Expanding roles for beta-arrestins as scaffolds and adapters in GPCR signaling and trafficking

beta-arrestins play previously unsuspected and important roles as adapters and scaffolds that localize signaling proteins to ligand-activated G-protein-coupled receptors. As with the paradigmatic role of the beta-arrestins in uncoupling receptors from G proteins (desensitization), these novel functions involve the interaction of beta-arrestin with phosphorylated heptahelical receptors. beta-arrestins interact with at least two main classes of signaling proteins. First, interaction with molecules such as clathrin, AP-2 and NSF directs the clathrin-mediated internalization of G-protein-coupled receptors. Second, interaction with molecules such as Src, Raf, Erk, ASK1 and JNK3 appears to regulate several pathways that result in the activation of MAP kinases. These recent discoveries indicate that the beta-arrestins play widespread roles as scaffolds and/or adapter molecules that organize a variety of complex signaling pathways emanating from heptahelical receptors. It is likely that additional roles for the beta-arrestins remain to be discovered.     

Bioinformatical analysis of G-protein-coupled receptors

G-protein-coupled receptors play a key role in cellular signaling networks that regulate various physiological processes, such as vision, smell, taste, neurotransmission, secretion, inflammatory, immune responses, cellular metabolism, and cellular growth. These proteins are very important for understanding human physiology and disease. Many efforts in pharmaceutical research have been aimed at understanding their structure and function. Unfortunately, because they are difficult to crystallize and most of them will not dissolve in normal solvents, so far very few G-protein-coupled receptor structures have been determined. In contrast, more than 1000 G-protein-coupled receptor sequences are known, and many more are expected to become known soon. In view of the extremely unbalanced state, it would be very useful to develop a fast sequence-based method to identify their different types. This would no doubt have practical value for both basic research and drug discovery because the function or binding specificity of a G-protein coupled receptor is determined by the particular type it belongs to. To realize this, a statistical analysis has been performed for 566 G-protein-coupled receptors classified into seven different types. The results indicate that the types of G-protein-coupled receptors are predictable to a considerable accurate extent if a good training data set can be established for such a goal.     

Corticotropin-releasing hormone receptors

Corticotropin-releasing hormone (CRH) and related peptides (urocortins, sauvagine, urotensin) play a central role in the co-ordination of autonomic, behavioural, cardiovascular, immune and endocrine responses to stressful stimuli. Their actions are mediated through activation of two types of G-protein-coupled receptors encoded by separate genes. In this review we focus on the diverse structural and functional characteristics of the family of CRH-like peptides and their receptors.     

The metabotropic glutamate G-protein-coupled receptors mGluR3 and mGluR1a are voltage-sensitive

G-protein-coupled receptors play a key role in signal transduction processes. Despite G-protein-coupled receptors being transmembrane proteins, the notion that they exhibit voltage sensitivity is rather novel. Here we examine whether two metabotropic glutamate receptors, mGluR3 and mGluR1a, both involved in fundamental physiological processes, exhibit, by themselves, voltage sensitivity. Measuring mGluR3-induced K(+) currents and mGluR1a-induced Ca(2+)-activated Cl(-) currents in Xenopus oocytes, we show that the apparent affinity toward glutamate decreases (mGluR3) or increases (mGluR1a) upon depolarization. Measurements of binding of [(3)H]glutamate to oocytes expressing either mGluR3 or mGluR1a corroborated the electrophysiological results. Using the chimeric Galpha subunit, we further show that the voltage sensitivity does not reside in the G-protein. To locate sites within the receptors that are involved in the voltage sensitivity, we used chimeric mGluR1a, where the intracellular loops that couple to the G-protein were replaced by those of mGluR3. The voltage sensitivity of the chimeric mGluR1a resembled that of mGluR3 and not that of the parental mGluR1a. The cumulative results indicate that the voltage sensitivity does not reside downstream to the activation of the receptors but rather in the mGluR3 and mGluR1a themselves. Furthermore, the intracellular loops play a crucial role in relaying changes in membrane potential to changes in the affinity of the receptors toward glutamate.     

Mechanisms of cross-talk between G-protein-coupled receptors

There is increasing evidence to suggest that 'cross-talk' occurs between G-protein-coupled receptors and their intracellular second messenger pathways. Cross-talk between different pathways may occur at the level of receptors, G-proteins, effectors or second messengers and may serve to fine-tune cell signalling. There is a growing body of evidence to suggest that cellular compartmentalization may play a crucial role in regulating these cross-talk interactions. Understanding the mechanisms of cross-talk may therefore be the key to the design and application of future therapeutics and the development of drug specificity.     

Heterologous regulation of chemokine receptor signaling by the lipid phosphatase SHIP in lymphocytes

The SH2 domain-containing inositol polyphosphate 5-phosphatase (SHIP) is known to play an important role in the negative regulation by FcgammaRIIB of PI3K-dependent signaling cascades activated by the B cell antigen receptor (BCR) as well as several tyrosine-kinase coupled cytokine receptors. However, to date the role of SHIP in the regulation of PI3K-dependent signals elicited by G-protein-coupled receptors (GPCR) such as chemokine receptors has not been investigated. In this study, we report that ligation of the G-protein-coupled chemokine receptor CXCR4 by SDF-1/CXCL12 has no effect on the tyrosine phosphorylation of SHIP in the murine B cell lymphoma A20. However, co-ligation of the B cell antigen receptor and FcgammaRIIB inhibits the PI3K-dependent phosphorylation of PKB and ERK1/2 in response to CXCL12. We have also utilised a constitutively active membrane-localised SHIP mutant expressed in the Jurkat leukaemic T cell line (which do not normally express SHIP), in order to investigate the effect of this mutant on CXCL12 stimulated PI3K-dependent signaling events. Experiments have revealed that CXCL12-mediated PKB phosphorylation, chemotaxis and lipid accumulation are inhibited in the presence of this SHIP mutant. Thus, it appears that heterologous activation of SHIP by non-G-protein-coupled receptor-mediated routes can impinge on PI3K-dependent signaling pathways activated by independently ligated G-protein-coupled chemokine receptors.     

An improved classification of G-protein-coupled receptors using sequence-derived features

Background  

G-protein-coupled receptors (GPCRs) play a key role in diverse physiological processes and are the targets of almost two-thirds of the marketed drugs. The 3 D structures of GPCRs are largely unavailable; however, a large number of GPCR primary sequences are known. To facilitate the identification and characterization of novel receptors, it is therefore very valuable to develop a computational method to accurately predict GPCRs from the protein primary sequences.     

Role of dynamin, Src, and Ras in the protein kinase C-mediated activation of ERK by gonadotropin-releasing hormone

G-protein-coupled receptors are a large group of integral membranal receptors, which in response to ligand binding initiate diverse downstream signaling. Here we studied the gonadotropin-releasing hormone (GnRH) receptor, which uses Gq for its downstream signaling. We show that extracellular signal-regulated kinase (ERK) activation is fully dependent on protein kinase C (PKC), but only partially dependent on Src, dynamin, and Ras. Receptor tyrosine kinases, FAK, Gbetagamma, and beta-arrestin, which were implicated in some G-protein-coupled receptor signaling to MAPK cascades, do not play a role in the GnRH to ERK pathway. Our results suggest that the activation of ERK by GnRH involves two distinct signaling pathways, which converge at the level of Raf-1. The main pathway involves a direct activation of Raf-1 by PKC, and this step is partially dependent on a second pathway consisting of Ras activation, which occurs in a dynamin-dependent manner, downstream of Src.     

Modulation of cancer cell signaling by long noncoding RNAs

Cellular signaling pathways play a very important role in almost all molecular processes in the cell, and are generally composed of a complex set of cascades in which enzymes and proteins play a key role. These signaling pathways include different types of cellular signaling classified based on their receptors and effector proteins such as enzyme-linked receptors, cytokine receptors, and G-protein-coupled receptors each of which is subdivided into different classes. Signaling pathways are tightly controlled by different mechanisms mostly thorough inhibiting/activating their receptors or effector proteins. In the last two decades, our knowledge of molecular biology has changed dramatically and today we know that more than 85% of the human genome expresses noncoding RNAs most of which are crucial in the cellular and molecular mechanisms of cells. One of these noncoding RNAs are long noncoding RNAs (lncRNA) containing more than 200 nucleotides. LncRNAs participate in the progression of cancer growth through several mechanism including signaling pathways. In this review, we summarize some of the most important of lncRNAs and their effect on important signaling pathways.     

Tethering of the platelet-derived growth factor beta receptor to G-protein-coupled receptors. A novel platform for integrative signaling by these receptor classes in mammalian cells

Here we provide evidence to show that the platelet-derived growth factor beta receptor is tethered to endogenous G-protein-coupled receptor(s) in human embryonic kidney 293 cells. The tethered receptor complex provides a platform on which receptor tyrosine kinase and G-protein-coupled receptor signals can be integrated to produce more efficient stimulation of the p42/p44 mitogen-activated protein kinase pathway. This was based on several lines of evidence. First, we have shown that pertussis toxin (which uncouples G-protein-coupled receptors from inhibitory G-proteins) reduced the platelet-derived growth factor stimulation of p42/p44 mitogen-activated protein kinase. Second, transfection of cells with inhibitory G-protein alpha subunit increased the activation of p42/p44 mitogen-activated protein kinase by platelet-derived growth factor. Third, platelet-derived growth factor stimulated the tyrosine phosphorylation of the inhibitory G-protein alpha subunit, which was blocked by the platelet-derived growth factor kinase inhibitor, tyrphostin AG 1296. We have also shown that the platelet-derived growth factor beta receptor forms a tethered complex with Myc-tagged endothelial differentiation gene 1 (a G-protein-coupled receptor whose agonist is sphingosine 1-phosphate) in cells co-transfected with these receptors. This facilitates platelet-derived growth factor-stimulated tyrosine phosphorylation of the inhibitory G-protein alpha subunit and increases p42/p44 mitogen-activated protein kinase activation. In addition, we found that G-protein-coupled receptor kinase 2 and beta-arrestin I can associate with the platelet-derived growth factor beta receptor. These proteins play an important role in regulating endocytosis of G-protein-coupled receptor signal complexes, which is required for activation of p42/p44 mitogen-activated protein kinase. Thus, platelet-derived growth factor beta receptor signaling may be initiated by G-protein-coupled receptor kinase 2/beta-arrestin I that has been recruited to the platelet-derived growth factor beta receptor by its tethering to a G-protein-coupled receptor(s). These results provide a model that may account for the co-mitogenic effect of certain G-protein-coupled receptor agonists with platelet-derived growth factor on DNA synthesis.     

Genome wide survey of G protein-coupled receptors in <Emphasis Type="Italic">Tetraodon nigroviridis</Emphasis>

Background  

The G-protein-coupled receptors (GPCRs) constitute one of the largest and most ancient superfamilies of membrane proteins. They play a central role in physiological processes affecting almost all aspects of the life cycle of an organism. Availability of the complete sets of putative members of a family from diverse species provides the basis for cross genome comparative studies.     

The trick of the tail: protein-protein interactions of metabotropic glutamate receptors

It was initially believed that G-protein-coupled receptors, such as metabotropic glutamate receptors, could simply be described as individual proteins that are associated with intracellular signal cascades via G-proteins. This view is no longer tenable. Today we know that metabotropic glutamate receptors (mGluRs) can dimerize and bind to a variety of proteins in addition to trimeric G-proteins. These newly identified protein interactions led to the discovery of new regulatory mechanisms that are independent of and sometimes synergistic with the classical G-protein-coupled second messenger pathways. Notably, several of these mechanisms connect mGluR-mediated signaling to other receptor classes, thereby creating a network of different receptor types and associated signal cascades. The intracellular C-termini of mGluRs play a key role in the regulation of these networks, and various new protein interactions of these domains were described recently. Because mGluRs are involved in a variety of physiological and pathophysiological processes, some of the proteins interacting with this receptor class have potential as valuable pharmaceutical targets. This review will give a comprehensive overview of proteins interacting with mGluR C-termini, highlight new evolving regulatory mechanisms for glutamatergic signal transduction and discuss possibilities for future drug development.     

New thoughts on the role of the beta-gamma subunit in G-protein signal transduction.

Heterotrimeric G proteins are involved in numerous biological processes, where they mediate signal transduction from agonist-bound G-protein-coupled receptors to a variety of intracellular effector molecules and ion channels. G proteins consist of two signaling moieties: a GTP-bound alpha subunit and a beta-gamma heterodimer. The beta-gamma dimer, recently credited as a significant modulator of G-protein-mediated cellular responses, is postulated to be a major determinant of signaling fidelity between G-protein-coupled receptors and downstream effectors. In this review we have focused on the role of beta-gamma signaling and have included examples to demonstrate the heterogeneity in the heterodimer composition and its implications in signaling fidelity. We also present an overview of some of the effectors regulated by beta-gamma and draw attention to the fact that, although G proteins and their associated receptors play an instrumental role in development, there is rather limited information on beta-gamma signaling in embryogenesis.     

A study on the correlation of G-protein-coupled receptor types with amino acid composition

G-protein-coupled receptors have become a target in utilizing bioinformatics and genomics technology to facilitate drug discovery for psychiatric diseases. In this study the covariant-discriminant algorithm [Chou and Elrod (1999) Protein Eng., 12, 107-118] has been used to analyze the correlation between the types of G-protein-coupled receptors and the amino acid composition. It has been found that different types of G-protein-coupled receptors are quite closely correlated with the amino acid composition, implying that the types of G-protein-coupled receptors are predictable to a considerably accurate extent if a good training data set can be established for that purpose. The method derived here can be also used to do preliminary classification of orphan G-protein-coupled receptors. This will significantly expedite the process of identifying proper G-protein-coupled receptors for drug discovery.     

The cytoplasmic end of transmembrane domain 3 regulates the activity of the Saccharomyces cerevisiae G-protein-coupled alpha-factor receptor

The binding of alpha-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the alpha-factor receptor that includes transmembrane domains 1-5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the alpha-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors.     

G-protein-coupled receptors and melanoma

G-protein-coupled receptors (GPCR) are the largest family of receptors with over 500 members. Evaluation of GPCR gene expression in primary human tumors identified over-expression of GPCR in several tumor types. Analysis of cancer samples in different disease stages also suggests that some GPCR may be involved in early tumor progression and others may play a critical role in tumor invasion and metastasis. Currently, >50% of drug targets to various human diseases are based on GPCR. In this review, the relationships between several GPCR and melanoma development and/or progression will be discussed. Finally, the possibility of using one or more of these GPCR as therapeutic targets in melanoma will be summarized.     

Regulation of protease-activated receptor signaling by post-translational modifications

Protease-activated receptors (PARs) are a unique family of G-protein-coupled receptors (GPCRs) that are irreversibly activated following proteolytic cleavage of their extracellular N-terminus. PARs play critical functions in hemostasis, thrombosis, inflammation, embryonic development, and cancer progression. Because of the irreversible proteolytic nature of PAR activation, signaling by the receptors is tightly regulated. Three distinct processes including desensitization, internalization, and lysosomal degradation, regulate the temporal and spatial aspects of activated PAR signaling. Post-translational modifications play a critical role in regulating each of these processes and here we review the nature of PAR post-translational modifications and their importance in signal regulation. The PARs are activated by numerous proteases, and some can elicit distinct cellular responses, how this biased agonism is determined is unknown. Further study of the function of post-translational modifications of the PARs will lead to a greater understanding of the physiological regulation of baised agonism and how PAR signaling is precisely controlled in different cellular contexts.     

Chemotactic activity of extracellular nucleotideson human immune cells.

Purinergic P2 receptors are a class of plasma membrane receptors that are express in many tissues and are ligated by extracellular nucleotides [such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP)], which are released as a consequence of cell damage, cell stress, bacterial infection or other noxious stimuli. According to the molecular structure, P2 receptors are divided into two subfamilies: P2X and P2Y receptors. The P2X receptors are ligand-gated channels, whereas P2Y receptors are G-protein-coupled seven-membrane-spanning receptors. Several studies indicate that nucleotides play an important role in immune response modulation through their action on multiple cell types, including monocytes, mast cells, dendritic cells, neutrophils, and eosinophils. Recent work by our group and others identified extracellular nucleotides as chemotaxins for various human immune cells, including eosinophils, neutrophils and dendritic cells. In this review, we summarise recent findings in this field and put forward a hypothesis on the role of P2 receptors in the early recruitment of human immune cells to the site of inflammation.     

NPY, ET-1, and Ang II nuclear receptors in human endocardial endothelial cells

Neuropeptide Y (NPY), endothelin-1 (ET-1), and angiotensin II (Ang II) are peptides that are known to play many important roles in cardiovascular homeostasis. The physiological actions of these peptides are thought to be primarily mediated by plasma membrane receptors that belong to the G-protein-coupled receptor superfamily. However, there is increasing evidence that suggests the existence of functional G-protein-coupled receptors at the level of the nucleus and that the nucleus could be a cell within a cell. Here, we review our work showing the presence in the nucleus of the NPY Y(1) receptor, the ET(A) and ET(B) receptors, as well as the AT(1) and AT(2) receptors and their respective ligands. This work was carried out in 20-week-old fetal human endocardial endothelial cells. Our results demonstrate that nuclear Y1, AT(1), and ET(A) receptors modulate nuclear calcium in these cells.     

Regulation of tyrosine kinase cascades by G-protein-coupled receptors

Mitogenic signaling by G-protein-coupled receptors (GPCRs) involves tyrosine phosphorylation of adaptor proteins and assembly of multiprotein Ras activation complexes. Over the past three years, three types of scaffolds for GPCR-directed complex assembly have been identified: transactivated receptor tyrosine kinases (RTKs), integrin-based focal adhesions, and GPCRs themselves. Nonreceptor tyrosine kinases play an important role in each case. The processes of GPCR desensitization and sequestration via clathrin-coated pits are also involved in signaling through the RTK- and GPCR-based scaffolds.