Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins

Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs.     

Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins

Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs.     

Novel roles for arrestins in the post-endocytic trafficking of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling molecules in the human genome. As such, they interact with numerous intracellular molecules, which can act either to propagate or curtail signaling from the receptor. Their primary mode of cellular activation occurs through heterotrimeric G proteins, which in turn can activate a wide spectrum of effector molecules, including phosphodiesterases, phospholipases, adenylyl cyclases and ion channels. Active GPCRs are also the target of G protein-coupled receptor kinases, which phosphorylate the receptors culminating in the binding of the protein arrestin. This results in rapid desensitization through inhibition of G protein binding, as well as novel mechanisms of cellular activation that involve the scaffolding of cellular kinases to GPCR-arrestin complexes. Arrestins can also serve to mediate the internalization of certain GPCRs, a process which plays an important role in regulating cellular activity both by mediating long-term desensitization through down regulation (degradation) of receptors and by recycling desensitized receptors back to the cell surface to initiate additional rounds of signaling. The mechanisms that regulate the subsequent intracellular trafficking of GPCRs following internalization are largely unknown. Recently however, it has become clear that the pattern of receptor phosphorylation and subsequent binding of arrestin play a critical role in the intracellular trafficking of internalized receptors, thereby dictating the ultimate fate of the receptor. In addition, arrestins have now been shown to be required for the recycling of GPCRs that are capable of internalizing through arrestin-independent mechanisms. This review will summarize recent advances in our understanding of the roles of arrestins in post-endocytic GPCR trafficking.     

Mechanisms regulating membrane trafficking of G protein-coupled receptors in the endocytic pathway

Endocytic membrane trafficking plays multiple roles in GPCR signaling and regulation. In the past several years much has been learned about molecular mechanisms that mediate and regulate endocytic trafficking of cloned GPCRs expressed in transfected cell lines, and there is accelerating progress toward elucidating the membrane trafficking of GPCRs in native tissues. Current views regarding ligand-induced endocytosis of adrenergic catecholamine and opioid neuropeptide receptors will be reviewed, focusing on recent data suggesting the existence of additional machinery controlling the endocytosis of specific GPCRs via clathrin-coated pits. Evidence that GPCRs are selectively 'sorted' between divergent downstream pathways after endocytosis will be discussed, focusing on recent insight to mechanisms controlling receptor sorting between distinct recycling and non-recycling membrane pathways.     

Ubiquitin-dependent regulation of G protein-coupled receptor trafficking and signaling

G protein-coupled receptors (GPCRs) belong to one of the largest family of signaling receptors in the mammalian genome [1]. GPCRs elicit cellular responses to multiple diverse stimuli and play essential roles in human health and disease. GPCRs have important clinical implications in various diseases and are the targets of approximately 25–50% of all marketed drugs [2], [3]. Understanding how GPCRs are regulated is essential to delineating their role in normal physiology and in the pathophysiology of several diseases. Given the vast number and diversity of GPCRs, it is likely that multiple mechanisms exist to regulate GPCR function. While GPCR signaling is typically regulated by desensitization and endocytosis mediated by phosphorylation and β-arrestins, it can also be modulated by ubiquitination. Ubiquitination is emerging an important regulatory process that may have unique roles in governing GPCR trafficking and signaling. Recent studies have revealed a mechanistic link between GPCR phosphorylation, β-arrestins and ubiquitination that may be applicable to some GPCRs but not others. While the function of ubiquitination is generally thought to promote receptor endocytosis and endosomal sorting, recent studies have revealed that ubiquitination also plays an important role in positive regulation of GPCR signaling. Here, we will review recent developments in our understanding of how ubiquitin regulates GPCR endocytic trafficking and how it contributes to signal transduction induced by GPCR activation.     

ADAMs as mediators of EGF receptor transactivation by G protein-coupled receptors

A disintegrin and metalloprotease (ADAM) is a membrane-anchored metalloprotease implicated in the ectodomain shedding of cell surface proteins, including the ligands for epidermal growth factor (EGF) receptors (EGFR)/ErbB. It has been well documented that the transactivation of the EGFR plays critical roles for many cellular functions, such as proliferation and migration mediated through multiple G protein-coupled receptors (GPCRs). Recent accumulating evidence has suggested that ADAMs are the key metalloproteases activated by several GPCR agonists to produce a mature EGFR ligand leading to the EGFR transactivation. In this review, we describe the current knowledge on ADAMs implicated in mediating EGFR transactivation. The major focus of the review will be on the possible upstream mechanisms of ADAM activation by GPCRs as well as downstream signal transduction and the pathophysiological significances of ADAM-dependent EGFR transactivation. ectodomain shedding; angiotensin II     

Molecular modelling of human 5-hydroxytryptamine receptor (5-HT2A) and virtual screening studies towards the identification of agonist and antagonist molecules

The serotonin receptors, also known as 5-hydroxytryptamine (5-HT) receptors, are a group of G protein-coupled receptors (GPCRs) and ligand-gated ion channels found in the central and peripheral nervous systems. GPCRs have a characteristic feature of activating different signalling pathways upon ligand binding and these ligands display several efficacy levels to differentially activate the receptor. GPCRs are primary drug targets due to their central role in several signal transduction pathways. Drug design for GPCRs is also most challenging due to their inherent promiscuity in ligand recognition, which gives rise to several side effects of existing drugs. Here, we have performed the ligand interaction study using the two prominent states of GPCR, namely the active and inactive state of the 5-HT_2A receptor. Active state of 5-HT_2A receptor model enhances the understanding of conformational difference which influences the ligand-binding site. A 5-HT_2A receptor active state model was constructed by homology modelling using active state β₂-adrenergic receptor (β₂-AR). In addition, virtual screening and docking studies with both active and inactive state models reveal potential small molecule hits which could be considered as agonist-like and antagonist-like molecules. The results from the all-atom molecular dynamics simulations further confirmed that agonists and antagonists interact in different modes with the receptor.     

GPCR-Kir Channel Signaling Complexes: Defining Rules of Engagement

Ion channels and G protein-coupled receptors (GPCRs) are integral transmembrane proteins vital to a multitude of cell signaling and physiological functions. Members of these large protein families are known to interact directly with various intracellular protein partners in a dynamic and isoform-dependent manner, ultimately shaping their life cycle and signal output. The family of G protein-gated inwardly rectifying potassium channels (Kir3 or GIRK) expressed in brain, heart, and endocrine tissues were recently shown to stably associate with several different GPCRs, forming the basis of a macromolecular ion channel-GPCR signaling complex. The molecular determinants that mediate and maintain GPCR-Kir3 channel complexes are currently not well understood. Recent findings and emerging hypotheses on the assembly and stability of multiprotein GPCR-Kir channel signaling complexes are discussed, highlighting distinct mechanisms used by different Kir channel families. These protein-protein interaction processes are crucial in determining both the synaptic response times and the extent of GPCR “cross-talk” in Kir3-mediated inhibitory synaptic transmission.     

GPCR-Kir channel signaling complexes: defining rules of engagement

Ion channels and G protein-coupled receptors (GPCRs) are integral transmembrane proteins vital to a multitude of cell signaling and physiological functions. Members of these large protein families are known to interact directly with various intracellular protein partners in a dynamic and isoform-dependent manner, ultimately shaping their life cycle and signal output. The family of G protein-gated inwardly rectifying potassium channels (Kir3 or GIRK) expressed in brain, heart, and endocrine tissues were recently shown to stably associate with several different GPCRs, forming the basis of a macromolecular ion channel-GPCR signaling complex. The molecular determinants that mediate and maintain GPCR-Kir3 channel complexes are currently not well understood. Recent findings and emerging hypotheses on the assembly and stability of multiprotein GPCR-Kir channel signaling complexes are discussed, highlighting distinct mechanisms used by different Kir channel families. These protein-protein interaction processes are crucial in determining both the synaptic response times and the extent of GPCR "cross-talk" in Kir3-mediated inhibitory synaptic transmission.     

GPCR signalling to the translation machinery

G protein-coupled receptors (GPCRs) are involved in most physiological processes, many of them being engaged in fully differentiated cells. These receptors couple to transducers of their own, primarily G proteins and β-arrestins, which launch intracellular signalling cascades. Some of these signalling events regulate the translational machinery to fine-tune general cell metabolism or to alter protein expression pattern. Though extensively documented for tyrosine kinase receptors, translational regulation by GPCRs is still poorly appreciated. The objective of this review paper is to address the following questions: i) is there a “GPCR signature” impacting on the translational machinery, and ultimately on the type of mRNA translated? ii) are the regulatory networks involved similar as those utilized by tyrosine kinase receptors? In particular, we will discuss the specific features of translational control mediated by GPCRs and highlight the intrinsic properties of GPCRs these mechanisms could rely on.     

Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis

The heptahelical G protein-coupled receptor (GPCR) family includes approximately 900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein-coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane and some internalized receptors continue to signal from intracellular compartments. In addition to rapid desensitization, receptor trafficking is critical for regulation of the temporal and spatial aspects of GPCR signaling. Indeed, GPCR internalization functions to control signal termination and propagation as well as receptor resensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin- and dynamin-dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin-mediated endocytosis of diverse cargo. Given the vast number and diversity of GPCRs, the complexity of clathrin-mediated endocytosis and the discovery of multiple clathrin adaptors, a single universal mechanism controlling endocytosis of all mammalian GPCRs is unlikely. Indeed, several recent studies now suggest that endocytosis of different GPCRs is regulated by distinct mechanisms and clathrin adaptors. In this review, we discuss the diverse mechanisms that regulate clathrin-dependent GPCR endocytosis.     

Consequences of lipid raft association on G-protein-coupled receptor function

GPCRs (G-protein-coupled receptors) play key roles in many cellular processes, and malfunction may lead to a range of pathologies, including psychiatric and neurological disorders. It is therefore not surprising that this group of receptors supplies a majority of the targets for pharmaceutical drug development. Despite their importance, the mechanisms that regulate their function and signalling still remain only partially understood. Recently, it has become evident that a subset of GPCRs is not homogeneously distributed in the plasma membrane, but localizes instead to specific membrane microdomains known as lipid rafts. Lipid rafts are characterized by their enrichment in cholesterol and sphingolipids, and have been suggested to serve as platforms for a range of cellular signalling complexes. In the present review, we will be discussing the effects of the lipid raft environment on trafficking, signalling and internalization of raft-associated GPCRs.     

GPCRs: an update on structural approaches to drug discovery

G-protein-coupled receptors (GPCRs) are a major opportunity for drug discovery in the post-genomic era. There are thought to be more than 500 therapeutically relevant GPCRs out of a total of over 700 identified to date, although only one, rhodopsin, has been the subject of a full 3D X-ray crystallography study. Two structurally related proteins, bacteriorhodopsin and sensory rhodopsin, which are not GPCRs but are part of the seven-helix membrane receptor family, have also been the subject of X-ray crystallographic studies and have been used in GPCR modeling studies. The significant differences between these rhodopsin structures, the relatively low sequence homology between individual GPCRs, and some difficulties in rationalizing point-mutation data suggests that homology-based molecular modeling alone will not provide the accurate structural information on individual receptors required for ligand design and in silico screening. In the absence of such structural information, several approaches can be used to assist in the discovery of ligands.     

Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins

Olfaction in Drosophila is mediated by a large family of membrane-bound odorant receptor proteins (Ors). In heterologous cells, we investigated whether the structural features and signalling mechanisms of ligand-binding Drosophila Ors are consistent with them being G protein-coupled receptors (GPCRs). The detailed membrane topology of Or22a was determined by inserting epitope tags into the termini and predicted loop regions. Immunocytochemistry experiments in Drosophila S2 cells imply that Or22a has seven transmembrane domains but that its membrane topology is opposite to that of GPCRs, with a cytoplasmic N-terminus and extracellular C-terminus. To investigate Or signalling mechanisms, we expressed Or43b in Sf9 and HEK293 cells, and show that inhibitors of heterotrimeric G proteins (GDP-beta-S), adenylate cyclase (SQ22536), guanylyl cyclase (ODQ), cyclic nucleotide phosphodiesterases (IBMX) and phospholipase C (U73122) have negligible impact on Or43b responses. Whole cell patching of Or43b/Or83b-transfected HEK293 cells revealed the opening of plasma membrane cation channels on addition of ligand. The response was blocked by lanthanum and by 2-APB, but not by Ruthenium red or SKF96365. Based on these data, we conclude that Drosophila Ors comprise a novel family of seven transmembrane receptors that in HEK293 cells signal by opening cation channels, through a mechanism that is largely independent of G proteins.     

Partial adenosine A1 receptor agonists for cardiovascular therapies

Extracellular purines and pyrimidines have emerged as key regulators of a wide range of physiological and pathophysiological cellular processes acting through P1 and P2 cell surface receptors. Increasing evidence suggests that purinergic receptors can interact with and/or modulate the activity of other classes of receptors and ion channels. This review will focus on the interactions of purinergic receptors with other GPCRs, ion channels, receptor tyrosine kinases, and steroid hormone receptors. Also, the signal transduction pathways regulated by these complexes and their new functional properties are discussed.     

Opioid Receptor Regulation of Neuronal Voltage-Gated Calcium Channels

Neuronal voltage-gated calcium channels play a pivotal role in the conversion of electrical signals into calcium entry into nerve endings that is required for the release of neurotransmitters. They are under the control of a number of cellular signaling pathways that serve to fine tune synaptic activities, including G-protein coupled receptors (GPCRs) and the opioid system. Besides modulating channel activity via activation of second messengers, GPCRs also physically associate with calcium channels to regulate their function and expression at the plasma membrane. In this mini review, we discuss the mechanisms by which calcium channels are regulated by classical opioid and nociceptin receptors. We highlight the importance of this regulation in the control of neuronal functions and their implication in the development of disease conditions. Finally, we present recent literature concerning the use of novel μ-opioid receptor/nociceptin receptor modulators and discuss their use as potential drug candidates for the treatment of pain.

     

Do orphan G-protein-coupled receptors have ligand-independent functions? New insights from receptor heterodimers

G-protein-coupled receptors (GPCRs) are important drug targets and are involved in virtually every biological process. However, there are still more than 140 orphan GPCRs, and deciphering their function remains a priority for fundamental and clinical research. Research on orphan GPCRs has concentrated mainly on the identification of their natural ligands, whereas recent data suggest additional ligand-independent functions for these receptors. This emerging concept is connected with the observation that orphan GPCRs can heterodimerize with GPCRs that have identified ligands, and by so doing regulate the function of the latter. Pairing orphan GPCRs with their potential heterodimerization partners will have a major impact on our understanding of the extraordinary diversity offered by GPCR heterodimerization and, in addition, will constitute a novel strategy to elucidate the function of orphan receptors that needs to be added to the repertoire of 'deorphanization' strategies.     

Mechanisms of selective G protein–coupled receptor localization and trafficking

The trafficking of G protein–coupled receptors (GPCRs) to different membrane compartments has recently emerged as being a critical determinant of the signaling profiles of activation. GPCRs, which share many structural and functional similarities, also share many mechanisms that traffic them between compartments. This sharing raises the question of how the trafficking of individual GPCRs is selectively regulated. Here, we will discuss recent studies addressing the mechanisms that contribute to selectivity in endocytic and biosynthetic trafficking of GPCRs.     

胞外酸性与肿瘤的浸润转移

组织缺氧是实体瘤的一个主要特征,它引起肿瘤细胞胞外酸性环境的形成.肿瘤细胞通过质子感知的G蛋白偶联受体（G protein-coupled receptors,GPCRs）或质子感知的离子通道感知其胞外的酸性环境,并激活多条细胞内信号通路,影响细胞功能. 肿瘤最致命的方面在于其转移能力,肿瘤转移程度与肿瘤细胞迁移能力呈正相关. 因此,对胞外酸性与肿瘤细胞迁移扩散之间关系的深入研究将有助于发现更多新的抗肿瘤转移药物.本文就肿瘤酸性微环境的形成、肿瘤细胞的质子感知制、胞外酸性环境对肿瘤浸润转移的影响及如何将肿瘤pH调节应用于癌症治疗等方面的内容予以综述.