Mark M. Rasenick

Lipid raft microdomains and neurotransmitter signalling

Lipid rafts are specialized structures on the plasma membrane that have an altered lipid composition as well as links to the cytoskeleton. It has been proposed that these structures are membrane domains in which neurotransmitter signalling might occur through a clustering of receptors and components of receptor-activated signalling cascades. The localization of these proteins in lipid rafts, which is affected by the cytoskeleton, also influences the potency and efficacy of neurotransmitter receptors and transporters. The effect of lipid rafts on neurotransmitter signalling has also been implicated in neurological and psychiatric diseases.

Chronic Electroconvulsive Treatment Augments Coupling of the GTP-Binding Protein Gs to the Catalytic Moiety of Adenylyl Cyclase in a Manner Similar to That Seen with Chronic Antidepressant Drugs

Abstract: A significant increase of guanylylimidodiphosphate (GppNHp)‐, fluoride‐, and forskolin‐stimulated adenylyl cyclase was observed in synaptic membrane preparations from rat cerebral cortex subsequent to chronic electroconvulsive shock (ECS) treatment. This effect required at least five treatments over a course of 10 days. The inhibition of adenylyl cyclase induced by GppNHp was not affected by these treatments. The dissociation constant (KD) and maximal binding for the photoaffinity GTP analog, [32P]P3‐(4‐azidoanilido)‐P1‐5′‐GTP ([32P]AAGTP), to each of the synaptic membrane G proteins also were unchanged after ECS treatment. Nonetheless, the transfer of [32P]AAGTP from Gi to Gs, which we suggest is indicative of the coupling between Gs and the adenylyl cyclase catalytic moiety, was accelerated by chronic ECS treatment but not by acute or sham treatment. Furthermore, chemical uncoupling of Gs from adenylyl cyclase rendered membranes from treated animals indistinguishable from controls. Finally, in all cases tested, membranes prepared from animals subjected to chronic treatment with amitriptyline or iprindole showed similar changes in the Gs‐mediated activation of adenylyl cyclase. Acute treatments produced effects similar to controls, and liver and kidney membranes from animals receiving chronic treatment showed no changes in adenylyl cyclase despite the marked changes seen in brain. These results suggest that chronic administration of ECS enhances coupling between Gs and adenylyl cyclase enzyme and modifies interactions between Gs and Gi.

G Protein β1γ2 Subunits Promote Microtubule Assembly

α and βγ subunits of G proteins are thought to transduce signals from cell surface receptors to intracellular effector molecules. Gα and Gβγ have also been implicated in cell growth and differentiation, perhaps due to their association with cytoskeletal components. In this report Gβγ is shown to modulate the cytoskeleton by regulation of microtubule assembly. Specificity among βγ species exists, as β1γ2 stimulates microtubule assembly, and β1γ1 is without any effect. Furthermore, a mutant β1γ2, β1γ2(C68S), which does not undergo prenylation and subsequent carboxyl-terminal processing on the γ subunit, does not stimulate the formation of microtubules. β immunoreactivity was detected exclusively in the microtubule fraction after assembly in the presence of β1γ2, suggesting a preferential association with microtubules rather than soluble tubulin. Crude microtubule fractions from ovine brain contain Gβγ, and electron microscopy reveals a specific association with microtubules. The decoration of microtubules by Gβγ appears to be strikingly similar to the periodic pattern observed for microtubule-associated proteins, suggesting a similar site of activation of microtubule assembly by both agents. It is suggested that reformation of the cytoskeleton represents an additional cellular process mediated by Gβγ.

Tubulin, Gq, and Phosphatidylinositol 4,5-Bisphosphate Interact to Regulate Phospholipase Cβ1 Signaling

The cytoskeletal protein, tubulin, has been shown to regulate adenylyl cyclase activity through its interaction with the specific G protein α subunits, Gαs or Gαi1. Tubulin activates these G proteins by transferring GTP and stabilizing the active nucleotide-bound Gα conformation. To study the possibility of tubulin involvement in Gαq-mediated phospholipase Cβ1 (PLCβ1) signaling, the m1 muscarinic receptor, Gαq, and PLCβ1 were expressed in Sf9 cells. A unique ability of tubulin to regulate PLCβ1 was observed. Low concentrations of tubulin, with guanine nucleotide bound, activated PLCβ1, whereas higher concentrations inhibited the enzyme. Interaction of tubulin with both Gαq and PLCβ1, accompanied by guanine nucleotide transfer from tubulin to Gαq, is suggested as a mechanism for the enzyme activation. The PLCβ1 substrate, phosphatidylinositol 4,5-bisphosphate, bound to tubulin and prevented microtubule assembly. This observation suggested a mechanism for the inhibition of PLCβ1 by tubulin, since high tubulin concentrations might prevent the access of PLCβ1 to its substrate. Activation of m1 muscarinic receptors by carbachol relaxed this inhibition, probably by increasing the affinity of Gαq for tubulin. Involvement of tubulin in the articulation between PLCβ1 signaling and microtubule assembly might prove important for the intracellular governing of a broad range of cellular events.

G protein signaling and the molecular basis of antidepressant action.

Over the past four decades, a variety of interventions have been used for the treatment of clinical depression and other affective disorders. Several distinct pharmacological compounds show therapeutic efficacy. There are three major classes of antidepressant drugs: monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), and tricyclic compounds. There are also a variety of atypical antidepressant drugs, which defy ready classification. Finally, there is electroconvulsive therapy, ECT. All require chronic (2-3 weeks) treatment to achieve a clinical response. To date, no truly inclusive hypothesis concerning a mechanism of action for these diverse therapies has been formed. This review is intended to give an overview of research concerning G protein signaling and the molecular basis of antidepressant action. In it, the authors attempt to discuss progress that has been made in this arena as well as the possibility that some point (or points) along a G protein signaling cascade represent a molecular target for antidepressant therapy that might lead toward a unifying hypothesis for depression. This review is not designed to address the clinical studies. Furthermore, as it is a relatively short paper, citations to the literature are necessarily selective. The authors apologize in advance to authors whose work we have failed to cite.

Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a constitutive activator of the Gs G protein

The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein Gs and an orphan member of the G protein–coupled receptor family, GPR3. To determine whether GPR3 activates Gs, the localization of Gαs in follicle-enclosed oocytes from Gpr3 +/+ and Gpr3 −/− mice was compared by using immunofluorescence and GαsGFP. GPR3 decreased the ratio of Gαs in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Gαs in the oocyte. Both of these properties indicate that GPR3 activates Gs. The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent Gs activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity.

Exchange of Guanine Nucleotides Between Tubulin and GTP‐Binding Proteins That Regulate Adenylate Cyclase: Cytoskeletal Modification of Neuronal Signal Transduction

Tubulin, the primary constituent of microtubules, is a GTP‐binding protein with structural similarities to other GTP‐binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis‐resistant GTP analog, 5′‐guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP‐binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis‐resistant photoaffinity GTP analog, [32P]P3(4‐azidoanilido)‐P1‐5′‐GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP‐binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin‐AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP‐binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin‐Gpp(NH)p could be overridden by the inclusion of 100 μM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23°C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.

Chronic Treatment of C6 Glioma Cells with Antidepressant Drugs Increases Functional Coupling Between a G Protein (GS) and Adenylyl Cyclase

Abstract: It has been reported that antidepressant treatment in rats results in a significant increase of Gs‐mediated stimulation of adenylyl cyclase and this effect correlates well with the clinical therapeutic response. This increased activity occurs despite a down‐regulation of several receptors linked normally to the stimulation of that enzyme. To distinguish between these effects and to determine whether presynaptic components of the cell are required, C6 glioma cells were treated with antidepressants. Tricyclic (amitriptyline and desipramine) or atypical (iprindole) antidepressant exposure to C6 cells for 5 days significantly increased guanylyl‐5′‐imidodiphosphate [Gpp(NH)p]‐stimulated adenylyl cyclase activity in membrane preparations in a manner similar to that seen for rat brain membranes after 21‐day treatment. This effect was drug dose and exposure time dependent. Nevertheless, stimulation of adenylyl cyclase by isoproterenol was decreased after antidepressant treatment. By comparison, the antidepressant‐induced β‐receptor desensitization occurred earlier than the enhancement of Gpp(NH)p‐activated adenylyl cyclase, and extensive desensitization of β receptors by isoproterenol treatment did not enhance the Gpp(NH)p‐stimulated adenylyl cyclase activity. These results indicated that the antidepressant has a direct effect on cell signaling and this enhanced Gpp(NH)p‐stimulated adenylyl cyclase activity is not correlated with desensitization of β‐adrenergic receptor stimulated adenylyl cyclase. These data contribute to the suggestion that G proteins (especially Gs) are the target of antidepressant actions. Immunoblotting showed that neither the number of G protein subunits (αs, αi, αo, and β) nor their association with the plasma membrane was changed after antidepressant treatment. Thus, these results are consistent with the hypothesis that chronic antidepressant treatment acts directly at the postsynaptic membrane to increase the coupling between Gs and adenylyl cyclase.

Postmortem Brain Tissue of Depressed Suicides Reveals Increased Gsα Localization in Lipid Raft Domains Where It Is Less Likely to Activate Adenylyl Cyclase

Recent in vivo and in vitro studies have demonstrated that Gsα migrates from a Triton X-100 (TX-100)-insoluble membrane domain (lipid raft) to a TX-100-soluble nonraft membrane domain in response to chronic, but not acute, treatment with tricyclic or selective serotonin reuptake inhibitor antidepressants. This migration resulted in a more facile association with adenylyl cyclase. Our hypothesis is that Gsα may be ensconced, to a greater extent, in lipid rafts during depression, and that one action of chronic antidepressant treatment is to reverse this. In this postmortem study, we examined Gsα membrane localization in the cerebellum and prefrontal cortex of brains from nonpsychiatric control subjects and suicide cases with confirmed unipolar depression. Sequential TX-100 and TX-114 detergent extractions were performed on the brain tissue. In the cerebellum, the ratio of TX-100/TX-114-soluble Gsα is ∼2:1 for control versus depressed suicides. Results with prefrontal cortex samples from each group demonstrate a similar trend. These data suggest that depression localizes Gsα to a membrane domain (lipid rafts) where it is less likely to couple to adenylyl cyclase and that antidepressants may upregulate Gsα signaling via disruption of membrane microenvironments. Raft localization of Gsα in human peripheral tissue may thus serve as a biomarker for depression and as a harbinger of antidepressant responsiveness.

Chronic antidepressant treatment prevents accumulation of Gsα in cholesterol-rich, cytoskeletal-associated, plasma membrane domains (lipid rafts)

Previous studies demonstrated that Gsα migrates from a Triton X-100 (TTX-100) insoluble membrane domain to a TTX-100 soluble membrane domain in response to chronic treatment with the antidepressants desipramine and fluoxetine. Antidepressant treatment also causes a Gsα redistribution in cells as seen by confocal microscopy. The current studies have focused on examining the possibility that the association between Gsα and the plasma membrane and/or cytoskeleton is altered in response to antidepressant treatment, and that this is relevant to both Gsα redistribution and the increased coupling between Gsα and adenylyl cyclase seen after chronic antidepressant treatment. Chronic treatment of C6 cells with two fuctionally and structurally distinct antidepressants, desipramine and fluoxetine, decreased the Gsα content of TTX-100 insoluble membrane domains by as much as 60%, while the inactive fluoxetine analog LY368514 had no effect. Disruption of these membrane domains with the cholesterol chelator methyl-β-cyclodextrin altered the localization of many proteins involved in the cAMP signaling cascade, but only Gsα localization was altered by antidepressant treatment. In addition, microtubule disruption with colchicine elicited the movement of Gsα out of detergent-resistant membrane domains in a manner identical to that seen with antidepressant treatment. The data presented here further substantiate the role of Gsα as a major player in antidepressant-induced modification of neuronal signaling and also raise the possibility that an interaction between Gsα and the cytoskeleton is involved in this process.