would presumably also decrease the population of receptors available for binding to hydrophilic radioligands. To advance interpretation of alterations in D2 radiotracer BP, direct methods of assessment of D2R internalization are required. Here, we describe a confocal microscopy-based approach for the quantification of agonist-dependent receptor internalization. The method relies upon double-labeling of the receptors with antibodies directed against intracellular as well as extracellular epitopes. Following agonist stimulation, DA D2R internalization was quantified by differentiating, in optical cell sections, the signal due to the staining of the extracellular from intracellular epitopes of D2Rs. Receptor internalization was increased in the presence of the D2 agonists DA and bromocriptine, but not the D1 agonist SKF38393. Pretreatment with either the D2 antagonist sulpiride, or inhibitors of internalization (phenylarsine oxide and high molarity sucrose), blocked D2-agonist induced receptor internalization, thus validating this method in vitro. This approach therefore provides a direct and streamlined methodology for investigating the pharmacological and mechanistic aspects of D2R internalization, and should inform the interpretation of results from in vivo receptor imaging studies.

Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking.

Glutathione-S-transferase (GST)-Grb2 fusion proteins have been used to identify the potential role of Grb2-binding proteins in platelet activation by the platelet low-affinity IgG receptor, Fc gamma RIIA. Two tyrosine phosphoproteins of 38 and 63 kD bind to the SH2 domain of Grb2 following Fc gamma RIIA stimulation of platelets. Both are located in the particulate fraction following platelet activation and are also able to bind to a GST-construct containing the SH2 and SH3 domains of phospholipase C gamma 1. p38 also forms a complex with the tyrosine kinase csk in stimulated cells and is a substrate for the kinase. The SH3 domains of Grb2 form a stable complex with SOS1 and two proteins of 75 kD and 120 kD, which undergo tyrosine phosphorylation in Fc gamma RIIA stimulated cells. The 75-kD protein is recognized by antibodies to SLP-76, which has recently been isolated from T cells and sequenced. Tyrosine phosphorylation of p38 and p63 is also observed in platelets stimulated by the tyrosine kinase-linked receptor agonist collagen and by the G protein-coupled receptor agonist thrombin, although phosphorylation of SLP-76 is only observed in collagen-stimulated platelets. p38 and p63 may provide a docking site for Grb2, thereby linking Grb2 SH3-binding proteins SOS1, SLP-76, and p120 to downstream signalling events.

Agonist-induced endocytosis of rat somatostatin receptor 1

Somatostatin-receptor 1 (sst1) is an autoreceptor in the central nervous system that regulates the release of somatostatin. Sst1 is present intracellularly and at the cell surface. To investigate sst1 trafficking, rat sst1 tagged with epitope was expressed in rat insulinoma cells 1046-38 (RIN-1046-38) and tracked by antibody labeling. Confocal microscopic analysis revealed colocalization of intracellularly localized rat sst1-human simplex virus (HSV) with Rab5a-green fluorescent protein and Rab11a-green fluorescent protein, indicating the distribution of the receptor in endocytotic and recycling organelles. Somatostatin-14 induced internalization of cell surface receptors and reduction of binding sites on the cell surface. It also stimulated recruitment of intracellular sst1-HSV to the plasma membrane. Confocal analysis of sst1-HSV revealed that the receptor was initially transported within superficial vesicles. Prolonged stimulation of the cells with the peptide agonist induced intracellular accumulation of somatostatin-14. Because the number of cell surface binding sites did not change during prolonged stimulation, somatostatin-14 was internalized through a dynamic process of continuous endocytosis, recycling, and recruitment of intracellularly present sst1-HSV. Accumulated somatostatin-14 bypassed degradation via the endosomal-lysosomal route and was instead rapidly released as intact and biologically active somatostatin-14. Our results show for the first time that sst1 mediates a dynamic process of endocytosis, recycling, and re-endocytosis of its cognate ligand.

Recycling and resensitization of the neurokinin 1 receptor: influence of agonist concentration and Rab GTPases

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.