GPCR-induced dissociation of G-protein subunits in early stage signal transduction

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Gα and Gβγ subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Gα and Gβγ) following agonist-induced GPCR (α_2A-adrenergic receptor; α_2A-AR) activation in a cell-free assay system. α_2A-AR membranes were reconstituted with the G-proteins (±hexahistidine-tagged) Gα_i1 and Gβ₁γ₂ and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [³⁵S]GTPγS. In the presence of Ni²⁺-coated agarose beads, the activated his-tagged Gα_i1his-[³⁵S]GTPγS complex was captured on the Ni²⁺-presenting surface. When his-tagged Gβ₁γ₂ (Gβ₁γ₂his) was used with Gα_i1, the [³⁵S]GTPγS-bound Gα_i1 was not present on the Ni²⁺-coated beads, but rather, it was separated from the β₁γ₂(his)-beads, demonstrating receptor-induced dissociation of Gα and Gβγ subunits. Treatment of the reconstituted α_2A-AR membranes containing Gβ₁γ₂his:Gα_i1 with imidazole confirmed the specificity for the Ni²⁺:G-protein surface dissociation of Gα_i1 from Gβ₁γ₂his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.     

Cannabinoid receptor stimulation of guanosine-5′-O-(3-[35S]thio)triphosphate binding in rat brain membranes

Cannabinoid receptors belong to the class of G-protein-coupled receptors which inhibit adenylyl cyclase. Coupling of receptors to G-proteins can be assessed by the ability of agonists to stimulate guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding in the presence of excess GDP. The present study examined the effect of cannabinoid agonists on [³⁵S]GTPγS binding in rat brain membranes. Assays were conducted with 0.05 nM [³⁵S]GTPγS, incubated with rat cerebellar membranes, 1–30 μM GDP and the cannabinoid agonist WIN 55212-2. Results showed that the ability of WIN 55212-2 to stimulate [³⁵S]GTPγS binding increased with increasing concentrations of GDP, with 10–30 μM GDP providing approximately 150–200% stimulation by the cannabinoid agonist. The pharmacology of cannabinoid agonist stimulation of [³⁵S]GTPγS binding paralleled that of previously reported receptor binding and adenylyl cyclase assays, and agonist stimulation of [³⁵S]GTPγS binding was blocked by the cannabinoid antagonist SR141716A. Brain regional studies revealed widespread stimulation of [³⁵S]GTPγS binding by WIN 55212-2 in a number of brain areas, consistent with in vitro [³⁵S]GTPγS autoradiography. These results demonstrate that [³⁵S]GTPγS binding in the presence of excess GDP is an effective measure of cannabinoid receptor coupling to G-proteins in brain membranes.     

Physical association between neuropeptide FF and micro-opioid receptors as a possible molecular basis for anti-opioid activity

Neuropeptide FF (NPFF) modulates the opioid system by exerting functional anti-opioid activity on neurons, the mechanism of which is unknown. By using a model of SH-SY5Y cells, we recently postulated that anti-opioid activity likely takes place upstream from the signaling cascade, suggesting that NPFF receptors could block opioid receptors by physical interaction. In the present study, fluorescence techniques were used to monitor the physical association and the dynamic of NPFF2 and micro-opioid (MOP) receptors tagged with variants of the green fluorescent protein. Importantly, cyan fluorescent protein-tagged NPFF2 receptors retained their capacity to antagonize opioid receptors. Fluorescence resonance energy transfer (FRET) and coimmunoprecipitation studies indicate that NPFF and MOP receptors are close enough to generate a basal FRET signal. The opioid agonist Tyr-D-Ala-Gly-NMe-Phe-Gly-ol disrupts by 20-30% this FRET signal, mainly because it concomitantly induces 40% internalization of receptors. In contrast, the NPFF analog 1DMe significantly increases by 10-15% the basal FRET signal, suggesting an association between both receptors. In addition, 1DMe reduces, by half, MOP receptor internalization, indicating that, besides a functional blockade of opioid receptors, the NPFF analog also inhibits their internalization. Finally, as a first report showing the modulation of the mobility of a G-protein-coupled receptor by another one, fluorescence recovery after photobleaching analysis reveals that 1DMe modifies the lateral diffusion of MOP receptors in the cell membrane, changing them from a confined to a freely diffusing state. By promoting NPFF-MOP receptor heteromerization, 1DMe could disrupt the domain organization of MOP receptors in the membrane, resulting in a reduction of opioid response.     

GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.     

Anti-opioid activities of NPFF1 receptors in a SH-SY5Y model

In order to elucidate the mechanisms of the neuronal anti-opioid activity of Neuropeptide FF, we have transfected the SH-SY5Y neuroblastoma cell line, which expresses mu- and delta-opioid receptors, with the human NPFF1 receptor. The SH1-C7 clone expresses high affinity NPFF1 receptors in the same range order of density as opioid receptors. Similarly to the opioids, acute stimulation with the NPFF1 agonist NPVF inhibits adenylyl cyclase activity and voltage-gated (N-type) Ca2+ currents and enhances the intracellular Ca2+ release triggered by muscarinic receptors activation. In contrast, preincubation of cells with NPVF decreases the response to opioids on both calcium signaling, thus reproducing the cellular anti-opioid activity described in neurons. SH1-C7 cells are therefore a suitable model to investigate the interactions between NPFF and opioid receptors.     

Modulation of calcium conductance by opioid and anti-opioid peptides

In the central nervous system, opening of voltage-gated Ca2+ channels triggers the release of neurotransmitters. Numerous membrane receptors, particularly those belonging to the superfamily of G-protein coupled receptors modulate, in most cases inhibit the activity of these channels. In the present review, we describe the modulation of calcium channels by opioid and anti-opioid peptides. Following a brief presentation of the opioid system, we describe the characteristics of the modulation of calcium channels by opioids. Recent major advances concerning neuropeptide FF (NPFF), taken as an example of anti-opioid systems, are reviewed. Results from our laboratory demonstrating the anti-opioid activity of NPFF, in the modulation of Ca2+ channels in isolated neurones, are described.     

Collision coupling, crosstalk, and compartmentalization in G-protein coupled receptor systems: can a single model explain disparate results?

The collision coupling model describes interactions between receptors and G-proteins as first requiring the molecules to find each other by diffusion. A variety of experimental data on G-protein activation have been interpreted as suggesting (or not) the compartmentalization of receptors and/or G-proteins in addition to a collision coupling mechanism. In this work, we use a mathematical model of G-protein activation via collision coupling but without compartmentalization to demonstrate that these disparate observations do not imply the existence of such compartments. In experiments with GTP analogs (commonly GTPγS), the extent of G-protein activation is predicted to be a function of both receptor number and the rate of GTP analog hydrolysis. The sensitivity of G-protein activation to receptor number is shown to be dependent upon the assay used, with the sensitivity of phosphate production assays (GTPase) >GTPγS-binding assays >cAMP inhibition assays. Finally, the amount of competition or crosstalk between receptor species activating the same type of G-proteins is predicted to depend on receptor and G-protein number, but in some (common) experimental regimes this dependence is expected to be minimal. Taken together, these observations suggest that the collision coupling model, without compartments of receptors and/or G-proteins, is sufficient to explain a variety of observations in literature data.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

Bell-shaped agonist activation of 5-HT1A receptor-coupled Gαi3 G-proteins: Receptor density-dependent switch in receptor signaling

A previous study observed bell-shaped concentration-response isotherms for activation of Gα_i3 G-protein subunits by high efficacy 5-HT_1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gα_i3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT_1A receptors in other cellular environments.Here, using an antibody capture-based [³⁵S]GTPγS binding assay for Gα_i3 activation, we investigated whether efficacious 5-HT_1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (−)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT_1A receptors.In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly ‘silent’ for Gα_i3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gα_i3 activation.These data indicate that Gα_i3 G-protein activation by 5-HT_1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.     

Synthesis of quinolinyl and isoquinolinyl phenyl ketones as novel agonists for the cannabinoid CB2 receptor

A series of quinolinyl and isoquinolinyl phenyl ketones was synthesized and their CB₂ receptor-dependent G-protein activities were determined using the [³⁵S]GTPγS binding assay. Both quinoline and isoquinoline derivatives exhibited similar CB₂ receptor agonist activity, the most potent ligands being the 2-(Me₂N)-phenyl substituted derivatives, which were also full agonists at the CB₂-receptor.     

Solubilization and functional reconstitution of human neuropeptide FF2 receptors

Neuropeptide FF (NPFF, FLFQPQRFamide) receptors modulate endogenous opioid functions. Here, we report the solubilization of the human NPFF₂ receptor expressed in Chinese hamster ovary (CHO) cells by the zwitterionic detergent Chaps. Chaps solubilization resulted in the abolishment of specific agonist binding activity, which was restored by a polyethylene glycol (PEG) precipitation method. Reincorporation after the precipitation step into liposomes made of endogenous lipids issued from CHO membranes or exogenous lipids significantly enhanced the specific agonist binding activity and G-protein coupling. This method of solubilization and lipid reconstitution could be useful for studies of NPFF receptors.     

Anti-analgesia of a selective NPFF2 agonist depends on opioid activity

NPFF agonists designed to be selective NPFF(2) receptor probes were synthesized. D.Asn-Pro-(N-Me)Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH(2) (dNPA) displays a very high affinity (0.027nM) for NPFF(2) receptors transfected in CHO cells, and a very high selectivity with a discrimination ratio greater than 100 versus NPFF(1) receptors. dNPA acts as a potent and selective agonist in [(35)S]GTPgammaS binding experiments and inhibits intracellular cAMP production with the same efficacy as NPA-NPFF. In SH-SY5Y cells expressing NPFF(2) receptors dNPA, in the presence of carbachol, stimulates Ca(2+) release from the intracellular stores. In vivo, after intracerebroventricular injection dNPA increases body temperature in mice and reverses the morphine-induced analgesia. Also, dNPA displays anti-opioid activity after systemic administration. So far, dNPA exhibits the highest affinity and selectivity for NPFF(2) receptors and reveals that its behavioral anti-opioid activity depends on the degree of opioid-induced analgesia.     

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [³⁵S]GTPγS functional assays were performed in the presence of putative δ₁-(DPDPE: agonist, BNTX: antagonist), δ₂-(agonist: deltorphin II, Ile^5,6-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ₂- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K_D = 0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [³H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [³H]Ile^5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49 ± 0.06 and 0.30 ± 0.01 nM potency against DPDPE and deltorphin II in the [³⁵S]GTPγS functional assay, respectively. The rank order of potency of putative δ₁- and δ₂-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ₁- and δ₂-opioid receptors could not be unequivocally distinguished in vitro.     

Activation of a pertussis toxin-sensitive, inhibitory G-protein is necessary for steroid-mediated oocyte maturation in spotted seatrout

Oocyte maturation (OM) is initiated in lower vertebrates and echinoderms when maturation-inducing substances (MIS) bind oocyte membrane receptors. This study tested the hypothesis that activation of a G_i protein is necessary for MIS-mediated OM in spotted seatrout. Addition of MIS significantly decreased adenylyl cyclase activity in a steroid specific, pertussis toxin (PTX)-sensitive manner in oocyte membranes and microinjection of PTX into oocytes inhibited MIS-induced OM, suggesting the steroid activates a G_i protein. MIS significantly increased [³⁵S]GTPγS binding to ovarian membranes, confirming that MIS receptor binding activates a G-protein, and immunoprecipitation studies showed the increased [³⁵S]GTPγS binding was associated with Gα_i1-3 proteins. Radioligand binding studies in ovarian membranes using GTPγS and PTX demonstrated that the MIS binds a receptor coupled to a PTX-sensitive G-protein. This study provides the first direct evidence in a vertebrate model that MIS-induced activation of a G_i protein is necessary for OM. These results support a mechanism of MIS action involving binding to a novel, G-protein coupled receptor and activation of an inhibitory G-protein, the most comprehensive and plausible model of MIS initiation of OM proposed to date.     

Breaking down the barriers: fMRI applications in pain, analgesia and analgesics

Background

The vanilloid receptor 1 (TRPV1) is critical in the development of inflammatory hyperalgesia. Several receptors including G-protein coupled prostaglandin receptors have been reported to functionally interact with the TRPV1 through a cAMP-dependent protein kinase A (PKA) pathway to potentiate TRPV1-mediated capsaicin responses. Such regulation may have significance in inflammatory pain. However, few functional receptor interactions that inhibit PKA-mediated potentiation of TRPV1 responses have been described.

Results

In the present studies we investigated the hypothesis that the μ opioid receptor (MOP) agonist morphine can modulate forskolin-potentiated capsaicin responses through a cAMP-dependent PKA pathway. HEK293 cells were stably transfected with TRPV1 and MOP, and calcium (Ca²⁺) responses to injection of the TRPV1 agonist capsaicin were monitored in Fluo-3-loaded cells. Pre-treatment with morphine did not inhibit unpotentiated capsaicin-induced Ca²⁺ responses but significantly altered capsaicin responses potentiated by forskolin. TRPV1-mediated Ca²⁺ responses potentiated by the direct PKA activator 8-Br-cAMP and the PKC activator Phorbol-12-myristate-13-acetatewere not modulated by morphine. Immunohistochemical studies confirmed that the TRPV1 and MOP are co-expressed on cultured Dorsal Root Ganglion neurones, pointing towards the existence of a functional relationship between the G-protein coupled MOP and nociceptive TRPV1.

Conclusion

The results presented here indicate that the opioid receptor agonist morphine acts via inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses. Targeting of peripheral opioid receptors may therefore have therapeutic potential as an intervention to prevent potentiation of TRPV1 responses through the PKA pathway in inflammation.     

Agonist Action at D2(short) Dopamine Receptors Determined in Ligand Binding and Functional Assays

Abstract: 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 µM). 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-propylpiperidine 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 [³⁵S]guanosine 5′-O-(3-thiotriphosphate) ([³⁵S]-GTPγS) binding for a range of agonist concentrations was measured and the EC₅₀ and maximal effects determined. The initial rates of [³⁵S]GTPγS binding induced by maximally stimulating agonist concentrations were also recorded. Simultaneous inhibition of agonist-stimulated [³⁵S]GTPγS 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.     

Alkamides and a neolignan from Echinacea purpurea roots and the interaction of alkamides with G-protein-coupled cannabinoid receptors

Multiple chromatographic separations of the CHCl₃-soluble extract of the roots of Echinacea purpurea led to the isolation of 19 compounds. Four natural products, three alkamides and nitidanin diisovalerianate, were identified, and five further compounds were detected for the first time in this species. Additionally, 10 known E. purpurea metabolites were isolated. The structures were determined by mass spectrometry and advanced 1D and 2D NMR techniques. The bioactivity of the isolated compounds was studied in [³⁵S]GTPγS-binding experiments performed on rat brain membrane preparations. Both partial and inverse agonist compounds for cannabinoid (CB1) receptors were identified among the metabolites, characterized by weak to moderate interactions with the G-protein signaling mechanisms. The G-protein-modulating activities of the Echinacea compounds are rather far from the full agonist effects seen with the CB1 receptor agonist reference compound arachidonyl-2′-chloroethylamide (ACEA). However, upon coadministration with ACEA, a number of them proved capable of inhibiting the stimulation of the pure agonist, thereby demonstrating cannabinoid receptor antagonist properties.     

Subtype differences in pre-coupling of muscarinic acetylcholine receptors

Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex "collides" with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to G(q/11), while even-numbered receptors prefer coupling to G(i/o). We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [(3)H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M(1) and M(3) receptors also pre-couple with non-preferential G(i/o) G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype and the class of G-protein.     

Use of plasmon waveguide resonance (PWR) spectroscopy for examining binding,signaling and lipid domain partitioning of membrane proteins

AimsDue to their anisotropic properties and other factors, it has been difficult to determine the conformational and dynamic properties of integral membrane proteins such as G-protein coupled receptors (GPCRs), growth factor receptors, ion channels, etc. in response to ligands and subsequent signaling. Herein a novel methodology is presented that allows such studies to be performed while maintaining the receptors in a membrane environment.Main methodPlasmon waveguide resonance (PWR) spectroscopy is a relatively new biophysical method which allows one to directly observe structural and dynamic changes which occur on interaction of GPCRs (and other integral membrane proteins) with ligands and signaling molecules. The delta opioid receptor (DOR) and its ligands serve as an excellent model system to illustrate the new insights into GPCR signaling that can be obtained by this method.Key findingsAmong our key findings are: 1) it is possible to obtain the following information directly and without any need for labels (radioactive, fluorescent, etc.): binding affinities, and the ability to distinguish between agonists, antagonists, inverse agonist, and partial agonists without a need for second messenger analysis; 2) it is possible to determine directly, again without a need for labels, G-protein binding to variously occupied or unoccupied DORs, and to determine which α-subtype is involved in allowing structurally different agonist ligands to have differential effects; 3) GTPγS binding can be examined directly; and 4) binding of the DOR with different ligands leads to differential segregation of the ligand-receptor complex into lipid rafts.SignificanceThe implications of these discoveries suggest a need to modify our current views of GPCR-ligand interactions and signaling.     

Intracellularly Truncated Human α2B-Adrenoceptors: Stable and Functional GPCRs for Structural Studies

All three α₂-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 α_2B-adrenoceptor (α_2B-AR) was investigated with a series of 3i truncated constructs (Δ 3i). We used a variety of agonists/antagonists in competition binding assays. We stimulated α_2B-AR Δ3i with various agonists and measured [³⁵S]GTPγS 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]GTPγS assay. Our results reveal that the carboxyl end residues of 3i, R360(6.24) to V372(6.36), are important for G_i/G_o protein activation. Deletions in regions from G206(5.72) to R245(5.110) altered the binding of some α_2B-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 α_2B-AR.