Heterotrimeric G proteins as emerging targets for network based therapy in cancer: End of a long futile campaign striking heads of a Hydra.

Most common diseases, e.g., cancer are driven by not one, but multiple cell surface receptors that trigger and sustain a pathologic signaling network. The largest fraction of therapeutic agents that target individual receptors/pathways eventually fail due to the emergence of compensatory mechanisms that reestablish the pathologic network. Recently, a rapidly emerging paradigm has revealed GIV/Girdin as a central platform for receptor cross-talk which integrates signals downstream of a myriad of cell surface receptors, and modulates several key pathways within downstream signaling network, all via non-canonical activation of trimeric G proteins. Unlike canonical signal transduction via G proteins, which is spatially and temporally restricted, the temporal and spatial features of non-canonical activation of G protein via GIV is unusually unrestricted. Consequently, the GIV●G protein interface serves as a central hub allowing for control over several pathways within the pathologic signaling network, all at once. The relevance of this new paradigm in cancer and other disease states and the pros and cons of targeting the GIV●G protein interface are discussed.

Heterotrimeric G protein signaling outside the realm of seven transmembrane domain receptors.

Heterotrimeric G proteins, consisting of the guanine nucleotide-binding Galpha subunits with GTPase activity and the closely associated Gbeta and Ggamma subunits, are important signaling components for receptors with seven transmembrane domains (7TMRs). These receptors, also termed G protein-coupled receptors (GPCRs), act as guanine nucleotide exchange factors upon agonist stimulation. There is now accumulating evidence for noncanonical functions of heterotrimeric G proteins independent of 7TMR coupling. Galpha proteins belonging to all 4 subfamilies, including G(s), G(i), G(q), and G(12) are found to play important roles in receptor tyrosine kinase signaling, regulation of oxidant production, development, and cell migration, through physical and functional interaction with proteins other than 7TMRs. Association of Galpha with non-7TMR proteins also facilitates presentation of these G proteins to specific cellular microdomains. This Minireview aims to summarize our current understanding of the noncanonical roles of Galpha proteins in cell signaling and to discuss unresolved issues including regulation of Galpha activation by proteins other than the 7TMRs.

Pasteurella multocida toxin activates Gbetagamma dimers of heterotrimeric G proteins.

The mitogenic Pasteurella multocida toxin (PMT) is a major virulence factor of P. multocida, which causes Pasteurellosis in man and animals. The toxin activates the small GTPase RhoA, the MAP kinase ERK and STAT proteins via the stimulation of members of two G protein families, G(q) and G(12/13). PMT action also results in an increase in inositol phosphates, which is due to the stimulation of PLCbeta via Galpha(q). Recent studies indicate that PMT additionally activates Galpha(i) to inhibit adenylyl cyclase. Here we show that PMT acts not only via Galpha but also through Gbetagamma signaling. Activation of Gbetagamma by PMT causes stimulation of phosphoinositide 3-kinase (PI3K) gamma and formation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) as indicated by the recruitment of a PIP(3)-binding pleckstrin homology (PH) domain-containing protein to the plasma membrane. Moreover, it is demonstrated that Gbetagamma is necessary for PMT-induced signaling via Galpha. Mutants of Galpha(q) incapable of binding or releasing Gbetagamma are not activated by PMT. Similarly, sequestration of Gbetagamma inhibits PMT-induced Galpha-signaling.

Modulating G-protein coupled receptor/G-protein signal transduction by small molecules suggested by virtual screening.

Modulation of interactions between activated GPCRs (G-protein coupled receptors) and the intracellular (IC) signal transducers, heterotrimeric G-proteins, is an attractive, yet essentially unexplored, paradigm for treatment of certain diseases. Regulating downstream signaling for treatment of congenital diseases due to constitutively active GPCRs, as well as tumors where GPCRs are often overexpressed, requires the development of new methodologies. Modeling, experimental data, docking, scoring, and experimental testing (MEDSET) was developed to discover inhibitors that target the IC loops of activated GPCRs. As proof-of-concept, MEDSET developed and utilized a model of the interface between photoactivated rhodopsin (R*) and transducin (Gt), its G-protein. A National Cancer Institute (NCI) compound library was screened to identify compounds that bound at the interface between R* and its G-protein. High-scoring compounds from this virtual screen were obtained and tested experimentally for their ability to stabilize R* and prevent Gt from binding to R*. Several compounds that modulate signal transduction have been identified.

[The receptor of serpentine type and the heterotrimeric G protein as targets of action of the polylysine dendrimers].

The molecular mechanisms of action of the polycationic peptides--polylysine homo- and heterodendrimers on functional activity of biogenic amines- and peptide hormones-sensitive adenylyl; cyclase signaling system (AC system) in the myocardium and the brain of rats were studied. These peptides are expected to be used as highly effective polymer carries for biologically active substances. The polylysine homodendrimers of the third [(NH2)16(Lys)8(Lys)4(Lys)2Lys-Ala-NH2] (I), fourth [(NH2)32(Lys)16(Lys)8(Lys)4(Lys)2Lys-Ala-NH2 (II) and fifth [(NH2)64(Lys)32(Lys)16(Lys)8(Lys)4(Lys)2Lys-Ala-NH2] (III) generations and the polylysine homodendrimers of fifth generation--[(NH2)64(Lys-Glu)32(Lys-Glu)16(Lys-Glu)8(Lys-Glu)4(Lys-Glu)2Lys-Ala-Ala-Lys (ClAc)-Ala-NH2] (IV), [(NH2)64(Lys-Ala)32(Lys-Ala)16(Lys-Ala)8(Lys-Ala)4(Lys-Ala)2Lys-Ala-Lys(ClAc)-Ala-Ala-NH2] (V) and [(NH2)64(Lys-Gly-Gly)32(Lys-Gly-Gly)16(Lys-Gly-Gly)8(Lys-Gly-Gly)4(Lys-Gly-Gly)2 Lys-Gly-Gly-Lys(ClAc)-Ala-Ala-NH2] (VI) showed receptor-independent mechanism of heterotrimeric G-proteins activity, preferably of inhibitory type, interacting with C-terminal regions of their alpha-subunits. The homodendrimers II and III and heterodendrimer V are more effective G-protein activators. The polylysine dendrimers disturbed the functional coupling of the receptors of biogenic amines and peptides hormones with Gi-proteins and, in a lesser extent, Gs-proteins. This is illustrated by the decrease in regulatory effects of the hormones on AX activity and G-protein GTP binding and by the decrease in receptor affinity to agonists in the presence of the polylysine dendrimers, as result of receptor--G-proteins complex dissociation. It was shown also that the molecular mechanisms and the selectivity of the action on the G-proteins of the polylysine dendrimers were similar to those of mastoparan and melittin, natural toxins of insect venom.

Pertussis-toxin-sensitive Galpha subunits selectively bind to C-terminal domain of neuronal GIRK channels: evidence for a heterotrimeric G-protein-channel complex.

Neuronal G-protein-gated inwardly rectifying potassium (Kir3; GIRK) channels are activated by G-protein-coupled receptors that selectively interact with PTX-sensitive (Galphai/o) G proteins. Although the Gbetagamma dimer is known to activate GIRK channels, the role of the Galphai/o subunit remains unclear. Here, we established that Galphao subunits co-immunoprecipitate with neuronal GIRK channels. In vitro binding studies led to the identification of six amino acids in the GIRK2 C-terminal domain essential for Galphao binding. Further studies suggested that the Galphai/obetagamma heterotrimer binds to the GIRK2 C-terminal domain via Galpha and not Gbetagamma. Galphai/o binding-impaired GIRK2 channels exhibited reduced receptor-activated currents, but retained normal ethanol- and Gbetagamma-activated currents. Finally, PTX-insensitive Galphaq or Galphas subunits did not bind to the GIRK2 C-terminus. Together, these results suggest that the interaction of PTX-sensitive Galphai/o subunit with the GIRK2 C-terminal domain regulates G-protein receptor coupling, and may be important for establishing specific Galphai/o signaling pathways.

Involvement of signal transducing GTP-binding proteins in renal artery alpha 1-adrenoceptor mediated smooth muscle contraction.

OBJECTIVE: To determine the participation of GTP-binding proteins (G-proteins) in the cellular mechanism of the phenylephrine-induced renal artery vasospasm by using swine renal artery smooth muscle rings in a standard organ baths, as increased noradrenaline release from perivasal and intramural sympathetic nerve endings during renal ischaemia results in increased vascular smooth muscle tone that is important in the loss of kidney function during renal transplantation and nephron-sparing surgery. MATERIALS AND METHODS: Fresh swine kidneys were transported in cold calcium-free Tyrode solution to the laboratory. Adipose tissue around the arteries was removed, the organ de-capsulated and interlobar arteries dissected. The contractile properties of renal artery smooth muscle rings were assessed in a standard organ bath, the rings pre-tensioned at 2 g. Contractions were evoked by applying the alpha 1-adrenoceptor selective agonist phenylephrine (1 nmol/L to 0.3 mmol/L). Isometric contractions of the tissue were registered and stored digitally. Dose-response curves were obtained sequentially with a wash-out of 20 min between each concentration; the maximum contractility of an individual muscle ring was set at 100%. Dose-response curves of inhibitory agents (e.g. WB4101, cholera and pertussis toxins) were determined by comparing the remaining contractility after incubating with the respective drug with a control contraction that was evoked three times (10 mumol/L phenylephrine) and the mean set at 100%. RESULTS: Phenylephrine induced dose-dependent and fully reversible isometric contractions with a threshold concentration of 100 nmol/L and an EC50 of 0.8 mumol/L. The receptor was identified as the alpha 1A-subtype by the selective antagonist WB4101. Pre-treatment of tissue rings with 5 micrograms/mL pertussis toxin (120 min, 37 degrees C) inhibited the control contraction by a mean (SEM) of 52.0 (4.6)%, whereas pre-treatment with 1 microgram/mL cholera toxin (60 min, 37 degrees C), leading to a permanent activation of the Gs-protein via blockade of the GTPase activity, decreased the response by 39.0 (8.2%). CONCLUSION: These results suggest a coupling of alpha 1A-adrenoceptors in renal vascular tissue to the heterotrimeric Gs-protein and to heterotrimeric G-proteins of the G1- and/or G0-family in the phenylephrine-induced contraction.

Coupling to Gs and G(q/11) of histamine H2 receptors heterologously expressed in adult rat atrial myocytes.

The predominant histamine receptor subtype in the supraventricular and ventricular tissue of various mammalian species is the H2 receptor (H2-R) subtype, which is known to couple to stimulatory G proteins (Gs), i.e. the major effects of this autacoid are an increase in sinus rate and in force of contraction. To investigate histamine effects in H2-R-transfected rat atrial myocytes, endogenous GIRK currents and L-type Ca2+ currents were used as functional assays. In H2-R-transfected myocytes, exposure to His resulted in a reversible augmentation of L-type Ca2+ currents, consistent with the established coupling of this receptor to the Gs-cAMP-PKA signalling pathway. Mammalian K+ channels composed of GIRK (Kir3.x) subunits are directly controlled by interaction with betagamma subunits released from G proteins, which couple to seven-helix receptors. In mock-transfected atrial cardiomyocytes, activation of muscarinic K+ channels (IK(ACh)) was limited to Gi-coupled receptors (M2R, A1R). In H2-R-overexpressing cells, histamine activated IK(ACh) via Gs-derived betagamma subunits since the histamine-induced current was insensitive to pertussis toxin. These data indicate that overexpression of Gs-coupled H2-R results in a loss of target specificity due to an increased agonist-induced release of Gs-derived betagamma subunits. When IK(ACh) was maximally activated by GTP-gamma-S, histamine induced an irreversible inhibition of the inward current in a fraction of H2-R-transfected cells. This inhibition is supposed to be mediated via a G(q/11)-PLC-mediated depletion of PIP2, suggesting a partial coupling of overexpressed H2-R to G(q/11). Dual coupling of H2-Rs to Gs and Gq is demonstrated for the first time in cardiac myocytes. It represents a novel mechanism to augment positive inotropic effects by activating two different signalling pathways via one type of histamine receptor. Activation of the Gs-cAMP-PKA pathway promotes Ca2+ influx through phosphorylation of L-type Ca2+ channels. Simultaneous activation of Gq-signalling pathways might result in phosphoinositide turnover and Ca2+ release from intracellular stores, thereby augmenting H2-induced increases in [Ca2+]i.

Gonadotropin-releasing hormone-desensitized LbetaT2 gonadotrope cells are refractory to acute protein kinase C, cyclic AMP, and calcium-dependent signaling.

Sustained exposure of gonadotropes to GnRH causes a pronounced desensitization of gonadotropin release, but the mechanisms involved are poorly understood. It is known that desensitization is associated with decreased GnRH receptor and Gq/11 levels in alphaT3-1 cells, but it is not known whether downstream signaling is impaired. We have shown previously that chronic stimulation of signaling via expression of an active form of Galphaq causes GnRH resistance in LbetaT2 cells. In this study we investigated whether chronic GnRH treatment could down-regulate protein kinase C (PKC), cAMP, or Ca2+-dependent signaling in LbetaT2 cells. We found that chronic GnRH treatment desensitizes cells to acute GnRH stimulation not only by reducing GnRH receptor and Gq/11 expression but also by down-regulating PKC, cAMP, and calcium-dependent signaling. Desensitization was observed for activation of ERK and p38 MAPK and induction of c-fos and LHbeta protein expression. Activation of individual signaling pathways was able to partially mimic the desensitizing effect of GnRH on ERK, p38 MAPK, c-fos, and LHbeta but not on Gq/11. Chronic stimulation with phorbol esters reduced GnRH receptor expression to the same extent as chronic GnRH. Sustained GnRH also desensitized PKC signaling by down-regulating the delta, epsilon, and theta isoforms of PKC. We further show that chronic GnRH treatment causes heterologous desensitization of other Gq-coupled receptors.

Regulators of G protein signalling: potential targets for treatment of allergic inflammatory diseases such as asthma.

Asthma, a disease that affects nearly 15% of the world's population, is characterised by lung inflammation and reversible airway obstruction, which leads to wheezing and dyspnoea. Asthma is a prototype for allergic processes initiated by tissue inflammatory leukocytes, such as mast cells, whose secreted mediators recruit lymphocytes and eosinophils to the lung parenchyma. Signals transmitted through G-protein-coupled receptors (GPCRs) contribute to both the development and perpetuation of allergic processes, and pharmacological agents that block or stimulate GPCR action have been a mainstay of allergic disease therapy. Despite the widespread use of GPCR-targeted agents, little is understood about intracellular regulation of G protein pathways in immune cells. Regulators of G protein signalling (RGS proteins) enhance G protein deactivation and may contribute to the specificity and precision characteristic of GPCR signalling pathways. This review discusses the emerging functions of RGS proteins in immune processes and inflammatory states such as asthma, and their potential value as therapeutic targets for the treatment of allergic disease.

Alkylation of sulfhydryl groups on Galpha(s/olf) subunits by N-ethylmaleimide: regulation by guanine nucleotides.

In rat striatum A(2A) adenosine receptors activate adenylyl cyclase through coupling to G(s)-like proteins, mainly G(olf) that is expressed at high levels in this brain region. In this study we report that the sulfhydryl alkylating reagent, N-ethylmaleimide (NEM), causes a concentration- and time-dependent inhibition of [3H] 2-p-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamido adenosine ([3H]CGS21680) binding to rat striatal membranes. Membrane treatment with [14C]N-ethylmaleimide ([14C]NEM) labels numerous proteins while addition of 5'-guanylylimidodiphosphate (Gpp(NH)p) reduces labeling of only three protein bands that migrate in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of approximately 52, 45 and 39 kDa, respectively. The 52- and 45-kDa labeled bands show electrophoretic motilities as Galpha(s)-long and Galpha(s)-short/Galpha(olf) subunits. An anti-Galpha(s/olf) antiserum immunoprecipitates two 14C labeled bands of 44 and 39 kDa. The band density decreases by 21-26% when membranes are treated with NEM in the presence of Gpp(NH)p. An anti-A(2A) receptor antibody also immunoprecipitates two 14C labeled bands of 40 and 38 kDa, respectively. However, such protein bands do not show any decrease of their density upon membrane treatment with NEM plus Gpp(NH)p. These results indicate that in rat striatal membranes NEM alkylates sulfhydryl groups of both Galpha(s/olf) subunits and A(2A) adenosine receptors. In addition, cysteine residues of Galpha(s/olf) are easily accessible to modification when the subunit is in the GDP-bound form. The 39- and 38-kDa labeled proteins may represent proteolytic fragments of Galpha(s/olf) and A(2A) adenosine receptor, respectively.

Non-hydrolyzable analog of GTP induces activity of Na+ channels via disassembly of cortical actin cytoskeleton.

The role of G proteins in regulation of non-voltage-gated Na+ channels in human myeloid leukemia K562 cells was studied by inside-out patch-clamp method. Na+ channels were activated by non-hydrolyzable analog of guanosine triphosphate (GTP), GTPgammaS, known to activate both heterotrimeric and small G proteins. Channel activity was not affected by aluminum fluoride that indiscriminately activates heterotrimeric G proteins. The effect of GTPgammaS was prevented by phalloidin and by G-actin, both interfering with actin disassembly, which indicates that GTPgammaS-induced channel activation was likely due to microfilament disruption. GTPgammaS-activated channels were inactivated by polymerizing actin. These data show, for the first time, that small G proteins can regulate Na+ channels, and an intracellular mechanism mediating their effect involves actin cytoskeleton rearrangements.

Concerted stimulation and deactivation of pertussis toxin-sensitive G proteins by chimeric G protein-coupled receptor-regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity of RGS4.

Agonists stimulated high-affinity GTPase activity in membranes of HEK293 cells following coexpression of the alpha 2A-adrenoceptor and a pertussis toxin-resistant mutant of Go1 alpha. Enzyme kinetic analysis of Vmax and Km failed to detect regulation of the effect of agonist by a GTPase activating protein. This did occur, however, when cells were also transfected to express RGS4. Both elements of a fusion protein in which the N-terminus of RGS4 was linked to the C-terminal tail of the alpha 2A-adrenoceptor were functional, as it was able to provide concerted stimulation and deactivation of the G protein. By contrast, the alpha 2A-adrenoceptor-RGS4 fusion protein stimulated but did not enhance deactivation of a form of Go1 alpha that is resistant to the effects of regulator of G protein signaling (RGS) proteins. Employing this model system, mutation of Asn128 but not Asn88 eliminated detectable GTPase activating protein activity of RGS4 against Go1 alpha. Mutation of all three cysteine residues that are sites of post-translational acylation in RGS4 also eliminated GTPase activating protein activity but this was not achieved by less concerted mutation of these sites. These studies demonstrate that a fusion protein between a G protein-coupled receptor and an RGS protein is fully functional in providing both enhanced guanine nucleotide exchange and GTP hydrolysis of a coexpressed G protein. They also provide a direct means to assess, in mammalian cells, the effects of mutation of the RGS protein on function in circumstances in which the spatial relationship and orientation of the RGS to its target G protein is defined and maintained.

Age-related decreases in the response of aquaporin-5 to acetylcholine in rat parotid glands.

Aquaporin-5 (AQP5) is important in salivary fluid secretion in response to cholinergic and adrenergic stimuli in rat parotid glands. We hypothesized that expression and function of AQP5 might change with age. Acetylcholine and epinephrine induced increases in AQP5 levels in the apical plasma membranes of both young adult and senescent rats. The stimulatory effect of acetylcholine, but not that of epinephrine, on AQP5 levels in the apical plasma membranes of the cells decreased markedly during aging. The quinuclidine derivative, SNI-2011, induced a persistent increase in AQP5 levels in the apical plasma membrane in the cells of both these rats. The amounts of M(3)-muscarinic receptor and Gq proteins did not decrease during aging. The age-related alteration in the responsiveness of AQP5 in the cells to these stimuli might account for the concomitant changes in nitric oxide synthase activity. These results suggest that SNI-2011 might have therapeutic benefit for the treatment of age-related xerostomia.

The beta2-adrenergic receptor specifically sequesters Gs but signals through both Gs and Gi/o in rat sympathetic neurons.

Beta(2)-adrenergic receptors (beta(2)-AR) and CB1 cannabinoid receptors share the property of being constitutively active. The CB1 cannabinoid receptor can also sequester G(i/o) proteins; however, it is not known whether the beta(2)-AR can also sequester G proteins. Beta(2)-ARs were heterologously expressed in rat superior cervical ganglion neurons by microinjection of cDNA and studied using the patch-clamp technique. The beta-AR agonist isoproterenol increased the Ca(2+) current 25.9+/-1.6% in neurons microinjected with 100 ng/microl beta(2)-AR cDNA but was without effect on control neurons. Pretreatment with cholera toxin (CTX) abolished the effect of isoproterenol, indicating coupling via G(s) proteins. In neurons microinjected with 200 ng/microl beta(2)-AR cDNA, isoproterenol had the opposite effect of inhibiting the Ca(2+) current 36.5+/-2.0%. Inhibition of the Ca(2+) current was sensitive to pertussis toxin, indicating beta(2)-AR coupling to G(i/o) proteins. Pretreatment with CTX resulted in a greater 54+/-3.8% inhibition of the Ca(2+) current, indicating that G(s) coupling masks the full effect of G(i/o) coupling. Expression of beta(2)-ARs abolished signaling by G(s)-coupled receptors for vasoactive intestinal polypeptide (VIP). VIP inhibited the Ca(2+) current 49.5+/-0.5% in control neurons but had no effect in neurons expressing beta(2)-ARs. In contrast, expression of beta(2)-ARs had no effect on signaling by the G(i/o)-coupled alpha(2)-adrenergic receptor. This study demonstrates that the beta(2)-AR couples to both G(s) and G(i/o) proteins but specifically sequesters G(s) proteins, preventing their interaction with another G(s)-coupled receptor. beta(2)-adrenergic receptors thus have the potential to prevent other G(s)-coupled receptors from transducing their biological signals.

Regulation of GRK 2 and 6, beta-arrestin-2 and associated proteins in the prefrontal cortex of drug-free and antidepressant drug-treated subjects with major depression.

G protein-coupled receptor kinases (GRKs) and beta-arrestin-2 play a crucial role in the regulation of neurotransmitter receptors in brain. In this study, GRK 2, GRK 6, beta-arrestin-2 and associated proteins (Gbeta proteins and protein phosphatase (PP)-2A) were quantitated in parallel (immunodensity with specific antibodies) in brains of depressed subjects (drug-free and antidepressant-treated) to investigate the effect of major depression and antidepressant drugs on these receptor regulatory proteins. Specimens of the prefrontal cortex (Brodmann's area 9) were collected from 19 suicide and non-suicide depressed subjects and 13 control subjects. In drug-free (n=9), but not in antidepressant-treated (n=10), depressed subjects an increase in the density of membrane-associated GRK 2 (30%, n=9, P=0.005) was found compared with that in sex-, age-, and PMD-matched controls. Comparison between drug-free and antidepressant-treated depressed subjects showed that GRK 2 was reduced in membrane (39%, n=10, P=0.008) and cytosolic (44%, n=10, P=0.09) preparations after antidepressant drug treatment. In contrast, membrane-associated GRK 6 (drug-free and antidepressant-treated depressed subjects) was found unchanged when compared with that in matched controls. Similarly, the densities of beta-arrestin-2, PP-2A, and Gbeta proteins were not significantly different from those in matched controls. There was a positive correlation between the immunodensities of GRK 2 and beta-arrestin-2 in membrane preparations (r=0.48, n=19, P=0.04), suggesting that both proteins are regulated in a coordinated manner in brains of depressed subjects. The results of this study indicate that major depression is associated with upregulation of brain GRK 2, but not GRK 6, and that antidepressant drug treatment appears to induce downregulation of GRK 2 protein.

Stimulation-induced modifications in Go proteins examined in giant fused synaptosomes.

Synaptoneurosomes (1-3 microm in diameter), prepared from rat brain stem or brain cortex, were fused with liposomes, producing a high yield of giant synaptosomes (10-60 microm in diameter). Single channel currents were measured by using the cell-attach patch-clamp technique. The membrane of the majority of these giant synaptosomes retained the cell membrane selective permeability. However, nonpermeating molecules, such as guanine nucleotides and antibodies directed against GTP-binding region in the alpha-subunit of trimeric GTP-binding proteins, were trapped in the giant synaptosomes during their preparation. Activation of Go proteins was assayed in high [K(+)]-depolarized giant synaptosomes, indicating the advantage of this preparation for tracing signal-transduction mechanisms in stimulated synaptic membranes. Stimulation-induced interactions between membrane proteins, either native or reconstituted, can be studied in the giant synaptosomes.

Opioid peptide receptor studies. 16. Chronic morphine alters G-protein function in cells expressing the cloned mu opioid receptor.

Chronic morphine treatment results in functional uncoupling of the mu opioid receptor and its G protein in both cell culture and animal models. In the present study, Chinese hamster ovary (CHO) cells stably expressing the cloned human mu opioid receptor (hMOR-CHO cells) were incubated with 1 microM of morphine (or no drug) for 20 h. Subsequently, we assessed DAMGO- and morphine-stimulated [(35)S]-GTP-gamma-S binding and agonist-mediated inhibition of forskolin-stimulated cAMP accumulation. Using a single concentration of [(35)S]-GTP-gamma-S (0.05 nM), chronic morphine treatment did not significantly change basal [(35)S]-GTP-gamma-S binding, shifted the morphine EC(50) from 59 nM to 146 nM, and decreased the maximal stimulation (E(max)) from 201% to 177%. Similar results were observed with DAMGO. Binding surface analysis resolved two [(35)S]-GTP-gamma-S binding sites (high-affinity and low-affinity sites). In control cells, morphine stimulated [(35)S]-GTP-gamma-S binding by increasing the B(max) of the high-affinity site. In morphine-treated cells, morphine stimulated [(35)S]-GTP-gamma-S binding by decreasing the high-affinity K(d) without changing the B(max). Morphine treatment increased the EC(50) (5-11-fold) for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation. These changes were not observed in cells expressing a mutant mu opioid receptor which does not develop morphine tolerance, suggesting that the changes in [(35)S]-GTP-gamma-S binding observed in hMOR-CHO cells result from the development of morphine tolerance.

Role of G(i)alpha2-protein in opioid tolerance and mu-opioid receptor downregulation in vivo.

Although opioid receptors are G-protein coupled, the role that specific G-protein subunits play in the development of opioid tolerance and the regulation of opioid receptor number is not well understood. In the present study, we used a G((i)alpha2) antisense oligodeoxynucleotide (ODN) to examine the contribution of G((i)alpha2) proteins to mu-opioid tolerance and receptor downregulation in the mouse. Mice were injected intracerebroventricularly (ICV) and into the spinal intrathecal space (IT) for 4-5 consecutive days (30 microg/site/day), with an antisense ODN or a mismatch ODN directed at mRNA for the G((i)alpha2) subunit of G-proteins. Controls were treated with dH(2)O. On the second day of ODN treatment continuous subcutaneous (SC) infusion of etorphine (200 microg/kg/day) or morphine (40 mg/kg/day + 25 mg pellet) was begun. Control mice were implanted with inert placebo pellets. Three days later, pumps and pellets were removed and mice were tested for morphine analgesia or mu-opioid receptor density was determined in whole brain. Etorphine produced significant tolerance (ED(50) shift = approximately 11-fold) and downregulation of mu-opioid receptors (approximately 25%). Morphine treatment produced significant tolerance (ED(50) shift approximately 9-fold), but no mu-opioid receptor downregulation. Antisense treatment reduced G((i)alpha2) protein levels in striatum and spinal cord by approximately 25%. G((i)alpha2) antisense reduced the acute potency of morphine. G((i)alpha2) antisense blocked the development of tolerance to morphine treatment and reduced the development of tolerance to etorphine treatment. Antisense did not have any effect on etorphine-induced mu-opioid receptor downregulation. In another experiment, 7-day treatment with morphine or etorphine similarly increased G((i)alpha2) mRNA and protein abundance in spinal cord. Overall, these results support an important role for G((i)alpha2)-protein in the acute effects of opioids and opioid tolerance. However, G((i)alpha2) is not required for agonist-induced mu-opioid receptor density regulation in vivo.