Synergism between fluoxetine and the mGlu2/3 receptor agonist, LY379268, in an in vitro model for antidepressant drug-induced neurogenesis.

We examined the interaction between the selective serotonin reuptake inhibitor, fluoxetine, and group-II metabotropic glutamate (mGlu) receptors using progenitor cells isolated from cultured cerebellar granule cells, considered as an in vitro model of antidepressant-drug induced neurogenesis. These cells expressed mGlu3 receptors negatively coupled to adenylyl cyclase. A 72-h treatment with either fluoxetine or low concentrations of mGlu2/3 receptor agonists (LY379268 or 2R,4R-APDC) enhanced cell proliferation. The action of fluoxetine was mediated by the activation of 5-HT(1A) receptors. We found a strong synergism between fluoxetine and LY379268 in enhancing cell proliferation and inhibiting cAMP formation. The increased cell proliferation induced by fluoxetine+LY379268 was abrogated by the cAMP analogue, 8-Br-cAMP, as well as by drugs that inhibit the mitogen-activated protein kinase and phosphatidyilinositol-3-kinase pathways. Interestingly, fluoxetine and LY379268 also acted synergistically in promoting neuronal differentiation when progenitor cells were incubated in the presence of serum. These data support the hypothesis that a combination between classical antidepressants and mGlu2/3 receptor agonists may be helpful in the experimental treatment of depression.

PHCCC, a specific enhancer of type 4 metabotropic glutamate receptors, reduces proliferation and promotes differentiation of cerebellar granule cell neuroprecursors.

Exposure of immature rat cerebellar granule cell cultures to the type 4 metabotropic glutamate (mGlu4) receptor enhancer N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) reduced [3H]thymidine incorporation. Its action was sensitive to the growth conditions and was attenuated by two mGlu4 receptor antagonists. An antiproliferative action of PHCCC was also seen in cultures from wild-type, but not mGlu4, knock-out mice. At least in rat cultures, PHCCC was not neurotoxic and enhanced neuritogenesis. Although PHCCC reduced the increase in cAMP formation and phospho-AKT levels induced by forskolin, none of these transduction pathways significantly contributed to the reduction of [3H]thymidine incorporation. Interestingly, PHCCC reduced the expression of Gli-1, a transcription factor that mediates the mitogenic action of Sonic hedgehog. Finally, we treated newborn rats with PHCCC either intracerebrally (infusion of 5 nmol/2 microl in the cerebellar region once every other day) or systemically (5 mg/kg, i.p., once daily) from postnatal days 3-9. Local infusion of PHCCC induced substantial changes in the morphology of the developing cerebellum. In contrast, systemic injection of PHCCC induced only morphological abnormalities of the cerebellar lobule V, which became visible 11 d after the end of the treatment. These data suggest that mGlu4 receptors are involved in the regulation of cerebellar development.

Activation of Fas receptor is required for the increased formation of the disialoganglioside GD3 in cultured cerebellar granule cells committed to apoptotic death.

Apoptosis was induced in cultured cerebellar granule cells by lowering extracellular K+ concentrations (usually from 25 to 10 mM). The apoptotic phenotype was preceded by an early and transient increase in the intracellular levels of the disialoganglioside, GD3, which behaves as a putative pro-apoptotic factor. We examined whether activation of Fas receptor mediates the increase in GD3 formation in granule cells committed to die. Degenerating granule cells showed increased expression of both Fas receptor and its ligand (Fas-L), at times that coincided with the increase in GD3 levels and the induction of GD3 synthase mRNA. Addition of neutralizing anti-Fas-L antibodies reduced the extent of 'low-K+'-induced apoptosis and abolished the increase in GD3 levels and GD3 synthase mRNA. Similar reductions were observed in cultures prepared from gld or lpr mice, which harbor loss-of-function mutations of Fas-L and Fas receptor, respectively. In addition, exogenous application of soluble Fas-L further enhanced both the increase in GD3 formation and cell death in cultured granule cells switched from 25 into 10 mM K+. We conclude that activation of Fas receptor is entirely responsible for the increase in GD3 levels and contributes to the development of apoptosis by trophic deprivation in cultured cerebellar granule cells.

Regulation of mGlu4 metabotropic glutamate receptor signaling by type-2 G-protein coupled receptor kinase (GRK2).

We examined the role of G-protein coupled receptor kinase-2 (GRK2) in the homologous desensitization of mGlu4 metabotropic glutamate receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Receptor activation with the agonist l-2-amino-4-phosphonobutanoate (l-AP4) stimulated at least two distinct signaling pathways: inhibition of cAMP formation and activation of the mitogen-activated protein kinase (MAPK) pathway [assessed by Western blot analysis of phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2]. Activation of both pathways was attenuated by pertussis toxin. Overexpression of GRK2 (but not GRK4) largely attenuated the stimulation of the MAPK pathway by l-AP4, whereas it slightly potentiated the inhibition of FSK-stimulated cAMP formation. Transfection with a kinase-dead mutant of GRK2 (GRK2-K220R) or with the C-terminal fragment of GRK2 also reduced the mGlu4-mediated stimulation of MAPK, suggesting that GRK2 binds to the Gbetagamma subunits to inhibit signal propagation toward the MAPK pathway. This was confirmed by the evidence that GRK2 coimmunoprecipitated with Gbetagamma subunits in an agonist-dependent manner. Finally, neither GRK2 nor its kinase-dead mutant had any effect on agonist-induced mGlu4 receptor internalization in HEK293 cells transiently transfected with GFP-tagged receptors. Agonist-dependent internalization was instead abolished by a negative-dominant mutant of dynamin, which also reduced the stimulation of MAPK pathway by l-AP4. We speculate that GRK2 acts as a "switch molecule" by inhibiting the mGlu4 receptor-mediated stimulation of MAPK and therefore directing the signal propagation toward the inhibition of adenylyl cyclase.

Presence of beta-arrestin in cellular inclusions in metamphetamine-treated PC12 cells.

Cellular inclusions containing ubiquitin and alpha-synuclein were observed in PC12 cells treated with metamphetamine (MA). To study the possible involvement of beta-arrestin in inclusion formation, we treated PC12 cells with MA for different times and analyzed the ubiquitin proteosome pathway (UPP). We found that beta-arrestin is ubiquitinated in the MA-treated PC12 cell line. The involvement of beta-arrestin in UPP was further supported by electron microscopy and by confocal microscopy, which documented the presence of beta-arrestin in these Lewy body-like inclusions. Our experiments reveal an interesting and previously unappreciated connection between beta-arrestin and ubiquitination and suggest that beta-arrestin could be involved in the development of the inclusion bodies.

Regulation of lysophosphatidic acid receptor-stimulated response by G-protein-coupled receptor kinase-2 and beta-arrestin1 in FRTL-5 rat thyroid cells.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that activates a variety of biological activities including cell proliferation. Three mammalian LPA receptor (LPAr) subtypes have been identified by molecular cloning, named lp(A1), lp(A2) and lp(A3), that are coupled to heterotrimeric G-proteins for signal transduction. The LPAr are endogenously expressed in the rat thyroid cell line FRTL-5 and we used the FRTL-5 cells permanently transfected to obtain moderate overexpression of G-protein-coupled receptor kinase-2 (GRK2) or beta-arrestin1 to study whether GRK2 and beta-arrestin1 desensitise LPAr-mediated signalling and regulate LPA-stimulated functional effects. Using RT-PCR we documented that lp(A1), lp(A2) and lp(A3) receptors are all expressed in FRTL-5 cells. We then analysed the signal transduction of the LPAr in FRTL-5 cells. Exposure to LPA did not stimulate inositol phosphate formation nor cAMP accumulation but reduced forskolin-stimulated cAMP. LPA was also able to stimulate MAP kinase activation and this effect was abolished by pertussis toxin pretreatment. These results suggest that LPAr are mainly coupled to a pertussis toxin-sensitive G-protein in FRTL-5 cells. In order to investigate whether GRKs and arrestins are involved in the regulation of LPAr-mediated signalling, we used the FRTL-5 cell line permanently transfected to overexpress GRK2 (named L5GRK2 cells) or beta-arrestin1 (L5betaarr1 cells). The ability of LPA to inhibit forskolin-stimulated cAMP accumulation was blunted in L5GRK2 and more markedly in L5betaarr1. The MAP kinase activation was also blunted in L5GRK2 and in L5betaarr1B cells. Exposure to 20 microM LPA increased the phosphorylation of extracellular signal-regulated kinases ERK1/2 by approximately 3-fold in L5pBJI cells (FRTL-5 cells transfected with the empty vector pBJI) while it induced a modest increase in L5betaarr1 and was ineffective in L5GRK2. We measured [3H]thymidine uptake in L5betaarr1B and in L5 GRK2 cells to test whether GRK2 and beta-arrestin1 could have a role in the regulation of LPAr-mediated cell proliferation. The mitogenic response induced by 35 microM LPA was substantially blunted in L5betaarr1 (-69+/-6%) and in L5GRK2 (-69.8+/-4.5%) cells as compared with L5pBJI. Our findings document that the receptor-mediated responses elicited by LPA are regulated by GRK2 and beta-arrestin1 in FRTL-5 cells and indicate that this mechanism is potentially important for the control of the LPA-stimulated proliferative response.

Native group-III metabotropic glutamate receptors are coupled to the mitogen-activated protein kinase/phosphatidylinositol-3-kinase pathways.

We used cultured cerebellar granule cells to examine whether native group-III metabotropic glutamate (mGlu) receptors are coupled to the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3-K) pathways. Cultured granule cells responded to the group-III mGlu receptor agonist, L-2-amino-4-phosphonobutanoate (l-AP4), with an increased phosphorylation and activity of MAPKs (ERK-1 and -2) and an increased phosphorylation of the PI-3-K target, protein kinase B (PKB/AKT). These effects were attenuated by the group-III antagonists, alpha-methyl-serine-O -phosphate (MSOP) and (R,S )-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), or by pretreatment of the cultures with pertussis toxin. l-AP4 also induced the nuclear translocation of beta-catenin, a downstream effector of the PI-3-K pathway. To assess the functional relevance of these mechanisms we examined the ability of l-AP4 to protect granule cells against apoptosis by trophic deprivation, induced by lowering extracellular K(+) from 25 to 10 mm. Neuroprotection by l-AP4 was attenuated by MSOP and abrogated by the compounds PD98059 and UO126, which inhibit the MAPK pathway, or by the compound LY294002, which inhibits the PI-3-K pathway. Taken together, these results show for the first time that native group-III mGlu receptors are coupled to MAPK and PI-3-K, and that activation of both pathways is necessary for neuroprotection mediated by this particular class of receptors.

Thyrotropin activates mitogen-activated protein kinase pathway in FRTL-5 by a cAMP-dependent protein kinase A-independent mechanism.

The involvement of mitogen-activated protein (MAP) kinases in the mitogenic effect of thyrotropin (TSH) is not fully elucidated. In FRTL-5 cells, we found that the MAP kinase kinase (MEK) inhibitors UO126 and PD98059 substantially decreased TSH-induced DNA synthesis, indicating that MAP kinases are involved in the TSH-stimulated proliferative response. Accordingly, TSH, forskolin (FSK) and 8-bromo-cAMP induced a rapid (3 min) and transient activation of ERK1/2, as assessed by phosphorylation of myelin basic protein and ERK1/2. This effect was cAMP-dependent and protein kinase A (PKA)-independent. The activation of Rap1 and B-Raf was involved in the mechanism of MAP kinase stimulation by TSH. TSH induced rapid (3 min) GDP/GTP exchange and activation of Rap1. After a 3-min exposure to FSK, B-Raf was recruited to a vesicular compartment, where it colocalized with Rap1. Both activation of Rap1 and translocation of B-Raf were PKA-independent. The Rap1 dominant negative Rap1N17 significantly reduced TSH-stimulated but not insulin-like growth factor 1-stimulated ERK1/2 phosphorylation, whereas the Ras dominant negative RasN17 inhibited the effect of both agonists. In conclusion, our results document that TSH increases intracellular cAMP, which rapidly stimulates MAP kinase cascade independent of PKA. This novel mechanism could integrate other pathways involved in TSH-stimulated proliferative response.

Regulation of G protein-coupled receptor kinase subtypes by calcium sensor proteins.

G protein-coupled receptor homologous desensitization is intrinsically related to the function of a class of S/T kinases named G protein-coupled receptor kinases (GRK). The GRK family is composed of six cloned members, named GRK1 to 6. Studies from different laboratories have demonstrated that different calcium sensor proteins (CSP) can selectively regulate the activity of GRK subtypes. In the presence of calcium, rhodopsin kinase (GRK1) is inhibited by the photoreceptor-specific CSP recoverin through direct binding. Several other recoverin homologues (including NCS 1, VILIP 1 and hippocalcin) are also able to inhibit GRK1. The ubiquitous calcium-binding protein calmodulin (CaM) can inhibit GRK5 with a high affinity (IC(50)=40-50 nM). A direct interaction between GRK5 and Ca(2+)/CaM was documented and this binding does not influence the catalytic activity of the kinase, but rather reduced GRK5 binding to the membrane. These studies suggest that CSP act as functional analogues in mediating the regulation of different GRK subtypes by Ca(2+). This mechanism is, however, highly selective with respect to the GRK subtypes: while GRK1, but not GRK2 and GRK5, is regulated by recoverin and other NCS, GRK4, 5 and 6, that belong to the GRK4 subfamily, are potently inhibited by CaM, which had little or no effect on members of other GRK subfamilies.

Selective regulation of Gq signaling by G protein-coupled receptor kinase 2: direct interaction of kinase N terminus with activated galphaq.

In this study, we investigated the regulation of different G protein-coupled receptor (GPCR)-stimulated signaling pathways by GPCR kinase 2 (GRK2). We used thyrotropin receptor, which is coupled to different G proteins, to investigate the regulation of Galphas- and Galphaq-mediated signaling (assessed by cAMP and inositol phosphate production, respectively). In transfected cells, both pathways were desensitized by GRK2. However a kinase-dead GRK2 mutant (GRK2-K220R) only decreased inositol phosphate production, indicating that GRK2 could regulate Galphaq signaling through a phosphorylation-independent mechanism. Similar results were obtained with serotonin receptor 5-hydroxytryptamine(2C), which is coupled to Galphaq. This effect was mimicked by the N-terminal domain of GRK2 (GRK2-Nter), but not by the C-terminal domain. In cells transfected with Galphaq, direct activation of Galphaq signaling (by AlF(4)(-)) was desensitized by GRK2-Nter, indicating an effect at the Galpha-level. For comparison, in parallel samples we studied a protein regulator of G protein signaling RGS4 and we found a similar regulatory profile. We therefore hypothesized that the GRK2-Nter could directly interact with the Galphaq subunit to regulate its signaling, as demonstrated for several RGS proteins. This hypothesis is further supported by the presence, within the GRK2-Nter, of an RGS homology domain. In direct binding experiments, we found that GRK2-Nter interacts with Galphaq (only when activated) but not with Galphas and Galphao. We conclude that GRK2, besides desensitizing the GPCR by phosphorylation, is able to selectively bind to Galphaq and to regulate its signaling.

Involvement of G protein-coupled receptor kinases and arrestins in desensitization to follicle-stimulating hormone action.

FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. In the present study, we investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. We found that these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 as detected by RT-PCR and by Western blotting, were rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells allowed us to demonstrate 1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase, P < 0.001); 2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition, P < 0.01) whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold (P < 0.001); 3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and 4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold, P < 0.05) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. Our results show that the desensitization of the FSH-induced response involves the GRK/arrestin system.

Selective regulation of G protein-coupled receptor-mediated signaling by G protein-coupled receptor kinase 2 in FRTL-5 cells: analysis of thyrotropin, alpha(1B)-adrenergic, and A(1) adenosine receptor-mediated responses.

G protein-coupled receptor kinases (GRKs) play a key role in the process of receptor homologous desensitization. In the present study, we address the question of whether a variety of receptors coupled to different G protein subtypes and naturally expressed on the same cell are selectively regulated by GRK2. The signaling stimulated by thyrotropin (TSH), alpha(1B)-adrenergic, and A(1) adenosine receptors was studied in FRTL-5 cells permanently transfected to overexpress GRK2 and GRK2-K220R, a kinase dead GRK dominant negative mutant. In FRTL-5 overexpressing GRK2, TSH-induced cyclic AMP response was attenuated, indicating that TSH receptor is desensitized by this kinase. Consistently, FRTL-5 cells overexpressing GRK2-K220R show increased TSH-induced cyclic AMP response, demonstrating that this receptor is under tonic control by GRK. Unlike TSH receptor, alpha(1B)-adrenergic receptor response was unaffected in FRTL-5 overexpressing GRK2 and GRK2-K220R. When A(1) adenosine receptors were stimulated, G(ialpha)-mediated cyclic AMP inhibition was totally unaffected by overexpression of either GRK2 or GRK2-K220R. By contrast, G(betagamma)-mediated response (activation of mitogen-activated protein kinases) was efficiently desensitized by GRK2 but was unaffected by GRK2-K220R overexpression. The present study documents that overexpression of GRK2 results in a selective regulation of different G protein-coupled receptors expressed on the same cell and that this kinase can regulate preferentially only one of the different pathways activated by the same receptor. The preferential regulation of the A(1) adenosine receptor-stimulated mitogen-activated protein kinases by GRK2 indicates that this kinase can have additional regulatory effects on G(betagamma)-stimulated pathways, possibly through direct binding and regulation of the receptor-G(betagamma) complex.

GRK2 and beta-arrestin 1 as negative regulators of thyrotropin receptor-stimulated response.

Arrestins are regulatory proteins for a number of G-coupled receptors. The binding of arrestin to receptor phosphorylated by G protein-coupled receptor kinase (GRK) quenches the activation of the G protein, thus resulting in receptor homologous desensitization. We have previously shown that the levels of beta-arrestin1 are regulated by intracellular cAMP and proposed that this may represent one homeostatic mechanism with which to regulate some cellular responses. To test this hypothesis, we focused on the TSH receptor using a rat thyroid cell line, FRTL5. We found that beta-arrestin1 is the only detectable isoform of arrestin expressed in FRTL5 and that its expression is regulated by TSH. To investigate the possible role of GRK2/beta-arrestin1 machinery in the mechanism of TSH receptor homologous desensitization, we used a cotransfection approach. The TSH-induced cAMP accumulation in COS7 cells transfected with TSH receptor was reduced by 35-45% when cotransfected with GRK2 and/or beta-arrestin1, indicating that the TSH receptor can be regulated by a GRK/arrestin mechanism. This raised the hypothesis that TSH increases the levels of beta-arrestin1, which in turn could regulate the TSH stimulation. To test this point a FRTL5-derived cell line overexpressing beta-arrestin1 was generated. In these cells the TSH-stimulated cAMP accumulation and, more importantly, the mitogenic activity were substantially blunted. Our results show that TSH receptor-stimulated cAMP accumulation and cell proliferation can be controlled by a GRK2/beta-arrestin1 mechanism.

Glycerophosphoinositol 4-phosphate, a putative endogenous inhibitor of adenylylcyclase.

In a continuous line of rat thyroid cells transformed by the k-ras oncogene (KiKi), the expression of ras-p21 correlates with an increased activity of a phosphoinositide-specific phospholipase A2, which leads to elevated levels of glycerophosphoinositols. In this study we have characterized the biological activities of these compounds. Growth and differentiation in thyroid cells are mainly regulated by the activation of adenylylcyclase. Therefore, we have studied the effects of glycerophosphoinositols on the activity of this enzyme using a normal thyroid cell line (FRTL5). Micromolar concentrations of glycerophosphoinositol 4-phosphate (GroPIns-4-P) caused a approximately 50% inhibition of the adenylylcyclase activity in FRTL5 membranes stimulated by the GTP-binding protein activator fluoroaluminate. Similar concentrations of GroPIns-4-P were detected in KiKi cells but not in the normal FRTL5 line. Micromolar GroPIns-4-P was found to be taken up by intact FRTL5 cells and to induce nearly 50% inhibition of the thyrotropin- and cholera toxin-induced increase in cAMP levels. Similar results were also observed in other cell lines (smooth muscle, pituitary cells, and pneumocytes). GroPIns-4-P inhibited cAMP-dependent cellular functions such as iodide uptake and thymidine incorporation in FRTL5 cells when stimulated by thyrotropin and cholera toxin but not when induced by forskolin. These results are consistent with GroPIns-4-P exerting an inhibitory effect on the GTP-binding protein that stimulates adenylycyclase. We propose that GroPIns-4-P might mediate a mechanism of cross-talk between adenylylcyclase and phospholipase A2 in thyroid as well as in other cell systems.

Phorbol ester effect on the hydrosmotic response to vasopressin in frog skin.

Serosal preincubation of frog skin with tetradecanoyl phorbol acetate, TPA, an activator of protein kinase C, inhibits the hydrosmotic response elicited by vasopressin (AVP) but not that induced by 8br-cAMP. This proves that serosal TPA primarily influences a pre-cAMP step. The TPA-induced inhibition of AVP response appears to be related to TPA-induced prostaglandin synthesis. The pretreatment with naproxen, in fact, prevents the inhibition induced by serosal TPA on the AVP response. On the contrary, mucosal TPA produces a more marked inhibition of the response to AVP and significantly diminishes the water flow induced by 8br-cAMP; this suggests that mucosal TPA interferes mainly with a post-cAMP step. Furthermore, naproxen is unable to completely prevent the inhibition induced by mucosal TPA on AVP response thus indicating that mucosal TPA may also activate a prostaglandin-independent mechanism able to inhibit one of the last steps of the hydrosmotic response to AVP.