Agonist-dependent trafficking of alpha2-adrenoceptor subtypes: dependence on receptor subtype and employed agonist

Many G protein-coupled receptors (GPCRs) are internalized from the plasma membrane after agonist exposure. Previously, marked agonist-induced internalization of human alpha2A- and alpha2B-adrenergic receptors (AR) was observed in transfected neuronal rat pheochromocytoma (PC12) cells; alpha2A- and alpha2B-AR were internalized into partly distinct intracellular vesicles (Olli-L?hdesm?ki et al., J. Neurosci. 19, 9281-9288, 1999). In this paper, the extent of alpha2-AR internalization was quantitated in human embryonic kidney (HEK-293) and PC12 cells by combined application of cell surface biotinylation and ELISA methods, which allow measurement of protein trafficking in intact, differentiated and undifferentiated cells. Significant subtype-specific (but not cell type-dependent) trafficking of human alpha2-AR was observed by quantitation and immunocytochemistry. Agonist-induced sequestration of alpha2B-AR was markedly reduced after blocking the formation of clathrin-coated vesicles by hyperosmotic sucrose pretreatment. The sequestration of alpha2A-AR was partly inhibited after sucrose pretreatment but could be further reduced after inhibiting the formation of both clathrin-coated and caveolin vesicles by combined pretreatment with hyperosmotic sucrose and filipin. Differences were also observed in the recycling of alpha2A- and alpha2B-AR. The extent of maximal agonist-induced sequestration in PC12 cells was not directly dependent on relative agonist efficacy.     

Intracellularly truncated human alpha2B-adrenoceptors: stable and functional GPCRs for structural studies

All three alpha2-adrenoceptor subtypes have a long third intracellular loop (3i), which is conserved by overall size and charge-hydrophobic properties but not by amino acid sequence similarity. These properties must be relevant for function and structure, because they have been preserved during hundreds of millions of years of evolution. The contribution of different loop portions to agonist/antagonist binding properties and G protein coupling of the human alpha2B-adrenoceptor (alpha2B-AR) was investigated with a series of 3i truncated constructs (delta3i). We used a variety of agonists/antagonists in competition binding assays. We stimulated alpha2B-AR delta3i with various agonists and measured [35S]GTPgammaS binding in isolated cell membranes with or without antagonist inhibition. We also evaluated the ability of oligopeptides, analogous to the amino and carboxyl terminal parts of 3i, to promote G protein activation, monitored with the [35S]GTPgammaS assay. Our results reveal that the carboxyl end residues of 3i, R360(6.24) to V372(6.36), are important for Gi/Go protein activation. Deletions in regions from G206(5.72) to R245(5.110) altered the binding of some alpha2B-AR agonists, indicating that agonist binding is dependent on the conformation of the 3i domain, possibly through the involvement of G protein interactions. The truncated receptor constructs may be more stable on purification and thus be useful for structural characterization of alpha2B-AR.     

Alpha2-adrenergic agonist modulation of [35S]GTPgammaS binding to guanine-nucleotide-binding-proteins in human platelet membranes.

Alpha2-adrenoceptor (alpha2-AR)-regulated binding of the labelled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. Under optimal conditions, the potent alpha2-AR agonist, 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), increased the binding of [35S]GTPgammaS up to approximately 1.8 fold, with half-maximal increase at 60 nM and was competitively inhibited by the alpha2-AR antagonist Rauwolscine. The actions of both UK 14304 and Rauwolscine were modulated by monovalent and divalent cation levels, as well as by the concentrations of GDP. [35S]GTPgammaS binding induced by UK 14304 had a Kd value of 4.5 +/- 0.3 nM and a Bmax value of 4.15 +/- 0.40 pmol/mg protein. The rank order of potencies of adrenergic ligands tested in stimulating [3S]GTPgammaS binding and inhibiting forskolin-stimulated c-AMP accumulation was UK 14304> Guanabenz acetate> Oxymetazoline hydrochloride> B-HT 920 dihydrochloride> p-Aminoclonidine hydrochloride> Clonidine hydrochloride. The data presented indicate that enhancement of [35S]GTPgammaS binding by alpha2-AR in human platelet membranes provides a simple functional measure for receptor activation and can be used for determination of potencies and efficacies of ligands at the alpha2-AR.     

G protein activation by endomorphins in the mouse periaqueductal gray matter

The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of mu-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated mu-opioid peptide endomorphins can activate G proteins through mu-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS). An autoradiographic [(35)S]GTPgammaS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 microM increased [(35)S]GTPgammaS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6+/-3.8 and 72.3+/-4.0%, respectively, at 10 microM. In contrast, the synthetic selective mu-opioid receptor agonist [D-Ala(2),NHPhe(4), Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6+/-5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [(35)S]GTPgammaS binding was verified by coincubating membranes with endomorphins in the presence of specific mu-, delta- or kappa-opioid receptor antagonists. Coincubation with selective mu-opioid receptor antagonists beta-funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) blocked both endomorphin-1 and-2-stimulated [(35)S]GTPgammaS binding. In contrast, neither delta- nor kappa-opioid receptor antagonist had any effect on the [(35)S]GTPgammaS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [(35)S]GTPgammaS binding by selectively stimulating mu-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for mu-opioid receptors in the mouse PAG.     

Functional interactions between the alpha1b-adrenoceptor and Galpha11 are compromised by de-palmitoylation of the G protein but not of the receptor

Both the alpha1b-adrenoceptor and Galpha11 are targets for post-translational thio-acylation that is regulated by agonist occupancy of the receptor [P.A. Stevens, J. Pediani, J.J. Carrillo, G. Milligan, J. Biol. Chem. 276 (2001) 35883]. In co-expression studies mutation of the sites of thio-acylation in the G protein or treatment of cell membranes with hydroxylamine greatly reduced agonist stimulation of guanosine 5'-[gamma-[35S]thio]triphosphate ([35S]GTPgammaS) binding. In alpha1b-adrenoceptor-Galpha11 fusion proteins mutation of thio-acylation sites in receptor or G protein did not alter the binding affinity of the antagonist [3H]prazosin or the agonist phenylephrine. Although the potency of phenylephrine to stimulate binding of [35S]GTPgammaS to alpha1b-adrenoceptor-Galpha11 fusion proteins was unaffected by the thio-acylation potential of either element, the maximal effect was reduced by some 50% when the G protein but not the receptor was mutated to prevent thio-acylation. This reflected lack of thio-acylation of the G protein rather than mutation of Cys9 and Cys10 to Ser because treatment with hydroxylamine mimicked this in fusions containing the wild type G protein but was without effect in those mutated to prevent thio-acylation. Mutation to reduce binding of beta/gamma to Galpha11 markedly reduced phenylephrine stimulation of [35S]GTPgammaS binding. Combination of mutations to prevent thio-acylation and beta/gamma binding did not, however, have an additive effect on [35S]GTPgammaS binding. These results indicate that the thio-acylation status of the alpha1b-adrenoceptor does not regulate G protein activation whereas thio-acylation of Galpha11 plays a key role in activation by the receptor beyond providing membrane association and proximity.     

Reciprocal inhibition of G-protein signaling is induced by CB(1) cannabinoid and GABA(B) receptor interactions in rat hippocampal membranes

Cannabinoid CB(1) and the metabotropic GABA(B) receptors have been shown to display similar pharmacological effects and co-localization in certain brain regions. Previous studies have reported a functional link between the two systems. As a first step to investigate the underlying molecular mechanism, here we show cross-inhibition of G-protein signaling between GABA(B) and CB(1) receptors in rat hippocampal membranes. The CB(1) agonist R-Win55,212-2 displayed high potency and efficacy in stimulating guanosine-5'-O-(3-[(35)S]thio)triphosphate, [(35)S]GTPgammaS binding. Its effect was completely blocked by the specific CB(1) antagonist AM251 suggesting that the signaling was via CB(1) receptors. The GABA(B) agonists baclofen and SKF97541 also elevated [(35)S]GTPgammaS binding by about 60%, with potency values in the micromolar range. Phaclofen behaved as a low potency antagonist with an ED(50) approximately 1mM. However, phaclofen at low doses (1 and 10nM) slightly but significantly attenuated maximal stimulation of [(35)S]GTPgammaS binding by the CB(1) agonist R-Win55,212-2. The observation that higher concentrations of phaclofen had no such effect rule out the possibility of its direct action on CB(1) receptors. The pharmacologically inactive stereoisomer S-Win55,212-3 had no effect either alone or in combination with phaclofen establishing that the interaction is stereospecific in hippocampus. The specific CB(1) antagonist AM251 at a low dose (1 nM) also inhibited the efficacy of G-protein signaling of the GABA(B) receptor agonist SKF97541. Cross-talk of the two receptor systems was not detected in either spinal cord or cerebral cortex membranes. It is speculated that the interaction might occur via an allosteric interaction between a subset of GABA(B) and CB(1) receptors in rat hippocampal membranes. Although the exact molecular mechanism of the reciprocal inhibition between CB(1) and GABA(B) receptors will have to be explored by future studies it is intriguing that the cross-talk might be involved in balance tuning the endocannabinoid and GABAergic signaling in hippocampus.     

Activation of G-proteins in the mouse pons/medulla by beta-endorphin is mediated by the stimulation of mu- and putative epsilon-receptors

The activation of mu-, delta- and kappa1-opioid receptors by their respective agonists increases the binding of the non-hydrolyzable GTP analog guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS) to G proteins. Beta-endorphin is an endogenous opioid peptide which binds nonselectively to mu-, delta- and putative epsilon-opioid receptors. The present experiment was designed to determine which opioid receptors are involved in the stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the mouse pons/medulla. The mouse pons/medulla membranes were incubated in an assay buffer containing 50 pM [35S]GTPgammaS, 30 microM GDP and various concentrations of beta-endorphin. Beta-endorphin (0.1 nM-10 microM) increased [35S]GTPgammaS binding in a concentration-dependent manner, and 10 microM beta-endorphin produced a maximal stimulation of approximately 260% over baseline. This stimulation of [35S]GTPgammaS binding by beta-endorphin was partially attenuated by the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA), but not by the delta-opioid receptor antagonist naltrindole (NTI) or the kappa1-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Beta-endorphin stimulated [35S]GTPgammaS binding by about 80% in the presence of 10 microM beta-FNA, 30 nM NTI and 100 nM nor-BNI. The same concentrations of these antagonists completely blocked the stimulation of [35S]GTPgammaS binding induced by 10 microM [D-Ala2,NHPhe4,Gly-ol]enkephalin, [D-Pen(2,5)]enkephalin and U50,488H, respectively. Moreover, the residual stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the presence of the three opioid receptor antagonists was significantly attenuated by 100 nM of the putative epsilon-opioid receptor partial agonist beta-endorphin (1-27). These results indicate that the stimulation of [35S]GTPgammaS binding induced by beta-endorphin is mediated by the stimulation of both mu- and putative epsilon-opioid receptors in the mouse pons/medulla.     

Dimers of class A G protein-coupled receptors function via agonist-mediated trans-activation of associated G proteins

The histamine H1 receptor and the alpha1b-adrenoreceptor are G protein-coupled receptors that elevate intracellular [Ca2+] via activation of Gq/G11. Assessed by co-immunoprecipitation and time-resolved fluorescence resonance energy transfer they both exist as homo-dimers. The addition of the G protein G11alpha to the C terminus of these receptors did not prevent dimerization. Agonists produced a large stimulation of guanosine 5'-3-O-([35S]thio)triphosphate ([35S]GTPgammaS) binding to receptor-G protein fusions containing wild type forms of both polypeptides. For both receptors this was abolished by incorporation of G208AG11alpha into the fusions. Mutation of a highly conserved leucine in intracellular loop 2 of each receptor also eliminated agonist function but not binding. Co-expression of the two non-functional but complementary fusion constructs reconstituted agonist-mediated binding of [35S]GTPgammaS in membranes of HEK293 cells and elevation of [Ca2+]i in mouse embryo fibroblasts lacking both Gq and G11. Co-expression of the histamine H1 receptor- and the alpha1b-adrenoreceptor-G11alpha fusions allowed detection of functional hetero-dimeric complexes, whereas co-expression of histamine H1 receptor-G11alpha with increasing amounts of L151Dalpha1b-adrenoreceptor resulted in decreasing levels of histamine-stimulated [35S]GTPgammaS binding. Co-expression of the alpha1b-adrenoreceptor with a fusion protein incorporating the N-terminal domain and transmembrane helix 1 of the alpha1b-adrenoreceptor and G11alpha did not result in agonist activation of the G protein but did indicate a role for transmembrane helix 1 in dimerization. These data demonstrate that dimers of these class A receptors function via trans-activation of associated G proteins.     

Sustained norepinephrine stimulation induces different regulation of expression in three alpha1-adrenoceptor subtypes.

The norepinephrine (NE)-induced regulation of alpha1-adrenoceptors (ARs) expression in human embryonic kidney (HEK) 293 cells stably expressing cloned alpha1-AR subtypes with similar receptor densities was investigated. In the presence of 10 microM propranolol, the treatment of cells with 10 microM NE for 4-72 h down-regulated alpha1A- and alpha1D-AR. but increased alpha1B-AR expression in a time-dependent manner. The down-regulation of alpha1A-AR reached maximum of 40.3 +/- 14.7 % at 48h. The down-regulation of alpha1D-AR reached maximum of 51.3 +/- 3.7% at 24h. With the stimulation of NE, alpha1B-AR density was increased maximally by 112.4 +/- 43.4% at 48h. The protein kinase C (PKC) inhibitor calphostin C or R0-31-8220 abolished the NE-induced down-regulation of alpha1A- and alpha1D-AR, but showed no effect on the up-regulation of alpha1B-AR. The PKC agonist PMA not only mimicked the NE-induced down-regulation of alpha1A- and alpha1D-AR, but also induced a down-regulation of alpha1B-AR. The endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) or thapsigargin, or the calcium chelator BAPTA/AM did not affect the down-regulation of alpha1A-AR, but inhibited the up-regulation of alpha1B-AR induced by NE. Calmodulin antagonist W-7. tyrosine kinase inhibitor genistein or tyrphostin A25 had no effect on NE-induced up-regulation of alpha1B-AR. The results suggest that three alpha1-AR subtypes are differently regulated by sustained NE stimulation with different signal transduction pathways.     

Allosteric modulation of adenosine A1 receptor coupling to G-proteins in brain

2-Amino-4,5,6,7-tetrahydrobenzo(beta)thiophen-3-yl 4-chlorophenylmethanone (T62) is a member of a group of allosteric modulators of adenosine A1 receptors tested in animal models of neuropathic pain to increase the efficacy of adenosine. To determine its mechanisms at the level of receptor-G-protein activation, the present studies examined the effect of T62 on A1-stimulated [35S]guanosine-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) binding in brain membranes, and by [35S]GTPgammaS autoradiography using the A1 agonist, phenylisopropyladenosine (PIA), to activate G-proteins. In hippocampal membranes, T62 increased both basal and PIA-stimulated [35S]GTPgammaS binding. The effect of T62 was non-competitive in nature, since it increased the maximal effect of PIA, with no effect on agonist potency. GTPgammaS saturation analysis showed that T62 increased the number of G-proteins activated by agonist but had no effect on the affinity of activated G-proteins for GTPgammaS. [35S]GTPgammaS autoradiography showed that the neuroanatomical localization of T62-stimulated [35S]GTPgammaS binding was identical to that of PIA-stimulated activity. The increase in PIA-stimulated activity by T62 varied between brain regions, with areas of lower A1 activation producing the largest percent modulation by T62. These results suggest a mechanism of allosteric modulators to increase the number of activated G-proteins per receptor, and provide a neuroanatomical basis for understanding potential therapeutic effects of such drugs.     

G Protein independent phosphorylation and internalization of the δ-opioid receptor

Agonist activation of the δ-opioid receptor leads to internalization via Gβγ recruitment of G protein coupled receptor kinase-2, which phosphorylates the receptor at several sites, including Ser363, allowing β-arrestin binding and localization to clathrin coated pits. Using human embryonic kidney cells expressing a δ-opioid receptor we tested the hypothesis that prevention of receptor coupling to G protein by treatment with pertussis toxin (PTX) will block these processes. PTX treatment did not reduce phosphorylation of δ-opioid receptor Ser363 in response to the agonist [ d -Pen2, d -Pen5]enkephalin, or recruitment of β-arrestin 2-green fluorescent protein to the membrane and only slowed, but did not prevent, [ d -Pen2, d -Pen5]enkephalin-induced internalization. Similarly, PTX treatment only partially prevented the ability of the δ-opioid peptide agonists deltorphin II and [Met5]enkephalin and the non-peptide agonist BW373U86 to induce receptor internalization. No internalization was seen with morphine, oxymorphindole or the putative δ₁-opioid agonist TAN-67 in the presence or absence of PTX, even though TAN-67 showed a strong activation of G protein, as measured by guanosine-5'-O-(3-[³⁵S]thio)triphosphate binding. The ability of an agonist to stimulate phosphorylation at Ser363 was predictive of its capacity to induce internalization. The results suggest a role for G protein in δ-opioid receptor internalization, but show that alternative G protein independent pathways exist.     

Agonists activate Gi1 alpha or Gi2 alpha fused to the human mu opioid receptor differently

As preferential coupling of opioid receptor to various inhibitory Galpha subunits is still under debate, we have investigated the selectivity of the human mu opioid receptor fused to a pertussis toxin insensitive C351I Gi1 alpha or C352I Gi2 alpha in stably transfected HEK 293 cells. Overall agonist binding affinities were increased for both fusion constructs when compared to the wild type receptor. [35 S]GTPgammaS binding was performed on pertussis toxin treated cells to monitor coupling efficiency of the fusion constructs. Upon agonist addition hMOR-C351I Gi1 a exhibited an activation profile similar to the non-fused receptor while hMOR-C352I Gi2 alpha was poorly activated. Interestingly no correlation could be drawn between agonist binding affinity and efficacy. Upon agonist addition, forskolin-stimulated cAMP production, as measured using a reporter gene assay, was inhibited by signals transduced via the fused Gi1 alpha and Gi2 alpha mainly. In contrast both fusion constructs were able to initiate ERK-MAPK phosphorylation via coupling to endogenous G proteins only. In conclusion our data indicate that hMOR couples more efficiently to Gi1 alpha than Gi2 alpha and that the coupling efficacy is clearly agonist-dependent.     

Dominance of the alpha1B-adrenergic receptor and its subcellular localization in human and TRAMP prostate cancer cell lines

The function and distribution of alpha1-adrenergic receptor (AR) subtypes in prostate cancer cells is well characterized. Previous studies have used RNA localization or low-avidity antibodies in tissue or cell lines to determine the alpha1-AR subtype and suggested that the alpha1A-AR is dominant. Two androgen-insensitive, human metastatic cancer cell lines DU145 and PC3 were used as well as the mouse TRAMP C1-C3 primary and clonal cell lines. The density of alpha1-ARs was determined by saturation binding and the distribution of the different alpha1-AR subtypes was examined by competition-binding experiments. In contrast to previous studies, the major alpha1-AR subtype in DU145, PC3 and all of the TRAMP cell lines is the alpha1B-AR. DU145 cells contained 100% of the alpha1B-AR subtype, whereas PC3 cells were composed of 21% alpha1 A-AR and 79% alpha1B-AR. TRAMP cell lines contained between 66% and 79% of the alpha1B-AR with minor fractions of the other two subtypes. Faster doubling time in the TRAMP cell lines correlated with decreasing alpha 1B-AR and increasing alpha1 A- and alpha1D-AR densities. Transfection with EGFP-tagged alpha1B-ARs revealed that localization was mainly intracellular, but the majority of the receptors translocated to the cell surface after extended preincubation (18 hr) with either agonist or antagonist. Localization was confirmed by ligand-binding studies and inositol phosphate assays where prolonged preincubation with either agonist and/or antagonist increased the density and function of alpha 1-ARs, suggesting that the native receptors were mostly intracellular and nonfunctional. Our studies indicate that alpha1B-ARs are the major alpha1-AR subtype expressed in DU145, PC3, and all TRAMP cell lines, but most of the receptor is localized in intracellular compartments in a nonfunctional state, which can be rescued upon prolonged incubation with any ligand.     

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.     

Dynorphin peptides differentially regulate the human kappa opioid receptor

Dynorphins, endogenous peptides for the kappa opioid receptor, play important roles in many physiological and pathological functions. Here, we examined how prolonged treatment with three major prodynorphin peptides, dynorphin A (1-17) (Dyn A), dynorphin B (1-13) (Dyn B) and alpha-neoendorphin (alpha-Neo), regulated the human kappa opioid receptor (hKOR) stably expressed in Chinese hamster ovary (CHO) cells. Results from receptor binding and [(35)S]GTPgammaS binding assays showed that these peptides were potent full agonists of the hKOR with comparable receptor reserve and intrinsic efficacy to stimulate G proteins. A 4-h incubation with alpha-Neo at a concentration of approximately 600xEC(50) value (from [(35)S]GTPgammaS binding) resulted in receptor down-regulation to a much lower extent than the incubation with Dyn A and Dyn B at comparable concentrations ( approximately 10% vs. approximately 65%). Extending incubation period and increasing concentrations did not significantly affect the difference. The plateau level of alpha-Neo-mediated receptor internalization (30 min) was significantly less than those of Dyn A and Dyn B. Omission of the serum from the incubation medium or addition of peptidase inhibitors into the serum-containing medium enhanced alpha-Neo-, but not Dyn A- or Dyn B-, mediated receptor down-regulation and internalization; however, the degrees of alpha-Neo-induced adaptations were still significantly less than those of Dyn A and Dyn B. Thus, these endogenous peptides differentially regulate KOR after activating the receptor with similar receptor occupancy and intrinsic efficacy. Both stability in the presence of serum and intrinsic capacity to promote receptor adaptation play roles in the observed discrepancy among the dynorphin peptides.     

delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains

Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor and cognate G proteins was also blocked by high-energy ultrasound and repeated freezing-thawing. Our data indicate, for the first time, that membrane domains isolated using 'detergent-free' procedures exhibit higher efficiency of coupling between a G protein-coupled receptor and its corresponding G protein(s) than bulk plasma membranes. Detergent-extraction diminishes these interactions, even when the receptor and G proteins are physically tethered together.     

Mechanisms of mu opioid receptor/G-protein desensitization in brain by chronic heroin administration

Previous studies have shown that chronic opiate treatment decreases mu opioid-stimulated [35S]GTPgammaS binding in specific brain regions. To extend these findings, the present study investigated DAMGO-stimulated [35S]GTPgammaS binding in membrane homogenates and coronal sections from rats non-contingently administered heroin. Rats were administered saline or increasing doses of heroin i.v. hourly up to 288 mg/kg/day over 40 days. In brain sections, chronic heroin administration decreased DAMGO-stimulated [35S]GTPgammaS binding in medial thalamus and amygdala, with no effect in cingulate cortex or nucleus accumbens. Chronic heroin administration also reduced [35S]GTPgammaS binding stimulated by the principal metabolite of heroin, 6-monoacetylmorphine. In contrast, no significant changes in mu opioid receptor binding were observed in amygdala or thalamus using [3H]DAMGO autoradiography. In membranes from amygdala and thalamus, chronic heroin treatment decreased the maximal effect of DAMGO in stimulating [35S]GTPgammaS binding, with no effect on DAMGO potency. GTPgammaS saturation analysis showed that chronic heroin treatment decreased the Bmax, and increased the K(D), of DAMGO-stimulated [35S]GTPgammaS binding. These data suggest potential mechanisms by which chronic agonist treatment produces opioid receptor/G-protein desensitization in brain.     

New 1,2,3,9-tetrahydro-4H-carbazol-4-one derivatives: analogues of HEAT as ligands for the alpha1-adrenergic receptor subtypes

With the aim to develop new ligands able to discriminate among the three subtypes of alpha1-adrenergic receptors (alpha1A-AR, alpha1B-AR, and alpha1D-AR), a series of new 1,2,3,9-tetrahydro-4H-carbazol-4-ones bearing a 3-[[[2-(4-hydroxyphenyl)ethyl]amino]methyl] or a 3-[[4-(2-substitutedphenyl)piperazin-1-yl]methyl] side chain were synthesized. The general structure of the new compounds is reminiscent of HEAT and RN5, two potent alpha1-AR antagonists which show high affinities for all three alpha1-AR subtypes. Some derivatives in which one ring of the tetrahydrocarbazolone system was opened were also prepared. Compounds were tested in radioligand binding assays on human cloned alpha1A-AR, alpha1B-AR, and alpha1D-AR subtypes stably expressed in HEK293 cells. They showed moderate to good affinities, although their selectivity among the receptor subtypes hardly reached one order of magnitude.     

The [35S]GTPgammaS binding assay: approaches and applications in pharmacology

Receptors of the of seven transmembrane spanning, heterotrimeric G protein coupled family (GPCR) play crucial roles in regulating physiological functions and consequently are targets for the action of many classes of drugs. Activation of receptor by agonist leads to the dissociation of GDP from Galpha of the Galphabetagamma heterotrimer, followed by the binding of GTP to Galpha and subsequent modulation of downstream effectors. The G protein heterotrimer is reformed by GTPase activity of the Galpha subunit, forming Galpha-GDP and so allowing Galpha and Gbetagamma to recombine. The [35S]GTPgammaS assay measures the level of G protein activation following agonist occupation of a GPCR, by determining the binding of the non-hydrolyzable analog [35S]GTPgammaS to Galpha subunits. Thus, the assay measures a functional consequence of receptor occupancy at one of the earliest receptor-mediated events. The assay allows for traditional pharmacological parameters of potency, efficacy and antagonist affinity, with the advantage that agonist measures are not subjected to amplification or other modulation that may occur when analyzing parameters further downstream of the receptor. In general the assay is experimentally more feasible for receptors coupled to the abundant G(i/o) proteins. Nevertheless, [35S]GTPgammaS binding assays are used with GPCRs that couple to the G(s) and G(q) families of G proteins, especially in artificial expression systems, or using receptor-Galpha constructs or immunoprecipitation of [35S]GTPgammaS-labeled Galpha. The relative simplicity of the assay has made it very popular and its use is providing insights into contemporary pharmacological topics including the roles of accessory proteins in signaling, constitutive activity of receptors and agonist specific signaling.     

Developmental regulation of GABA(B) receptor function in rat spinal cord

GABA(B) receptors are heterodimers coupled to G-proteins. The present study was undertaken to investigate activation of GABA(B) receptors in cerebral cortex and spinal cord using [35S]GTPgammaS binding assays, a direct measure of G-protein activity. The results revealed that the GABA(B) agonist baclofen stimulates GTPgammaS binding in cerebral cortex, with an ED50 of 50microM. This response is blocked by the GABA(B) receptor antagonist CGP 55845A (100nM). In contrast, baclofen-stimulated GTPgammaS binding was not observed in adult spinal cord tissue under similar incubation conditions, or after varying magnesium, calcium, GDP, [35S]GTPgammaS, or membrane concentrations in the assay medium. Stimulation of adult rat spinal cord muscarinic receptors did result in a concentration-related increase in [35S]GTPgammaS binding. Baclofen-stimulated GTPgammaS binding in adult spinal cord did not appear after peripheral inflammation, despite significant increases in GABA(B) subunit mRNA levels. As opposed to adult, appreciable GTPgammaS binding was observed in membranes prepared from spinal cords of rats within the first 14 days of postnatal development, suggesting that GABA(B) receptor function in the rat spinal cord is developmentally regulated. The results indicate that GABA(B) receptors may not be coupled to G-proteins in the adult rat spinal cord, or couple in a way that differs from that in newborns or adult cerebral cortex.