Lateral allosterism in the glucagon receptor family: glucagon-like peptide 1 induces G-protein-coupled receptor heteromer formation.

Activation of G-protein-coupled receptors (GPCRs) results in a variety of cellular responses, such as binding to the same receptor of different ligands that activate distinct downstream cascades. Additional signaling complexity is achieved when two or more receptors are integrated into one signaling unit. Lateral receptor interactions can allosterically modulate the receptor response to a ligand, which creates a mechanism for tissue-specific fine tuning, depending on the cellular receptor coexpression pattern. GPCR homomers or heteromers have been explored widely for GPCR classes A and C but to lesser extent for class B. In the present study, we used bioluminescence resonance energy transfer (BRET) techniques, calcium flux measurements, and microscopy to study receptor interactions within the glucagon receptor family. We found basal BRET interactions for some of the receptor combinations tested that decreased upon ligand binding. A BRET increase was observed exclusively for the gastric inhibitory peptide (GIP) receptor and the glucagon-like peptide 1 (GLP-1) receptor upon binding of GLP-1 that could be reversed with GIP addition. The interactions of GLP-1 receptor and GIP receptor were characterized with BRET donor saturation studies, shift experiments, and tests of glucagon-like ligands. The heteromer displayed specific pharmacological characteristics with respect to GLP-1-induced ß-arrestin recruitment and calcium flux, which suggests a form of allosteric regulation between the receptors. This study provides the first example of ligand-induced heteromer formation in GPCR class B. In the body, the receptors are functionally related and coexpressed in the same cells. The physiological evidence for this heteromerization remains to be determined.

Identification of insulin-sensitizing molecules acting by disrupting the interaction between the Insulin Receptor and Grb14.

Metabolic diseases are characterized by a decreased action of insulin. During the course of the disease, usual treatments frequently fail and patients are finally submitted to insulinotherapy. There is thus a need for innovative therapeutic strategies to improve insulin action. Growth factor receptor-bound protein 14 (Grb14) is a molecular adapter that specifically binds to the activated insulin receptor (IR) and inhibits its tyrosine kinase activity. Molecules disrupting Grb14-IR binding are therefore potential insulin-sensitizing agents. We used Structure-Based Virtual Ligand Screening to generate a list of 1000 molecules predicted to hinder Grb14-IR binding. Using an acellular bioluminescence resonance energy transfer (BRET) assay, we identified, out of these 1000 molecules, 3 compounds that inhibited Grb14-IR interaction. Their inhibitory effect on insulin-induced Grb14-IR interaction was confirmed in co-immunoprecipitation experiments. The more efficient molecule (C8) was further characterized. C8 increased downstream Ras-Raf and PI3-kinase insulin signaling, as shown by BRET experiments in living cells. Moreover, C8 regulated the expression of insulin target genes in mouse primary hepatocytes. These results indicate that C8, by reducing Grb14-IR interaction, increases insulin signalling. The use of C8 as a lead compound should allow for the development of new molecules of potential therapeutic interest for the treatment of diabetes.

Parkinson's disease: from causes to mechanisms.

Parkinson's disease (PD) is a common age-related, progressive neurodegenerative disease of unknown etiology. Environmental factors have long been suspected to participate in the pathogenesis of PD due to the existence of neurotoxins that preferentially damage the dopaminergic nigrostriatal pathway. In the past few years, novel insights into the degenerative process have been provided by the discovery of genes responsible for rare monogenic parkinsonian syndromes. Compelling evidence is accumulating, suggesting that the products of several of these genes can interact with environmental toxins and intervene in molecular pathways controlling the functional integrity of mitochondria.

O-GlcNAcylation-inducing treatments inhibit estrogen receptor α expression and confer resistance to 4-OH-tamoxifen in human breast cancer-derived MCF-7 cells.

O-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine residues) is a post-translational modification that regulates stability, activity or localization of cytosolic and nuclear proteins. O-linked N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc from proteins is catalyzed by the ß-N-Acetylglucosaminidase (OGA). Recent evidences suggest that O-GlcNAcylation may affect the growth of cancer cells. However, the consequences of O-GlcNAcylation on anti-cancer therapy have not been evaluated. In this work, we studied the effects of O-GlcNAcylation on tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells. Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT expression by siRNA potentiated the effect of tamoxifen on cell death. Since the PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to evaluate the effect of PUGNAc+glucosamine on PIP3 production. We observed that these treatments stimulated PIP3 production in MCF-7 cells. This effect was associated with an increase in Akt phosphorylation. However, the PI-3 kinase inhibitor LY294002, which abolished the effect of PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects of PUGNAc+glucosamine against tamoxifen-induced cell death. These results suggest that the protective effects of O-GlcNAcylation are independent of the PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine on the expression of this receptor. We observed that O-GlcNAcylation-inducing treatment significantly reduced the expression of ERα mRNA and protein, suggesting a potential mechanism for the decreased tamoxifen sensitivity induced by these treatments. Therefore, our results suggest that inhibition of O-GlcNAcylation may constitute an interesting approach to improve the sensitivity of breast cancer to anti-estrogen therapy.

Effect of insulin analogues on insulin/IGF1 hybrid receptors: increased activation by glargine but not by its metabolites M1 and M2.

BACKGROUND: In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R), present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids. METHODOLOGY: To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET) assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP(3)) production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP(3) production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells. RESULTS: Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP(3) production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP(3) production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin. CONCLUSION: Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in MCF-7 cells.

Insulin receptor substrates-5 and -6 are poor substrates for the insulin receptor.

Insulin receptor substrates (IRS)-5 and -6 are two recently identified members of the IRS family. We investigated their roles as insulin receptor substrates and compared them with Src-homology-2-containing (Shc) protein, a well-established substrate. Bioluminescence resonance energy transfer (BRET) experiments showed no interaction between the receptor and IRS-5, while interaction with IRS-6 was not enhanced by insulin. By contrast, Shc showed an insulin-induced BRET response, as did a truncated form of IRS-1 (1-262). While Shc was heavily phosphorylated after stimulation of the insulin receptor, IRS-5 and -6 showed very weak phosphorylation levels. These results suggest that, although these two adaptors have previously been proposed as substrates for the insulin receptor, they are poor substrates for the insulin receptor. This calls into question their relevance to insulin signalling.

Molecular determinants of Grb14-mediated inhibition of insulin signaling.

Grb14 belongs to the Grb7 family of molecular adapters and was identified as an inhibitor of insulin signaling. Grb14 binds to activated insulin receptors (IR) and inhibits their catalytic activity. To gain more insight into the Grb14 molecular mechanism of action, we generated various mutants and studied the Grb14-IR interaction using coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments. Biological activity was further analyzed using the Xenopus oocyte model and a functional complementation assay measuring cellular proliferation rate in Grb14 knockout mouse embryonic fibroblasts. These studies identified two important interaction sites, Grb14 L404-IR L1038 and Grb14 R385-IR K1168, involving the IR alphaC-helix and activation loop, respectively. Interestingly, the former involves residues that are likely to be crucial for the specificity of IR binding with regard to other members of the Grb7 family. In addition, mutation of the Grb14-S370 residue suggested that its phosphorylation status controlled the biological activity of the protein. We further demonstrated that insulin-induced Grb14-PDK1 interaction is required in addition to Grb14-IR binding to mediate maximal inhibition of insulin signaling. This study provides important insights into the molecular determinants of Grb14 action by demonstrating that Grb14 regulates insulin action at two levels, through IR binding and by interfering with downstream pathways. Indeed, a precise knowledge of the molecular mechanism of insulin signaling inhibition by Grb14 is a prerequisite for the development of insulin-sensitizing molecules to treat pathophysiological states such as obesity or type 2 diabetes.

Biochemical analysis of Parkinson's disease-causing variants of Parkin, an E3 ubiquitin-protein ligase with monoubiquitylation capacity.

Mutations in the parkin gene, encoding an E3 ubiquitin-protein ligase, are a frequent cause of autosomal recessive parkinsonism and are also involved in sporadic Parkinson's disease. Loss of Parkin function is thought to compromise the polyubiquitylation and proteasomal degradation of specific substrates, leading to their deleterious accumulation. Several studies have analyzed the effects of parkin gene mutations on the biochemical properties of the protein. However, the absence of a cell-free system for studying intrinsic Parkin activity has limited the interpretation of these studies. Here we describe the biochemical characterization of Parkin and 10 pathogenic variants carrying amino-acid substitutions throughout the sequence. Mutations in the RING fingers or the ubiquitin-like domain decreased the solubility of the protein in detergent and increased its tendency to form visible aggregates. None of the mutations studied compromised the binding of Parkin to a series of known protein partners/substrates. Moreover, only two variants with substitutions of conserved cysteine residues of the second RING finger were inactive in a purely in vitro ubiquitylation assay, demonstrating that loss of ligase activity is a minor pathogenic mechanism. Interestingly, in this in vitro assay, Parkin catalyzed the linkage of single ubiquitin molecules only, whereas the ubiquitin-protein ligases CHIP and Mdm2 promoted the formation of polyubiquitin chains. Similarly, in mammalian cells Parkin promoted the multimonoubiquitylation of its substrate p38, rather than its polyubiquitylation. Thus, Parkin may mediate polyubiquitylation or proteasome-independent monoubiquitylation depending on the protein context. The discovery of monoubiquitylated Parkin species in cells hints at a novel post-translational modification potentially involved in the regulation of Parkin function.

Coordinate control and selective expression of the full complement of replication-dependent histone H4 genes in normal and cancer cells.

The replication of eukaryotic genomes necessitates the coordination of histone biosynthesis with DNA replication at the onset of S phase. The multiple histone H4 genes encode identical proteins, but their regulatory sequences differ. The contributions of these individual genes to histone H4 mRNA expression have not been described. We have determined, by real-time quantitative PCR and RNase protection, that the human histone H4 genes are not equally expressed and that a subset contributes disproportionately to the total pool of H4 mRNA. Differences in histone H4 gene expression can be attributed to observed unequal activities of the H4 gene promoters, which exhibit variations in gene regulatory elements. The overall expression pattern of the histone H4 gene complement is similar in normal and cancer cells. However, H4 genes that are moderately expressed in normal cells are sporadically silenced in tumor cells with compensation of expression by other H4 gene copies. Chromatin immunoprecipitation analyses and in vitro DNA binding assays indicated that 11 of the 15 histone H4 genes interact with the cell cycle regulatory histone nuclear factor P, which forms a complex with the cyclin E/CDK2-responsive co-regulator p220(NPAT). These 11 H4 genes account for 95% of the histone H4 mRNA pool. We conclude that the cyclin E/CDK2/p220(NPAT)/histone nuclear factor P signaling pathway is the principal regulator of histone H4 biosynthesis.

Parkin prevents mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death.

Parkin gene mutations have been implicated in autosomal-recessive early-onset parkinsonism and lead to specific degeneration of dopaminergic neurons in midbrain. To investigate the role of Parkin in neuronal cell death, we overproduced this protein in PC12 cells in an inducible manner. In this cell line, neuronally differentiated by nerve growth factor, Parkin overproduction protected against cell death mediated by ceramide, but not by a variety of other cell death inducers (H(2)O(2), 4-hydroxynonenal, rotenone, 6-OHDA, tunicamycin, 2-mercaptoethanol and staurosporine). Protection was abrogated by the proteasome inhibitor epoxomicin and disease-causing variants, indicating that it was mediated by the E3 ubiquitin ligase activity of Parkin. Interestingly, Parkin acted by delaying mitochondrial swelling and subsequent cytochrome c release and caspase-3 activation observed in ceramide-mediated cell death. Subcellular fractionation demonstrated enrichment of Parkin in the mitochondrial fraction and its association with the outer mitochondrial membrane. Together, these results suggest that Parkin may promote the degradation of substrates localized in mitochondria and involved in the late mitochondrial phase of ceramide-mediated cell death. Loss of this function may underlie the degeneration of nigral dopaminergic neurons in patients with Parkin mutations.

The p38 subunit of the aminoacyl-tRNA synthetase complex is a Parkin substrate: linking protein biosynthesis and neurodegeneration.

Parkinson's disease (PD) is a severe neurological disorder, characterized by the progressive degeneration of the dopaminergic nigrostriatal pathway and the presence of Lewy bodies (LBs). The discovery of genes responsible for familial forms of the disease has provided insights into its pathogenesis. Mutations in the parkin gene, which encodes an E3 ubiquitin-protein ligase involved in the ubiquitylation and proteasomal degradation of specific protein substrates, have been found in nearly 50% of patients with autosomal-recessive early-onset parkinsonism. The abnormal accumulation of substrates due to loss of Parkin function may be the cause of neurodegeneration in parkin-related parkinsonism. Here, we demonstrate that Parkin interacts with, ubiquitylates and promotes the degradation of p38, a key structural component of the mammalian aminoacyl-tRNA synthetase complex. We found that the ubiquitylation of p38 is abrogated by truncated variants of Parkin lacking essential functional domains, but not by the pathogenic Lys161Asn point mutant. Expression of p38 in COS7 cells resulted in the formation of aggresome-like inclusions in which Parkin was systematically sequestered. In the human dopaminergic neuroblastoma-derived SH-SY5Y cell line, Parkin promoted the formation of ubiquitylated p38-positive inclusions. Moreover, the overexpression of p38 in SH-SY5Y cells caused significant cell death against which Parkin provided protection. Analysis of p38 expression in the human adult midbrain revealed strong immunoreactivity in normal dopaminergic neurons and the labeling of LBs in idiopathic PD. This suggests that p38 plays a role in the pathogenesis of PD, opening the way for a detailed examination of its potential non-canonical role in neurodegeneration.

The C289G and C418R missense mutations cause rapid sequestration of human Parkin into insoluble aggregates.

Mutations in the parkin gene are responsible for autosomal recessive parkinsonism. The disease-linked missense mutations are highly concentrated in the RING-IBR-RING domains of Parkin. In this study, we investigated the consequences of several missense parkin gene mutations in cell culture. We have demonstrated that two of these mutations (C289G and C418R), which replace consensus cysteine residues in the RING domains, significantly decrease the solubility of Parkin in cells. Upon overexpression, the presumably misfolded proteins formed cytoplasmic aggregates that concentrated into large perinuclear inclusion bodies when proteasome activity was inhibited. This process required active microtubule-dependent retrograde transport, as previously reported for aggresome formation. These results provide information on the molecular basis of the loss of function caused by mutations of critical residues in Parkin. They also contribute to our understanding of the cellular mechanism underlying the aggregation of mutant Parkin.