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Background Perinatal inflammation is a key factor of brain vulnerability in neonates born preterm or with intra-uterine growth restriction (IUGR), two leading conditions associated with brain injury and responsible for neurocognitive and behavioral disorders. Systemic inflammation is recognized to activate microglia, known to be the critical modulators of brain vulnerability. Although some evidence supports a role for metabotropic glutamate receptor 3 (mGlu3 receptor) in modulation of neuroinflammation, its functions are still unknown in the developing microglia. Methods We used a double-hit rat model of perinatal brain injury induced by a gestational low-protein diet combined with interleukin-1β injections (LPD/IL-1β), mimicking both IUGR and prematurity-related inflammation. The effect of LPD/IL-1β on mGlu3 receptor expression and the effect of mGlu3 receptor modulation on microglial reactivity were investigated using a combination of pharmacological, histological, and molecular and genetic approaches. Results Exposure to LPD/IL-1β significantly downregulated Grm3 gene expression in the developing microglia. Both transcriptomic analyses and pharmacological modulation of mGlu3 receptor demonstrated its central role in the control of inflammation in resting and activated microglia. Microglia reactivity to inflammatory challenge induced by LPD/IL-1β exposure was reduced by an mGlu3 receptor agonist. Conversely, both specific pharmacological blockade, siRNA knock-down, and genetic knock-out of mGlu3 receptors mimicked the pro-inflammatory phenotype observed in microglial cells exposed to LPD/IL-1β. Conclusions Overall, these data show that Grm3 plays a central role in the regulation of microglial reactivity in the immature brain. Selective pharmacological activation of mGlu3 receptors may prevent inflammatory-induced perinatal brain injury.

The metabotropic glutamate receptor mGlu5 has been implicated in the neuropathology of several debilitating disorders. In schizophrenia, mGlu5 receptor hypofunction has been linked with neuropathology and cognitive deficits, making it an attractive therapeutic target. The cognitive impairment associated with schizophrenia remains an unmet clinical need, with existing antipsychotics primarily targeting positive symptoms and failing to induce consistent, substantial improvements in cognition, thus providing incomplete treatment. Using the sub-chronic phencyclidine (scPCP) rat model, widely shown to mimic the cognitive impairment and neuropathology of schizophrenia, we have investigated two mGlu5 receptor positive allosteric modulators (PAMs), VU0409551 and VU0360172. We compared the efficacy of these compounds in restoring cognitive deficits and, since these two PAMs have reportedly distinct signalling mechanisms, changes in mGlu5 receptor signalling molecules AKT and MAPK in the prefrontal cortex. Although not effective at 0.05 and 1mg/kg, cognitive deficits were significantly alleviated by both PAMs at 10 and 20 mg/kg. The compounds appeared to have differential effects on the scPCP-induced increases in AKT and MAPK phosphorylation: VU0409551 induced a significant decrease in expression of p-AKT, whereas VU0360172 had this effect on p-MAPK levels. Thus, the beneficial effects of PAMs on scPCP-induced cognitive impairment are accompanied by at least partial reversal of scPCP-induced elevated levels of p-MAPK and p-AKT, dysfunction of which is strongly implicated in schizophrenia pathology. These promising data imply an important role for mGlu5 receptor signalling pathways in improving cognition in the scPCP model and provide support for mGlu5 receptor PAMs as a possible therapeutic intervention for schizophrenia.

Contains fulltext : 235294.pdf (Publisher’s version ) (Closed access) Absence epilepsy is frequently associated with cognitive dysfunction, although the underlying mechanisms are not well understood. Here we report that some forms of hippocampal synaptic plasticity are abnormal in symptomatic Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. Metabotropic Glu 1/5 receptor-mediated long term depression (LTD) at Schaffer collateral CA1 synapses is significantly reduced in symptomatic, 5-6 months old WAG/Rij rats compared to age-matched non epileptic control rats. There were no significant changes in mGlu1/5-dependent LTD in pre-symptomatic, 4–6 weeks old WAG/Rij rats compared to age matched controls. The changes in LTD found in symptomatic WAG/Rij forms are not indicative of general deficits in all forms of synaptic plasticity as long term potentiation (LTP) was unchanged. Immunoblot analysis of hippocampal tissue showed a significant reduction in mGlu5 receptor expression, a trend to an increase in pan Homer protein levels and a decrease in GluA1 receptor expression in the hippocampus of symptomatic WAG/Rij rats vs non-epileptic control rats. There were no changes in mGlu1α receptor or GluA2 protein levels. These findings suggest that abnormalities in hippocampal mGlu5 receptor-dependent synaptic plasticity are associated with the pathological phenotype of WAG/Rij rats. This lays the groundwork for the study of mGlu5 receptors as a candidate drug target for the treatment of cognitive dysfunction linked to absence epilepsy. 12 p.

Cinnabarinic acid (CA) is a kynurenine metabolite that activates mGlu4 metabotropic glutamate receptors. Using a highly sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS-MS) method, we found that CA is present in trace amounts in human brain tissue. CA levels were largely reduced in the prefrontal cortex (PFC) of individuals affected by schizophrenia. This reduction did not correlate with age, sex, duration of the disease, and duration and type of antipsychotic medication and might, therefore, represent a trait of schizophrenia. Interestingly, systemic treatment with low doses of CA (

Background: Perinatal inflammation is a key factor of brain vulnerability in neonates born preterm or with intra-uterine growth restriction (IUGR), two leading conditions associated with brain injury and responsible for neurocognitive and behavioral disorders. Systemic inflammation is recognized to activate microglia, known to be the critical modulators of brain vulnerability. Although some evidence support a role for metabotropic glutamate receptor 3 (mGlu3 receptor) in modulation of neuroinflammation, its functions are still unknown in the developing microglia.Methods: We used a double-hit rat model of perinatal brain injury induced by a gestational low-protein diet combined with interleukin-1β injections (LPD/IL-1β), mimicking both IUGR and prematurity-related inflammation. The effect of LPD/IL-1β on mGlu3 receptor expression and the effect of mGlu3 receptor modulation on microglial reactivity were investigated using a combination of pharmacological, histological, and molecular approaches.Results: Exposure to LPD/IL-1β significantly down-regulated Grm3 gene expression in the developing microglia. Both transcriptomic analyses and pharmacological modulation of mGlu3 receptor demonstrated its central position in the control of inflammation in resting and activated microglia. Microglia hyper-reactivity to inflammatory challenge induced by LPD/IL-1β exposure was reduced by an mGlu3 receptor agonist. Conversely, both specific pharmacological blockade and siRNA knock-down of mGlu3 receptors in control microglia mimicked the pro-inflammatory phenotype observed in microglial cells exposed to LPD/IL-1β.Conclusions: Overall, these data show that Grm3 plays a central role in the regulation of microglial reactivity in the immature brain. Selective pharmacological activation of mGlu3 receptors may prevent inflammatory-induced perinatal brain injury.

We studied the interaction between mGlu7 and alpha(1)-adrenergic receptors in heterologous expression systems, brain slices, and living animals. L-2-Amino-4-phosphonobutanoate (L-AP4), and L-serine-O-phosphate (L-SOP), which activate group III mGlu receptors, restrained the stimulation of polyphosphoinositide (PI) hydrolysis induced by the alpha(1)-adrenergic receptor agonist, phenylephrine, in HEK 293 cells co-expressing alpha(1)-adrenergic and mGlu7 receptors. The inibitory action of L-AP4 was abrogated by (i) the mGlu7 receptor antagonist, XAP044; (ii) the C-terminal portion of type-2 G protein coupled receptor kinase; and (iii) the MAP kinase inhibitors, U0126 and PD98059. This suggests that the functional interaction between mGlu7 and alpha(1)-adrenergic receptors was mediated by the beta gamma-subunits of the G(i) protein and required the activation of the MAP kinase pathway. Remarkably, activation of neither mGlu2 nor mGlu4 receptors reduced alpha(1)-adrenergic receptor-mediated PI hydrolysis. In mouse cortical slices, both L-AP4 and L-SOP were able to attenuate norepinephrine- and phenylephrine-stimulated PI hydrolysis at concentrations consistent with the activation of mGlu7 receptors. L-AP4 failed to affect norepinephrine-stimulated PI hydrolysis in cortical slices from mGlu7(-/-) mice, but retained its inhibitory activity in slices from mGlu4(-/-) mice. At behavioural level, i.c.v. injection of phenylephrine produced antidepressant-like effects in the forced swim test. The action of phenylephrine was attenuated by L-SOP, which was inactive per se. Finally, both phenylephrine and L-SOP increased corticosterone levels in mice, but the increase was halved when the two drugs were administered in combination. Our data demonstrate that alpha(1)-adrenergic and mGlu7 receptors functionally interact and suggest that this interaction might be targeted in the treatment of stress-related disorders. (C) 2016 Elsevier Ltd. All rights reserved.

Epigenetic mechanisms can mediate gene-environment interactions relevant for complex disorders. The BDNF gene is crucial for development and brain plasticity, is sensitive to environmental stressors, such as hypoxia, and harbors the functional SNP rs6265 (Val(66)Met), which creates or abolishes a CpG dinucleotide for DNA methylation. We found that methylation at the BDNF rs6265 Val allele in peripheral blood of healthy subjects is associated with hypoxia-related early life events (hOCs) and intermediate phenotypes for schizophrenia in a distinctive manner, depending on rs6265 genotype: in ValVal individuals increased methylation is associated with exposure to hOCs and impaired working memory (WM) accuracy, while the opposite is true for ValMet subjects. Also, rs6265 methylation and hOCs interact in modulating WM-related prefrontal activity, another intermediate phenotype for schizophrenia, with an analogous opposite direction in the 2 genotypes. Consistently, rs6265 methylation has a different association with schizophrenia risk in ValVals and ValMets. The relationships of methylation with BDNF levels and of genotype with BHLHB2 binding likely contribute to these opposite effects of methylation. We conclude that BDNF rs6265 methylation interacts with genotype to bridge early environmental exposures to adult phenotypes, relevant for schizophrenia. The study of epigenetic changes in regions containing genetic variation relevant for human diseases may have beneficial implications for the understanding of how genes are actually translated into phenotypes.

In spite of the recent advancement in the molecular characterization of malignant gliomas and medulloblastomas, the treatment of primary brain tumors remains suboptimal. The use of small molecule inhibitors of intracellular signaling pathways, inhibitors of angiogenesis, and immunotherapic agents is limited by systemic adverse effects, limited brain penetration, and, in some cases, lack of efficacy. Thus, adjuvant chemo-therapy and radiotherapy still remain the gold standard in the treatment of grade-IV astrocytoma (glioblastoma multiforme) and medulloblastoma. We review evidence that supports the development of mGlu3 receptor antagonists as add-on drugs in the treatment of malignant gliomas. These drugs appear to display pleiotropic effect on tumor cells, affecting proliferation, differentiation, and response to chemotherapy. mGlu1 and mGlu4 receptors could also be targeted by potential anticancer agents in the treatment of malignant gliomas and medulloblastoma, but extensive research is required for target validation.

mGlu5 receptors are involved in mechanisms of activity-dependent synaptic plasticity, and are targeted by drugs developed for the treatment of CNS disorders. We report that mGlu3 receptors, which are traditionally linked to the control of neurotransmitter release, support mGlu5 receptor signaling in neurons and largely contribute to the robust mGlu5 receptor-mediated polyphosphoinositide hydrolysis in the early postnatal life. In cortical pyramidal neurons, mGlu3 receptor activation potentiated mGlu5 receptor-mediated somatic Ca2+ mobilization, and mGlu3 receptor-mediated long-term depression in the prefrontal cortex required the endogenous activation of mGlu5 receptors. The interaction between mGlu3 and mGlu5 receptors was also relevant to mechanisms of neuronal toxicity, with mGlu3 receptors shaping the influence of mGlu5 receptors on excitotoxic neuronal death. These findings shed new light into the complex role played by mGlu receptors in physiology and pathology, and suggest reconsideration of some of the current dogmas in the mGlu receptor field.

The purpose of the present article is to review our actual knowledge on the desensitization of metabotropic glutamate receptors based on the literature available so far, with the attempt to emphasize all converging data and to give a possible explanation to those evidences that still remain controversial. 1. We review our knowledge on the regulation of mGlu receptors based on the available literature 2. We report converging data and we comment on issues that still remain controversial. This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’.

Paradoxical sleep deprivation in rats is considered as an experimental animal model of mania endowed with face, construct, and pharmacological validity. We induced paradoxical sleep deprivation by placing rats onto a small platform surrounded by water. This procedure caused the animal to fall in the water at the onset of REM phase of sleep. Control rats were either placed onto a larger platform (which allowed them to sleep) or maintained in their home cage. Sleep deprived rats showed a substantial reduction in type-2 metabotropic glutamate (mGlu2) receptors mRNA and protein levels in the hippocampus, but not in the prefrontal cortex or corpus striatum, as compared to both groups of control rats. No changes in the expression of mGlu3 receptor mRNA levels or mGlu1α and mGlu5 receptor protein levels were found with exception of an increase in mGlu1α receptor levels in the striatum of SD rats. Moving from these findings we treated SD and control rats with the selective mGlu2 receptor enhancer, BINA (30mg/kg, i.p.). SD rats were also treated with sodium valproate (300mg/kg, i.p.) as an active comparator. Both BINA and sodium valproate were effective in reversing the manic-like phenotype evaluated in an open field arena in SD rats. BINA treatment had no effect on motor activity in control rats, suggesting that our findings were not biased by a non-specific motor-lowering activity of BINA. These findings suggest that changes in the expression of mGlu2 receptors may be associated with the enhanced motor activity observed with mania.

We studied the interaction between mGlu7 and α

We examined the regulation of mGlu2 and mGlu3 metabotropic glutamate receptor signaling prompted by the emerging role of these receptor subtypes as therapeutic targets for psychiatric disorders, such as anxiety and schizophrenia. In transfected human embryonic kidney 293 cells, G-protein-coupled receptor kinase (GRK) 2 and GRK3 fully desensitized the agonist-dependent inhibition of cAMP formation mediated by mGlu3 receptors. In contrast, GRK2 or other GRKs did not desensitize the cAMP response to mGlu2 receptor activation. Desensitization of mGlu3 receptors by GRK2 required an intact kinase activity, as shown by the use of the kinase-dead mutant GRK2-K220R or the recombinant GRK2 C-terminal domain. Overexpression of beta-arrestin1 also desensitized mGlu3 receptors and did not affect the cAMP signaling mediated by mGlu2 receptors. The difference in the regulation of mGlu2 and mGlu3 receptors was signal-dependent because GRK2 desensitized the activation of the mitogen-activated protein kinase pathway mediated by both mGlu2 and mGlu3 receptors. In vivo studies confirmed the resistance of mGlu2 receptor-mediated cAMP signaling to homologous desensitization. Wild-type, mGlu2(-/-), or mGlu3(-/-) mice were treated intraperitoneally with saline or the mixed mGlu2/3 receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]-exhane-4,6-dicarboxylic acid (LY379268; 1 mg/kg) once daily for 7 days. Inhibition of forskolin-stimulated cAMP formation by LY379268 was measured in cortical slices prepared 24 h after the last injection. Agonist pretreatment fully desensitized the cAMP response in wild-type and mGlu2(-/-) mice but had no effect in mGlu3(-/-) mice, in which LY379268 could only activate the mGlu2 receptor. We predict the lack of tolerance when mixed mGlu2/3 receptor agonists or selective mGlu2 enhancers are used continually in patients.

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.

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.

WNT factors represent key mediators of many processes in animal development and homeostasis and act through a receptor complex comprised of members of the Frizzled and low density lipoprotein-related receptors (LRP). In mammals, 19 genes encoding Wingless and Int-related factor (WNTs), 10 encoding Frizzled, and 2 encoding LRP proteins have been identified, but little is known of the identities of individual Frizzled-LRP combinations mediating the effects of specific WNT factors. Additionally, several secreted modulators of WNT signaling have been identified, including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate central nervous system capable of modulating aspects of neuronal plasticity. Gene knock-out models in the mouse have revealed that WNT7A plays a role in cerebellar maturation, although its function in the development of distal limb structures and of the reproductive tract have been more intensely studied. To identify a receptor complex for this WNT family member, we have analyzed the response of the rat pheochromocytoma cell line PC12 to WNT7A. We find that PC12 cells are capable of responding to WNT7A as measured by increased beta-catenin stability and activation of a T-cell factor-based luciferase reporter construct and that these cells express three members of the Frizzled family (Frizzled-2, -5, and -7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5.LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3.

Activation regulates the responsiveness of G-protein-coupled receptors (GPCRs) on T cells, and modifications in the activity of GPCRs characterize lymphocytes from some immune disorders such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Some lines of evidence suggest that such an effect is connected with the altered expression of some GPCRs regulatory proteins. Herein we demonstrate that phitoemagglutinin (PHA)-induced activation leads to differential expression of G-protein-coupled receptor kinase (GRK) 2, GRK3, beta-arrestin-1, regulators of G-protein signalling (RGS) 2, and RGS16 and decreases responsiveness of mononuclear leukocytes (MNL) to the beta-adrenergic agonist isoproterenol. Interferon beta-1a (IFN beta-1a), which is known to ameliorate the course of MS, counteracts the activation-induced effects on the expression of these GPCR regulatory proteins in MNL. Furthermore, IFN beta-1a quenches the effects of PHA on the isoproterenol-induced accumulation of cyclic AMP (cAMP). We suggest that regulation of GPCRs responsiveness may be a relevant property of IFN beta-1a in MS.

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.

The membrane-associated Intercellular Adhesion Molecule 1 (mICAM 1) is fundamental for adhesion of leukocytes to endothelial cells. A soluble form of ICAM 1 (sICAM 1) exists in the human serum, and is seen as marker of disease activity in patients suffering from Multiple Sclerosis (MS). High levels of sICAM 1 have been detected in MS patients benefiting from interferon beta (IFNbeta) treatment, but little is known on the molecular origins of sICAM 1. This study investigated the interrelationship and the mechanisms of production of sICAM 1 and mICAM 1 in human endothelium (Human Umbilical Vein Endothelial Cells, HUVECs) and mononuclear leukocytes (MNL) upon stimulation with IFNbeta-1a and other inducers. We found that the expression of mICAM 1 and the release of sICAM 1 are differentially regulated in both these cytotypes. HUVECs and MNL express specific mRNA for both mICAM 1 and sICAM 1, and modification of the content of each of these transcripts results in regulation of both the ICAM 1 isoforms. We show that IFNbeta-1a is strong regulator of the ICAM 1 RNA splicing machinery. Effect of IFNbeta-1a over expression of the ICAM 1 isoforms might have a relevant immunomoregulatory role in Multiple Sclerosis.

This chapter describes the protocol for the preparation of recombinant adenoviruses and infection of target cells to transiently express G protein-coupled receptors (GPCRs) or other proteins of interest. Adenoviruses are non-enveloped viruses containing a linear double-stranded DNA genome. Their life cycle does not normally involve integration into the host genome, rather they replicate as episomal elements in the nucleus of the host cell, and consequently there is no risk of insertional mutagenesis. Up to 30 kb out of the 35 kb of the wild-type adenovirus genome can be replaced by foreign DNA. Adenoviral vectors are very efficient in transducing target cells in vitro and in vivo and can be produced at high titers (>1011/ml). The viral infection has a number of useful features: (1) the efficiency of gene transduction is very high (up to 100 % in sensitive cells); (2) the infection is easy and does not physically alter the cell membrane for gene transduction; (3) it is possible to infect cells that are resistant to transfection with plasmids (including nondividing cells); and (4) the viral vectors can be used for infection in vivo (including gene therapy) and can potentially be targeted to specific cells or tissues.

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.

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.

mGlu7 receptors are coupled to Gi/Go-proteins and activate multiple transduction pathways, including inhibition of adenylyl cyclase activity and stimulation of ERK1/2 and JNK pathways. mGlu7 receptors play an important role in cognition and emotion and are involved in stress-related disorders such as anxiety and depression and in susceptibility to convulsive seizures. In spite of these potential clinical implications, little is known on the mechanisms that regulate mGlu7-receptor signaling. Here we show that mGlu7 receptor-dependent signaling pathways were regulated in a complementary manner by different GRK subtypes, with GRK4 affecting the adenylyl cyclase and the JNK pathways, and GRK2 selectively affecting the ERK1/2 pathway. Additionally we found that the two isoforms of non-visual arrestins, i.e. β-arrestin1 and β-arrestin2, exerted opposite effects on mGlu7-receptor signaling, with β-arrestin1 positively modulating ERK1/2 and inhibiting JNK, and β-arrestin2 doing the opposite. This represents a remarkable example of "reciprocal regulation" of receptor signaling by the two isoforms of β-arrestin. Finally we found that β-arrestin1 amplified mGlu7 receptor-dependent ERK1/2 activation in response to L-AP4 (an orthosteric agonist), but not in response to AMN082 (an atypical mGlu7-receptor allosteric agonist). The different effect of β-arrestin1 on L-AP4- and AMN082-stimulated ERK1/2 phosphorylation is in line with the emerging concept of β-arrestin-biased agonists. The present study may open new perspectives in elucidating the physio-pathological roles of the mGlu7 receptor and may provide new insights for the possibility to develop specific (biased) agonists that can selectively activate different signaling pathways.

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.

This chapter describes the protocol for the preparation of recombinant adenoviruses and infection of target cells to transiently express G protein-coupled receptors (GPCRs) or other proteins of interest. Adenoviruses are non-enveloped viruses containing a linear double-stranded DNA genome. Their life cycle does not normally involve integration into the host genome, rather they replicate as episomal -elements in the nucleus of the host cell, and consequently there is no risk of insertional mutagenesis. Up to 30 kb out of the 35 kb of the wild-type adenovirus genome can be replaced by foreign DNA. Adenoviral vectors are very efficient in transducing target cells in vitro and in vivo and can be produced at high titers (10¹¹/mL). The viral infection has a number of useful features: (1) the efficiency of gene transduction is very high (up to 100% in sensitive cells); (2) the infection is easy and does not physically alter the cell membrane for gene transduction; (3) it is possible to infect cells that are resistant to transfection with plasmids (including nondividing cells); and (4) the viral vectors can be used for infection in vivo (including gene therapy) and can potentially be targeted cell-specifically.

DNA methylation at CpG dinucleotides is associated with gene silencing, stress, and memory. The catechol-O-methyltransferase (COMT) Val158allele in rs4680 is associated with differential enzyme activity, stress responsivity, and prefrontal activity during working memory (WM), and it creates a CpG dinucleotide. We report that methylation of the Val158allele measured from peripheral blood mononuclear cells (PBMCs) of Val/Val humans is associated negatively with lifetime stress and positively with WM performance; it interacts with stress to modulate prefrontal activity during WM, such that greater stress and lower methylation are related to reduced cortical efficiency; and it is inversely related to mRNA expression and protein levels, potentially explaining thein vivoeffects. Finally, methylation ofCOMTin prefrontal cortex and that in PBMCs of rats are correlated. The relationship of methylation of theCOMTVal158allele with stress, gene expression, WM performance, and related brain activity suggests that stress-related methylation is associated with silencing of the gene, which partially compensates the physiological role of the high-activity Val allele in prefrontal cognition and activity. Moreover, these results demonstrate how stress-related DNA methylation of specific functional alleles impacts directly on human brain physiology beyond sequence variation.

This chapter describes the protocol for the preparation of recombinant adenoviruses and infection of target cells to transiently express G protein-coupled receptors (GPCRs) or other proteins of interest. Adenoviruses are non-enveloped viruses containing a linear double-stranded DNA genome. Their life cycle does not normally involve integration into the host genome, rather they replicate as episomal -elements in the nucleus of the host cell, and consequently there is no risk of insertional mutagenesis. Up to 30 kb out of the 35 kb of the wild-type adenovirus genome can be replaced by foreign DNA. Adenoviral vectors are very efficient in transducing target cells in vitro and in vivo and can be produced at high titers (>10¹¹/mL). The viral infection has a number of useful features: (1) the efficiency of gene transduction is very high (up to 100% in sensitive cells); (2) the infection is easy and does not physically alter the cell membrane for gene transduction; (3) it is possible to infect cells that are resistant to transfection with plasmids (including nondividing cells); and (4) the viral vectors can be used for infection in vivo (including gene therapy) and can potentially be targeted cell-specifically.

The D2/AKT1/GSK-3β signaling pathway has been involved in the downstream intracellular effects of dopamine, in the pathophysiology of cognitive deficits and related brain activity in schizophrenia, as well as in response to treatment with antipsychotics. Polymorphisms in the D2 ( DRD2 rs1076560) and AKT1 ( AKT1 rs1130233) genes have been associated with their respective protein expression and with higher-order cognition and brain function, including attention. Given the strong potential for their relationship, we investigated the interaction of these polymorphisms on multiple molecular and in vivo phenotypes associated with this signaling pathway. We measured AKT1 and GSK-3β proteins and phosphorylation in human peripheral blood mononuclear cells, functional MRI cingulate response during attentional control, behavioral accuracy during sustained attention, and response to 8 wk of treatment with olanzapine in a total of 190 healthy subjects and 66 patients with schizophrenia. In healthy subjects, we found that the interaction between the T allele of DRD2 rs1076560 and the A allele of AKT1 rs1130233 was associated with reduced AKT1 protein levels and reduced phosphorylation of GSK-3β, as well as with altered cingulate response and reduced behavioral accuracy during attentional processing. On the other hand, interaction of these two alleles was associated with greater improvement of Positive and Negative Syndrome Scale scores in patients with schizophrenia after treatment with olanzapine. The present results indicate that these functional polymorphisms are epistatically associated with multiple phenotypes of relevance to schizophrenia. Our results also lend support to further investigation of this downstream molecular pathway in the etiology and treatment of this disorder.

Exposure of PC12 cells to metamphetamine (MA) induces the formation of multilamellar structures (whorls) resembling autophagic granules that subsequently develop as intracellular inclusions. These inclusions stain for a variety of antigens belonging to the ubiquitin proteasome pathway. Since MA-induced intracellular bodies require the presence of dopamine in the present study we analyzed the role of dopamine (DA) receptors in producing neuronal inclusions. Moreover, we investigated potential signaling pathways which could lead to ubiquitination in the presence of MA. Based on recent reports that ubiquitination of beta-adrenergic receptors is promoted by beta-arrestin which shuttles proteins from the plasma membrane to the ubiquitin proteasome system we investigated whether beta-arrestin is involved in MA-induced inclusion formation. Our experiments document that (i) beta-arrestin was associated with MA-induced intracellular bodies; (ii) MA induced a rapid and reversible ubiquitination of beta-arrestin; (iii) dopamine antagonists reduced both MA-induced beta-arrestin ubiquitination and intracellular whorls formation; (iv) the number of MA-induced intracellular bodies was reduced in cells transfected with the beta-arrestin dominant negative mutant, betaarrV53D and was increased by the persistently ubiquitinated beta-arrestin-ubiquitin fusion protein. In conclusion, the present study demonstrates the involvement of beta-arrestin in MA-induced intracellular bodies and the participation of dopamine receptors in this process.

Cancer stem cells are currently a target for the treatment of malignant tumors. Transformed neural stem–progenitor cells of the brain subventricular zone and the external granular layer of the cerebellum are the putative cells of origin of malignant gliomas and medulloblastomas, which are the most frequent malignant brain tumors in adults and children, respectively. The proliferation of neural stem–progenitor cells is regulated by metabotropic glutamate (mGlu) receptors, which are G-protein-coupled receptors that are activated by glutamate, the major excitatory neurotransmitter of the CNS. At least two receptor subtypes – mGlu 3 and mGlu 4 receptors – control the proliferation of brain tumor cells, whereas mGlu 1 receptors have been implicated in the development of melanomas. We believe that individual mGlu receptor subtypes represent new potential targets for the treatment of several malignant tumors, including brain tumors.

Group-II metabotropic glutamate receptor ligands as adjunctive drugs in the treatment of depression: a new strategy to shorten the latency of antidepressant medication?

Metabotropic glutamate (mGlu) receptors are G-protein coupled receptors activated by glutamate, the major excitatory neurotransmitter of the CNS. A growing body of evidence suggests that the function of mGlu receptors is not restricted to the regulation of synaptic transmission. mGlu receptors are expressed in a variety of peripheral cells, including inter alia hepatocytes, pancreatic cells, osteoblasts and immune cells. Within the immunological synapses, mGlu receptors expressed by T cells might contribute to the vast array of signals generated by the antigen-presenting cells. mGlu receptors are also found in embryonic and neural stem cells. This suggests their involvement in the pathophysiology of brain tumors, which likely originates from cancer stem cells similar to neural stem cells. Ligands of mGlu3 and mGlu4 receptors are potential candidates for the experimental treatment of malignant gliomas and medulloblastomas, respectively.

Amphetamine derivatives are the most commonly abused drugs. These compounds have been known for many years to induce neurotoxicity. However, recent findings have highlighted novel alterations produced by amphetamines in the central nervous system consisting of neuronal inclusions and the involvement of proteins belonging to a multi-enzymatic complex known as the ubiquitin-proteasome system. These ultrastructural and molecular changes are similar to those that occur during degenerative processes that affect the basal ganglia, and in particular Parkinson's disease, which is characterized by ubiquitin-containing neuronal inclusions in the subtantia nigra. This is recently confirmed by the occurrence of ubiquitin immunoreactive structures in the substantia nigra of humans abusing methamphetamines. In this article, we propose that the neurotoxicity of amphetamines and degenerative disorders share a number of steps in their mechanism of action involving the ubiquitin-proteasome system. The fine tuning of this ubiquitous proteolytic pathway is now being elucidated because G-protein-coupled receptors and signaling proteins such as beta-arrestin regulate access to this catalytic machinery. The identification of the ubiquitin-proteasome pathway and beta-arrestin as molecular targets of neurotoxicity is expected to provide novel therapeutic strategies both for the treatment of drug addiction and the treatment of neurodegenerative disorders.

We examined the effect of the three human isoforms of apolipoprotein E (ApoE2, ApoE3, and ApoE4) on the canonical Wnt signaling pathway in undifferentiated PC12 cells. Addition of recombinant ApoE4 reduced Wingless-Int7a-stimulated gene expression at concentrations of 80 and 500 nm. Recombinant ApoE2 and ApoE3 were virtually inactive. Recombinant ApoE4 also inhibited Wnt signaling when combined with very low density lipoproteins (VLDLs) or in cells over-expressing the low density lipoprotein receptor-related protein, LRP6. In contrast, the enforced expression of LRP5 unmasked an inhibition by ApoE2 and ApoE3, which, however, were less effective than ApoE4 in inhibiting Wnt signaling. We also transfected PC12 cells with constructs encoding for the three human ApoE isoforms to examine whether endogenously expressed ApoE isoforms could modulate the Wnt pathway. Under these conditions, all three ApoE isoforms were able to inhibit Wnt signaling, although ApoE4 showed the greatest efficacy. Only the conditioned medium collected from cultures transfected with ApoE4 induced a significant inhibition of Wnt7a-stimulated gene expression, confirming that ApoE4 has an extracellular action that is not shared by the other ApoE isoforms. We conclude that ApoE4 behaves as an inhibitor of the canonical Wnt pathway in a context-independent manner.

The use of neural progenitor cells (NPCs) is limited by the incomplete knowledge of the extracellular signals regulating their proliferation and survival. We report that cultured mouse NPCs express functional mGlu3 and mGlu5 metabotropic glutamate receptors. Pharmacological blockade of both receptors reduced NPC proliferation and survival, whereas activation of mGlu5 receptors substantially enhanced cell proliferation. Adult mice lacking mGlu5 receptors or treated with mGlu5 or mGlu3 receptor antagonists showed a dramatic reduction in the number of dividing neuroprogenitors present in the subventricular zone and in the dentate gyrus of the hippocampus. These data disclose a novel function of mGlu receptors and offer new potential strategies for the optimization of cell replacement therapy in neurodegenerative disorders.

G-protein-coupled receptor kinases (GRK) contain a regulator of G-protein signaling (RGS)-like domain located at the N terminus (GRK-Nter) of their sequence. This domain is present in all the GRK subtypes, but the RGS-like domain of GRK2 was documented to be functionally active, as it is able to interact selectively with Galphaq (both in vitro and in cells) and to inhibit Galphaq-dependent signaling. In contrast GRK4, GRK5, and GRK6 are unable to interact with Galphaq. This article describes the methodology to investigate the modulatory activity of GRK2 and GRK4 on GPCR-stimulated Galphaq signaling. This analysis is essentially based on three types of experiments: (a) study of the effect of the GRK-Nter on GPCR-dependent signaling; (b) analysis of the binding of GRK-Nter to Galphaq in vitro; and (c) analysis of the interaction of GRK with Galphaq in cells.

Exposure of immature rat cerebellar granule cell cultures to the type 4 metabotropic glutamate (mGlu4) receptor enhancerN-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 μl 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.

Endothelial cells from rat brain microvessels, human aortic artery and human umbilical vein were examined, together with ex vivo rat brain capillaries and rat aortic ring sections, for the expression of opioid receptor-like OP-4 mRNA and protein. High levels of mRNA expression and an immunopositive reaction for the receptor protein were detected in the endothelial cells from primary and from established in vitro cultures, as well as in the intima of ex vivo rat aortic rings, where the signal was limited to the endothelial layer. Interaction of the OP4 receptor with its physiological ligand nociceptin caused, in cultured endothelial cells, the activation of a mitogen-activated protein (MAP) kinase cascade. Taken together, these results show that the OP4 receptor is synthesised and functionally expressed in endothelial cells, presumably as a starting point for some vasoactive mechanism(s).

The metabotropic glutamate 1 (mGlu(1)) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu(1) receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu(1) receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Galpha(q). In cells transfected with GRK4 and exposed to agonist, beta-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu(1) receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of beta-arrestin V53D dominant negative mutant, which did not affect the mGlu(1) receptor internalization, reduced by 70-80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu(1) receptor. The agonist-stimulated differential sorting of the mGlu(1) receptor and beta-arrestin as well as the activation of MAP kinases by mGlu(1) agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of beta-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing beta-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu(1) receptor by different mechanisms and that beta-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.

WNT factors represent key mediators of many processes in animal development and homeostasis and act through a receptor complex comprised of members of the Frizzled and low density lipoprotein-related receptors (LRP). In mammals, 19 genes encoding Wingless and Int-related factor (WNTs), 10 encoding Frizzled, and 2 encoding LRP proteins have been identified, but little is known of the identities of individual Frizzled-LRP combinations mediating the effects of specific WNT factors. Additionally, several secreted modulators of WNT signaling have been identified, including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate central nervous system capable of modulating aspects of neuronal plasticity. Gene knock-out models in the mouse have revealed that WNT7A plays a role in cerebellar maturation, although its function in the development of distal limb structures and of the reproductive tract have been more intensely studied. To identify a receptor complex for this WNT family member, we have analyzed the response of the rat pheochromocytoma cell line PC12 to WNT7A. We find that PC12 cells are capable of responding to WNT7A as measured by increased beta-catenin stability and activation of a T-cell factor-based luciferase reporter construct and that these cells express three members of the Frizzled family (Frizzled-2, -5, and -7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5.LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3.

Beta-amyloid protein (betaAP) is thought to cause neuronal loss in Alzheimer's disease (AD). Applied to neurons in culture, betaAP induces neuronal death and hyperphosphorylation of tau protein, which forms neurofibrillary tangles (NFTs) in AD brains. Neurons also undergo rapid apoptotic death following reactivation of a mitotic cycle. However, the molecular events that determine the fate of neurons challenged with betaAP (apoptotic death, formation of NFTs and survival) are unclear. We discuss a scenario for the pathogenesis of AD. This links betaAP-induced changes to the Wnt signaling pathway that promotes proliferation of progenitor cells and directs cells into a neuronal phenotype during brain development. We propose that betaAP-mediated facilitation of mitogenic Wnt signaling activates unscheduled mitosis in differentiated neurons. Furthermore, late downregulation of Wnt signaling by betaAP might lead to NFT formation. We propose that drugs that both inhibit the cell cycle and rescue Wnt activity could provide novel AD therapeutics.

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.

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 (IC50=40–50 nM). A direct interaction between GRK5 and Ca2+/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 Ca2+. 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.