Cellulose-bound Peptide Arrays: Preparation and Applications

Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery.     

Structural biology of G protein‐coupled receptor signaling complexes

G protein‐coupled receptors (GPCRs) constitute the largest family of cell surface receptors that mediate numerous cell signaling pathways, and are targets of more than one‐third of clinical drugs. Thanks to the advancement of novel structural biology technologies, high‐resolution structures of GPCRs in complex with their signaling transducers, including G‐protein and arrestin, have been determined. These 3D complex structures have significantly improved our understanding of the molecular mechanism of GPCR signaling and provided a structural basis for signaling‐biased drug discovery targeting GPCRs. Here we summarize structural studies of GPCR signaling complexes with G protein and arrestin using rhodopsin as a model system, and highlight the key features of GPCR conformational states in biased signaling including the sequence motifs of receptor TM6 that determine selective coupling of G proteins, and the phosphorylation codes of GPCRs for arrestin recruitment. We envision the future of GPCR structural biology not only to solve more high‐resolution complex structures but also to show stepwise GPCR signaling complex assembly and disassembly and dynamic process of GPCR signal transduction.     

Quantitative Analysis of Purine Nucleotides Indicates That Purinosomes Increase de Novo Purine Biosynthesis

Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome.     

植物G蛋白偶联受体及其在信号转导中的作用

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质超家族.GPCRs能调控细胞周期,参与多种植物信号通路以及影响一系列的代谢和分化活动.简要介绍了GPCR和G蛋白介导的信号转导机制,GPCRs的结构和植物GPCR及其在植物跨膜信号转导中的作用,并对GPCR的信号转导机制及植物抗病反应分子机制的研究提出展望.     

Adaptor Protein-2 Interaction with Arrestin Regulates GPCR Recycling and Apoptosis

G protein-coupled receptors (GPCRs) are integral to cellular function in nearly all physiologic and many pathologic processes. GPCR signaling represents an intricate balance between receptor activation, inactivation (desensitization, internalization and degradation) and resensitization (recycling and de novo synthesis). Complex formation between phosphorylated GPCRs, arrestins and an ever-increasing number of effector molecules is known to regulate cellular function. Previous studies have demonstrated that, although N -formyl peptide receptor (FPR) internalization occurs in the absence of arrestins, FPR recycling is arrestin-dependent. Furthermore, FPR stimulation in the absence of arrestins leads to receptor accumulation in perinuclear endosomes and apoptosis. In this study, we show that the interaction of GPCR-bound arrestin with adaptor protein-2 (AP-2) is a critical anti-apoptotic event. In addition, AP-2 associates with the receptor-arrestin complex in perinuclear endosomes and is required for proper post-endocytic GPCR trafficking. Finally, we observed that depletion of endogenous AP-2 results in the initiation of apoptosis upon stimulation of multiple GPCRs, including P2Y purinergic receptors and CXCR2, but not CXCR4. We propose a model in which the abnormal accumulation of internalized GPCR-arrestin complexes in recycling endosomes, resulting from defective arrestin-AP-2 interactions, leads to the specific initiation of aberrant signaling pathways and apoptosis.     

Implications of non-canonical G-protein signaling for the immune system

Heterotrimeric guanine nucleotide-binding proteins (G proteins), which consist of three subunits α, β, and γ, function as molecular switches to control downstream effector molecules activated by G protein-coupled receptors (GPCRs). The GTP/GDP binding status of Gα transmits information about the ligand binding state of the GPCR to intended signal transduction pathways. In immune cells heterotrimeric G proteins impact signal transduction pathways that directly, or indirectly, regulate cell migration, activation, survival, proliferation, and differentiation. The cells of the innate and adaptive immune system abundantly express chemoattractant receptors and lesser amounts of many other types of GPCRs. But heterotrimeric G-proteins not only function in classical GPCR signaling, but also in non-canonical signaling. In these pathways the guanine exchange factor (GEF) exerted by a GPCR in the canonical pathway is replaced or supplemented by another protein such as Ric-8A. In addition, other proteins such as AGS3-6 can compete with Gβγ for binding to GDP bound Gα. This competition can promote Gβγ signaling by freeing Gβγ from rapidly rebinding GDP bound Gα. The proteins that participate in these non-canonical signaling pathways will be briefly described and their role, or potential one, in cells of the immune system will be highlighted.     

Systematic protein–protein interaction mapping for clinically relevant human GPCRs

G‐protein‐coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR‐mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two‐hybrid (MYTH) approach and identified interacting partners for 48 selected full‐length human ligand‐unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5‐HT4d, and adenosine ADORA2A receptors. Our data represent the first large‐scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.     

Engineering GPCR signaling pathways with RASSLs

We are creating families of designer G protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (G(s), G(i) and G(q)). We review these advances here to facilitate the use of these powerful and diverse tools.     

Ins and outs of GPCR signaling in primary cilia

Primary cilia are specialized microtubule‐based signaling organelles that convey extracellular signals into a cellular response in most vertebrate cell types. The physiological significance of primary cilia is underscored by the fact that defects in assembly or function of these organelles lead to a range of severe diseases and developmental disorders. In most cell types of the human body, signaling by primary cilia involves different G protein‐coupled receptors (GPCRs), which transmit specific signals to the cell through G proteins to regulate diverse cellular and physiological events. Here, we provide an overview of GPCR signaling in primary cilia, with main focus on the rhodopsin‐like (class A) and the smoothened/frizzled (class F) GPCRs. We describe how such receptors dynamically traffic into and out of the ciliary compartment and how they interact with other classes of ciliary GPCRs, such as class B receptors, to control ciliary function and various physiological and behavioral processes. Finally, we discuss future avenues for developing GPCR‐targeted drug strategies for the treatment of ciliopathies.     

Biased signaling in naturally occurring mutations of G protein-coupled receptors associated with diverse human diseases

G protein-coupled receptors (GPCRs) play critical roles in transmitting a variety of extracellular signals into the cells and regulate diverse physiological functions. Naturally occurring mutations that result in dysfunctions of GPCRs have been known as the causes of numerous diseases. Significant progresses have been made in elucidating the pathophysiology of diseases caused by mutations. The multiple intracellular signaling pathways, such as G protein-dependent and β-arrestin-dependent signaling, in conjunction with recent advances on biased agonism, have broadened the view on the molecular mechanism of disease pathogenesis. This review aims to briefly discuss biased agonism of GPCRs (biased ligands and biased receptors), summarize the naturally occurring GPCR mutations that cause biased signaling, and propose the potential pathophysiological relevance of biased mutant GPCRs associated with various endocrine diseases.     

Heterotrimeric G Proteins and the Single-Transmembrane Domain IGF-II/M6P Receptor: Functional Interaction and Relevance to Cell Signaling

The G protein-coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors. Classical GPCR signaling constitutes ligand binding to a seven-transmembrane domain receptor, receptor interaction with a heterotrimeric G protein, and the subsequent activation or inhibition of downstream intracellular effectors to mediate a cellular response. However, recent reports on direct, receptor-independent G protein activation, G protein-independent signaling by GPCRs, and signaling of nonheptahelical receptors via trimeric G proteins have highlighted the intrinsic complexities of G protein signaling mechanisms. The insulin-like growth factor-II/mannose-6 phosphate (IGF-II/M6P) receptor is a single-transmembrane glycoprotein whose principal function is the intracellular transport of lysosomal enzymes. In addition, the receptor also mediates some biological effects in response to IGF-II binding in both neuronal and nonneuronal systems. Multidisciplinary efforts to elucidate the intracellular signaling pathways that underlie these effects have generated data to suggest that the IGF-II/M6P receptor might mediate transmembrane signaling via a G protein-coupled mechanism. The purpose of this review is to outline the characteristics of traditional and nontraditional GPCRs, to relate the IGF-II/M6P receptor’s structure with its role in G protein-coupled signaling and to summarize evidence gathered over the years regarding the putative signaling of the IGF-II/M6P receptor mediated by a G protein.     

Signaling and regulation of G protein-coupled receptors in airway smooth muscle

Signaling through G protein-coupled receptors (GPCRs) mediates numerous airway smooth muscle (ASM) functions including contraction, growth, and "synthetic" functions that orchestrate airway inflammation and promote remodeling of airway architecture. In this review we provide a comprehensive overview of the GPCRs that have been identified in ASM cells, and discuss the extent to which signaling via these GPCRs has been characterized and linked to distinct ASM functions. In addition, we examine the role of GPCR signaling and its regulation in asthma and asthma treatment, and suggest an integrative model whereby an imbalance of GPCR-derived signals in ASM cells contributes to the asthmatic state.     

Cellular mechanisms that determine selective RGS protein regulation of G protein-coupled receptor signaling

Regulators of G protein signaling (RGS proteins) bind directly to activated Galpha subunits to inhibit their signaling. However, recent findings show that RGS proteins selectively regulate signaling by certain G protein-coupled receptors (GPCRs) in cells, irrespective of the coupled G protein. New studies support an emerging model that suggests RGS proteins utilize both direct and indirect mechanisms to form stable functional pairs with preferred GPCRs to selectively modulate the signaling functions of those receptors and linked G proteins. Here, we discuss these findings and their implications for established models of GPCR signaling.     

Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR

G-protein-coupled receptors (GPCRs) mostly signal through heterotrimeric G proteins. Increasing evidence suggests that GPCRs could function in a G-protein-independent manner. Here, we show that at low concentrations of an agonist, beta(2)-adrenergic receptors (beta(2)-ARs) signal through Galpha(s) to activate the mitogen-activated protein kinase pathway in mouse embryonic fibroblast cells. At high agonist concentrations, signals are also transduced through beta(2)-ARs via an additional pathway that is G-protein-independent but tyrosine kinase Src-dependent. This new dosage-dependent switch of signaling modes of GPCRs has significant implications for GPCR intrinsic properties and desensitization.     

When a G protein-coupled receptor does not couple to a G protein

Classically, G protein-coupled receptors (GPCRs) relay signals by directly activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Increasing evidence indicates that GPCRs may also signal through G protein-independent pathways. JAK/STATs, Src-family tyrosine kinases, GRKs/beta-arrestins, and PDZ domain-containing proteins have been suggested to directly relay signals from GPCRs independent of G proteins. In addition, our laboratory recently reported that the beta(2) adrenergic receptor (beta(2)AR) could switch from G protein-coupled to G protein-independent ERK (extracellular signal-regulated kinase) activation in an agonist dosage-dependent manner. This finding provides a novel mechanism for G protein-independent GPCR signaling. This review focuses on recent progress in understanding the mechanisms by which G protein-independent GPCR signaling occurs.     

Effects of methotrexate on nucleotide pools in normal human T cells and the CEM T cell line

It has been proposed that the clinical utility of methotrexate (MTX) in the treatment of rheumatoid arthritis may be due, in part, to inhibition of 5-amino imidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT) by polyglutamated forms of MTX. AICARFT is the second folate dependent enzyme in de novo purine biosynthesis. In this study, the effects of MTX on de novo purine biosynthesis as well as total nucleotide pools were evaluated in both the human T cell line, CEM, and phytohemagglutinin-activated normal human T lymphocytes. De novo synthesized purines were metabolically labeled with 14C-glycine after MTX treatment and analyzed by HPLC. In normal T cells, MTX produced a dose-dependent reduction in de novo adenosine and guanosine pools with maximal effects (>50%) at 1 microM MTX. In CEM cells, de novo purine synthesis was almost completely blocked by 1 microM MTX. Total purine pools were also reduced in both cell types after MTX treatment. Since 1 microM MTX caused almost complete growth inhibition in CEM cells, we evaluated whether growth could be reconstituted with exogenous purine bases and pyrimidine nucleosides which can be utilized via salvage pathways. The combination of hypoxanthine and thymidine substantially reversed growth inhibition with 1 microM MTX in CEM cells. Taken together, these results demonstrate that MTX inhibits de novo nucleotide synthesis in T cells and suggest that AICARFT inhibition may be one aspect of the multi-site mechanism of MTX action in the treatment of rheumatoid arthritis.     

G-protein-coupled receptors function as oligomers in vivo

Hormones, sensory stimuli, neurotransmitters and chemokines signal by activating G-protein-coupled receptors (GPCRs) [1]. Although GPCRs are thought to function as monomers, they can form SDS-resistant dimers, and coexpression of two non-functional or related GPCRs can result in rescue of activity or modification of function [2-10]. Furthermore, dimerization of peptides corresponding to the third cytoplasmic loops of GPCRs increases their potency as activators of G proteins in vitro [11], and peptide inhibitors of dimerization diminish beta(2)-adrenergic receptor signaling [3]. Nevertheless, it is not known whether GPCRs exist as monomers or oligomers in intact cells and membranes, whether agonist binding regulates monomer-oligomer equilibrium, or whether oligomerization governs GPCR function. Here, we report that the alpha-factor receptor, a GPCR that is the product of the STE2 gene in the yeast Saccharomyces cerevisiae, is oligomeric in intact cells and membranes. Coexpression of receptors tagged with the cyan or yellow fluorescent proteins (CFP or YFP) resulted in efficient fluorescence resonance energy transfer (FRET) due to stable association rather than collisional interaction. Monomer-oligomer equilibrium was unaffected by binding of agonist, antagonist, or G protein heterotrimers. Oligomerization was further demonstrated by rescuing endocytosis-defective receptors with coexpressed wild-type receptors. Dominant-interfering receptor mutants inhibited signaling by interacting with wild-type receptors rather than by sequestering G protein heterotrimers. We suggest that oligomerization is likely to govern GPCR signaling and regulation.     

Cytoskeletal reorganization dependence of signaling by the gonadotropin-releasing hormone receptor

Activation of classical G protein-coupled receptors (GPCRs) like the mammalian gonadotropin-releasing hormone receptor (GnRHR) typically stimulates heterotrimeric G protein molecules that subsequently activate downstream effectors. Receptor activation of heterotrimeric G protein pathways primarily controls intermediary cell metabolism by elevation or diminution of soluble cytoplasmic second messenger molecules. We have demonstrated here that stimulation of the GnRHR also results in a dramatic change in both cell adhesion and superstructural morphology. Gonadotropin-releasing hormone (GnRH) receptor activation rapidly increases the capacity of HEK293 cells expressing the GnRHR to remain matrix-adherent in the face of fluid insults. Coinciding with this profound elevation in matrix adherence, we demonstrated a GnRH-induced alteration in both cell morphology and the de novo generation of polymerized actin structures. GnRH induction of cytoskeletal remodeling was correlated with significant increases in the tyrosine phosphorylation status of a series of cytoskeletal associated proteins, e.g. focal adhesion kinase (FAK), c-Src, and microtubule-associated protein kinase (MAPK or ERK1/2). The activation of the distal downstream effector ERK1/2 was demonstrated to be sensitive to the disrupters of cytoskeletal rearrangement, cytochalasin D and latrunculin B. In addition to the sensitivity of ERKs to cytoskeletal integrity, GnRH-induced FAK and c-Src kinase activation were sensitive to these agents and the fibronectin-integrin antagonistic RGDS peptide. Activation of ERK was dependent on its protein-protein assembly with FAK and c-Src at focal adhesion complexes. Induction of the cell remodeling event leading to this signaling complex assembly occurred primarily via GnRHR activation of the monomeric G protein Rac but not RhoA. These findings demonstrated a clear divergence of GnRHR signaling via the Rac monomeric G protein focal adhesion signaling complex assembly and cytoskeletal remodeling independent of the classical heterotrimeric G protein-controlled phospholipase C-beta pathway.     

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.     

Purine nucleotide metabolism in phytohemagglutinin-induced human T lymphocytes

The comprehensive studies of purine nucleotide metabolism were done in nonstimulated and phytohemagglutinin (PHA)-stimulated human peripheral blood T lymphocytes. Nonstimulated lymphocytes synthesize nucleotides in two alternative pathways: via biosynthesis de novo and salvage pathways. Although synthesis of triphosphonucleosides in unstimulated lymphocytes was the predominant pathway, interconversion of monophosphonucleosides was also active. Exposure of cells to PHA affects differently various pathways of nucleotide metabolism. The most marked changes observed were rapid activation of purine salvage within minutes after exposure to PHA, and significant increase of 5-phosphoribosyl-1-pyrophosphate levels. In addition, significant increases were found in de novo purine biosynthesis, nucleotide interconversions, and RNA and DNA synthesis, whereas catabolism of nucleotides remained unchanged. These results indicate that PHA activation of T lymphocytes causes a rapid synthesis of nucleotides which may be required immediately for increases in energy metabolism and later as the precursors of nucleic acid synthesis.