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[beta]-arrestin 1 and 2 (Barr1 and Barr2, respectively) are intracellular signaling molecules that regulate many important metabolic functions. We previously demonstrated that mice lacking Barr2 selectively in pancreatic [beta] cells showed pronounced metabolic impairments. Here we investigated whether Barr1 plays a similar role in regulating [beta] cell function and whole-body glucose homeostasis. Initially, we inactivated the Barrl gene in [beta] cells of adult mice ([beta]-barr1-KO mice). [beta]-barr1-KO mice did not display any obvious phenotypes in a series of in vivo and in vitro metabolic tests. However, glibenclamide and tolbutamide, 2 widely used antidiabetic drugs of the sulfonylurea (SU) family, showed greatly reduced efficacy in stimulating insulin secretion in the KO mice in vivo and in perifused KO islets in vitro. Additional in vivo and in vitro studies demonstrated that Barr1 enhanced SU-stimulated insulin secretion by promoting SU-mediated activation of Epac2. Pull-down and coimmunoprecipitation experiments showed that Barr1 can directly interact with Epac2 and that SUs such as glibenclamide promote Barr1/Epac2 complex formation, triggering enhanced Rap1 signaling and insulin secretion. These findings suggest that strategies aimed at promoting Barr1 signaling in [beta] cells may prove useful for the development of efficacious antidiabetic drugs., Introduction Accumulating evidence suggests that [beta]-arrestins play key roles in regulating many important metabolic functions including [beta] cell activity (1). The 2 [beta]-arrestin isoforms ([beta]-arrestin 1 and 2; referred to [...]

G protein-coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacologic profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype-selective and nearly all are cationic in nature. Using structure-based docking against the M2 and M3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype selectivities. Of 19 docking-prioritized molecules tested against the M2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar Ki values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M2 and M3 receptors, we selected molecules predicted by docking to bind to the M3 and but not the M2 receptor. Of 16 molecules tested, 8 bound to the M3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M3 receptor without measurable M2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse β-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biologic functions, even in an area as heavily explored as muscarinic pharmacology.

An increase in hepatic glucose production (HGP) is a key feature of type 2 diabetes. Excessive signaling through hepatic [G.sub.s]-linked glucagon receptors critically contributes to pathologically elevated HGP. Here, we tested the hypothesis that this metabolic impairment can be counteracted by enhancing hepatic [G.sub.i] signaling. Specifically, we used a chemogenetic approach to selectively activate [G.sub.i]-type G proteins in mouse hepatocytes in vivo. Unexpectedly, activation of hepatic [G.sub.i] signaling triggered a pronounced increase in HGP and severely impaired glucose homeostasis. Moreover, increased [G.sub.i] signaling stimulated glucose release in human hepatocytes. A lack of functional [G.sub.i]- type G proteins in hepatocytes reduced blood glucose levels and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Additionally, we delineated a signaling cascade that links hepatic [G.sub.i] signaling to ROS production, JNK activation, and a subsequent increase in HGP. Taken together, our data support the concept that drugs able to block hepatic G-coupled GPCRs may prove beneficial as antidiabetic drugs., Introduction Type 2 diabetes (T2D) has emerged as a major health problem worldwide (1). In addition to other pathophysiological features, T2D is characterized by an increase in hepatic glucose production [...]

Unstructured regions in functional proteins have gained attention in recent years due to advancements in informatics tools and biophysical methods. G protein-coupled receptors (GPCRs), a large family of cell surface receptors, contain unstructured regions in the form of the i3 loop and C-terminus. This review provides an overview of the functional significance of these regions in GPCRs. GPCRs transmit signals from the extracellular environment to the cell interior, regulating various physiological processes. The i3 loop, located between the fifth and sixth transmembrane helices, and the C-terminus, connected to the seventh transmembrane helix, are determinant of interactions with G proteins and with other intracellular partners such as arrestins. Recent studies demonstrate that the i3 loop and C-terminus play critical roles in allosterically regulating GPCR activation. They can act as autoregulators, adopting conformations that, by restricting G protein access, modulate receptor coupling specificity. The length and unstructured nature of the i3 loop and C-terminus provide unique advantages in GPCR interactions with intracellular protein partners. They act as "fishing lines", expanding the radius of interaction and enabling GPCRs to tether scaffolding proteins, thus facilitating receptor stability during cell membrane movements. Additionally, the i3 loop may be involved in domain swapping between GPCRs, generating novel receptor dimers with distinct binding and coupling characteristics. Overall, the i3 loop and C-terminus are now widely recognized as crucial elements in GPCR function and regulation. Understanding their functional roles enhances our comprehension of GPCR structure and signaling complexity and holds promise for advancements in receptor pharmacology and drug development. [ABSTRACT FROM AUTHOR]

An increase in hepatic glucose production (HGP) represents a key feature of type 2 diabetes. This deficiency in metabolic control of glucose production critically depends on enhanced signaling through hepatic glucagon receptors (GCGRs). Here, we have demonstrated that selective inactivation of the GPCR-associated protein [beta]-arrestin 2 in hepatocytes of adult mice results in greatly increased hepatic GCGR signaling, leading to striking deficits in glucose homeostasis. However, hepatocyte-specific [beta]-arrestin 2 deficiency did not affect hepatic insulin sensitivity or p -adrenergic signaling. Adult mice lacking [beta]-arrestin 1 selectively in hepatocytes did not show any changes in glucose homeostasis. Importantly, hepatocyte-specific overexpression of [beta]-arrestin 2 greatly reduced hepatic GCGR signaling and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Our data support the concept that strategies aimed at enhancing hepatic [beta]-arrestin 2 activity could prove useful for suppressing HGP for therapeutic purposes., Introduction One key pathophysiological feature of type 2 diabetes (T2D) is an increase in hepatic glucose production (HGP) (1-3). HGP is modulated by the activity of several GPCRs, including hepatic [...]

Autophagy is a tightly regulated catabolic process involved in the degradation and recycling of proteins and organelles. Ubiquitination plays an important role in the regulation of autophagy. Vacuole Membrane Protein 1 (VMP1) is an essential autophagy protein. The expression of VMP1 in pancreatic cancer stem cells carrying the activated Kirsten rat sarcoma viral oncogene homolog (KRAS) triggers autophagy and enables therapy resistance. Using biochemical and cellular approaches, we identified ubiquitination as a post-translational modification of VMP1 from the initial steps in autophagosome biogenesis. VMP1 remains ubiquitinated as part of the autophagosome membrane throughout autophagic flux until autolysosome formation. However, VMP1 is not degraded by autophagy, nor by the ubiquitin–proteasomal system. Mass spectrometry and immunoprecipitation showed that the cell division cycle protein cdt2 (Cdt2), the substrate recognition subunit of the E3 ligase complex associated with cancer, cullin–RING ubiquitin ligase complex 4 (CRL4), is a novel interactor of VMP1 and is involved in VMP1 ubiquitination. VMP1 ubiquitination decreases under the CRL inhibitor MLN4924 and increases with Cdt2 overexpression. Moreover, VMP1 recruitment and autophagosome formation is significantly affected by CRL inhibition. Our results indicate that ubiquitination is a novel post-translational modification of VMP1 during autophagy in human tumor cells. VMP1 ubiquitination may be of clinical relevance in tumor-cell-therapy resistance. [ABSTRACT FROM AUTHOR]

Many dementias are propagated through the spread of "prion-like" misfolded proteins. This includes prion diseases themselves (such as Creutzfeldt-Jakob disease) and Alzheimer's disease (AD), for which no treatments are available to slow or stop progression. The M1 acetylcholine muscarinic receptor (M1 receptor) is abundant in the brain, and its activity promotes cognitive function in preclinical models and in patients with AD. Here, we investigated whether activation of the M1 receptor might slow the progression of neurodegeneration associated with prion-like misfolded protein in a mouse model of prion disease. Proteomic and transcriptomic analysis of the hippocampus revealed that this model had a molecular profile that was similar to that of human neurodegenerative diseases, including AD. Chronic enhancement of the activity of the M1 receptor with the positive allosteric modulator (PAM) VU0486846 reduced the abundance of prion-induced molecular markers of neuroinflammation and mitochondrial dysregulation in the hippocampus and normalized the abundance of those associated with neurotransmission, including synaptic and postsynaptic signaling components. PAM treatment of prion-infected mice prolonged survival and maintained cognitive function. Thus, allosteric activation of M1 receptors may reduce the severity of neurodegenerative diseases caused by the prion-like propagation of misfolded protein. Protecting against prions: Various neurodegenerative diseases are caused by misfolded proteins that aggregate and propagate through the brain and lack effective treatments. Because loss of acetylcholine signaling is associated with cognitive deficits in patients, Dwomoh et al. investigated the effect of enhancing acetylcholine signaling with positive allosteric modulators (PAMs) targeting the M1 acetylcholine receptor in a mouse model of prion disease. Systemic administration of PAMs reduced the abundance of prion-induced molecular markers in the hippocampus, restored various cognitive functions, and slowed disease progression in the mice. The proteomic and transcriptomic data from the prion mouse model substantially overlapped with those associated with Alzheimer's disease, suggesting that M1 receptor PAMs may be therapeutic in patients with prion and prion-like neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The architecture of eukaryotic cells is defined by extensive membrane-delimited compartments, which entails separate metabolic processes that would otherwise interfere with each other, leading to functional differences between cells. G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors, and their signal transduction is traditionally viewed as a chain of events initiated from the plasma membrane. Furthermore, their intracellular trafficking, internalization, and recycling were considered only to regulate receptor desensitization and cell surface expression. On the contrary, accumulating data strongly suggest that GPCRs also signal from intracellular compartments. GPCRs localize in the membranes of endosomes, nucleus, Golgi and endoplasmic reticulum apparatuses, mitochondria, and cell division compartments. Importantly, from these sites they have shown to orchestrate multiple signals that regulate different cell pathways. In this review, we summarize the current knowledge of this fascinating phenomenon, explaining how GPCRs reach the intracellular sites, are stimulated by the endogenous ligands, and their potential physiological/pathophysiological roles. Finally, we illustrate several mechanisms involved in the modulation of the compartmentalized GPCR signaling by drugs and endogenous ligands. Understanding how GPCR signaling compartmentalization is regulated will provide a unique opportunity to develop novel pharmaceutical approaches to target GPCRs and potentially lead the way towards new therapeutic approaches. [ABSTRACT FROM AUTHOR]

BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas (1,2). By binding specific DNA sequences, BCL6 controls the transcription of a variety of genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells develop DLBCLs similar to the human disease (3,4). In many DLBCL patients, BCL6 overexpression is achieved through translocation (~40%) or hypermutation of its promoter (~15%). However, many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1-CUL1-Fbox protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11 (refs 5, 6). The gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly, FBXO11was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11 mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11 expression in FBXOll-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation, inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11 reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization. The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a haplo-insufficient tumour suppressor gene., Because the degradation of BCL6 has been reported to be phosphorylation- dependent (7,8), we examined the hypothesis that BCL6 is degraded by an SCF ubiquitin ligase complex, as most SCF [...]