Use of Kaede fusions to visualize recycling of G protein-coupled receptors

The heptahelical G protein-coupled receptors (GPCRs) are internalized following agonist treatment and either recycle rapidly to the plasma membrane or enter the lysosomal degradation pathway. Many conventional GPCR recycling assays suffer from the fact that receptors arriving from the secretory pathway may interfere with recycling receptors. In this study, we introduce a new methodology to study post-endocytotic GPCR trafficking using fusions with the recently cloned Kaede protein. In contrast to the widely used green fluorescent protein, the fluorescence of Kaede can be converted from green to red using ultraviolet irradiation. Our methodology allows to study recycling of GPCRs microscopically in real-time bypassing problems with secretory pathway receptors. Initially, receptors are internalized using an agonist. Fluorescence signals in endosomes are switched, and trafficking of the receptors to the plasma membrane can be easily visualized by monitoring their new fluorescence. Using this methodology, we show that the corticotropin-releasing factor receptor type 1 belongs to the family of recycling GPCRs. Moreover, we demonstrate by fluorescence correlation spectroscopy that Kaede does not oligomerize when fused to membrane proteins, representing an additional advantage of this technique. The Kaede technology may be a powerful tool to study membrane protein trafficking in general.     

Ubiquitination-independent trafficking of G protein-coupled receptors to lysosomes

Ubiquitination of cytoplasmic lysine residues can target G protein-coupled receptors (GPCRs) to proteasomes and has recently been shown to also be required for sorting of certain GPCRs to lysosomes following ligand-induced endocytosis. We addressed the generality of this mechanism by examining regulated proteolysis of the murine delta opioid receptor (DOR) expressed in human embryonic kidney 293 cells, a well characterized model system in which receptors are sorted to lysosomes. Incubation of cells in the presence of the highly specific proteasome inhibitor lactacystin did not detectably affect ligand-induced proteolysis of DOR but significantly delayed ligand-induced proteolysis of epidermal growth factor receptors. Mutation of all cytoplasmic lysine residues in DOR, creating a mutant opioid receptor that is unable to be ubiquitinated, did not detectably inhibit either ligand-induced endocytosis or proteolytic degradation of endocytosed receptors. Furthermore, the lysine-mutated DOR, like its wild type counterpart, colocalized extensively with lysosomes after ligand-induced endocytosis. These results demonstrate that ubiquitination of DOR is not required either for its ligand-induced endocytosis or for postendocytic trafficking to lysosomes.     

The regulatory mechanisms of export trafficking of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are a superfamily of cell-surface receptors that regulate a variety of cell functions by responding to a myriad of ligands. The magnitude of the response elicited by a ligand is dictated by the level of receptor available at the plasma membrane. GPCR expression levels at the cell surface are a balance of three highly regulated, dynamic intracellular trafficking processes, namely export, internalization and degradation. This review will cover recent advances in understanding the mechanism underlying GPCR export trafficking by focusing on specific motifs required for ER export and the role of the Ras-like Rab1 GTPase and glycosylation in regulating ER–Golgi-cell-surface transport. The manifestation of diseases due to the disruption of GPCR export is also discussed.     

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.     

Regulation of G protein-coupled receptor export trafficking

G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling.     

Regulation of G protein-coupled receptor export trafficking

G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling.     

G蛋白偶联受体计算研究的进展和前瞻

G蛋白偶联受体(G protein-coupled receptor,GPCR)是含有七个跨膜螺旋的一类重要蛋白,是迄今为止发现的最大的多药物靶标受体超蛋白家族。例如,目前上市药物中有超过30%是以GPCR为靶点的。然而,与GPCR重要性形成强烈反差的是科学界对于其结构与功能的了解非常贫乏,主要原因是通过实验手段来获得GPCR的结构与功能信息极其困难。利用生物信息学方法从基因组规模的数据中识别GPCR并预测三维结构是可行途径之一。基于生物信息学的GPCR研究将为新型药物靶标的筛选和药物的开发提供一定的帮助。本文论述了几种较为典型的GPCR计算方法,并基于已有研究提出可能的创新性研究策略来解决GPCR蛋白识别、跨膜区定位、以及结构和功能预测等问题。     

Molecular chaperones and subcellular trafficking of steroid receptors

Unliganded steroid receptors exist as heteromeric complexes comprised of heat shock and immunophilin proteins that associate either directly or indirectly with receptor carboxyl–terminal ligand-binding domains. Molecular chaperons, and other proteins associated with steroid receptors, play an important role in the maturation of receptors to a hormone-binding competent state. Steroid receptor-associated 90 and 70 kDa heat shock proteins, hsp90 and hsp70, respectively, have well established roles in protein folding in addition to participating in numerous subcellular trafficking pathways. In this review, we discuss the possible roles that molecular chaperons, such as hsp90, hsp70 and DnaJ proteins, have in steroid receptor trafficking within two distinct subcellular compartments, i.e. the cytoplasm and nucleus.     

Downregulation of G protein-coupled receptors

Major advances have been made in understanding mechanisms mediating downregulation of G protein-coupled receptors. Recent studies emphasize the role of multiple proteolytic mechanisms in downregulation. A specific mechanism of downregulation, mediated by endocytosis of receptors via clathrin-coated pits followed by sorting to lysosomes, has been examined in detail. Specific protein interactions that control the specificity of G-protein-coupled receptor trafficking in this pathway are beginning to be elucidated.     

Use of fluorescence polarization detection for the measurement of fluopeptidetm binding to G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the single largest molecular target of therapeutic drugs currently on the market, and are also the most common target in high throughput screening assays designed to identify potential new drug candidates. A large percentage of these assays are now formatted as radioligand binding assays. Fluorescence polarization ligand binding assays can offer a non-rad alternative to radioligand binding assays. In addition, fluorescence polarization assays are a homogenous format that is easy to automate for high throughput screening. We have developed a series of peptide ligands labeled with the fluorescent dye BODIPY TMR whose binding to GPCRs can be detected using fluorescence polarization methodology. BODIPY TMR has advantages over the more commonly used fluorescein dye in high throughput screening (HTS) assays due to the fact that its excitation and emission spectra are red-shifted approximately 50 nm relative to fluorescein. Assays based on BODIPY TMR ligands are therefore less susceptible to interference from tissue auto-fluorescence in the assay matrix, or the effects of colored or fluorescent compounds in the screening libraries. A series of BODIPY TMR labeled peptides have been prepared that bind to a range of GPCRs including melanin concentrating hormone, bradykinin, and melanocortin receptors. Conditions have been optimized in order to utilize a comparable amount of receptor membrane preparation as is used in a radioligand binding assay. The assays are formatted in 384-well microplates with a standard volume of 40 microL. We have compared the assays across the different fluorescence polarization (FP) readers available to determine the parameters for each instrument necessary to achieve the required precision.     

Innovative functional cAMP assay for studying G protein-coupled receptors: application to the pharmacological characterization of GPR17

In this work, an innovative and non-radioactive functional cAMP assay was validated at the GPR17 receptor. This assay provides a simple and powerful new system to monitor G protein-coupled receptor activity through change in the intracellular cAMP concentration by using a mutant form of Photinus pyralis luciferase into which a cAMP-binding protein moiety has been inserted. Results, expressed as EC₅₀ or IC₅₀ values for agonists and antagonists, respectively, showed a strong correlation with those obtained with [³⁵S]GTPγS binding assay, thus confirming the validity of this approach in the study of new ligands for GPR17. Moreover, this method allowed confirming that GPR17 is coupled with a G_αi.     

Methods to monitor the quaternary structure of G protein-coupled receptors

A wide range of approaches has been applied to examine the quaternary structure of G protein-coupled receptors, the basis of such protein-protein interactions and how such interactions might modulate the pharmacology and function of these receptors. These include co-immunoprecipitation, various adaptations of resonance energy transfer techniques, functional complementation studies and the analysis of ligand-binding data. Each of the available techniques has limitations that restrict interpretation of the data. However, taken together, they provide a coherent body of evidence indicating that many, if not all, G protein-coupled receptors exist and function as dimer/oligomers. Herein we assess the widely applied techniques and discuss the relative benefits and limitations of these approaches.     

Endosomal trafficking of the G protein-coupled receptor somatostatin receptor 3

Intracellular trafficking of G protein-coupled receptors (GPCRs) regulates their surface availability and determines cellular response to agonists. Rab GTPases regulate membrane trafficking and identifying Rab networks controlling GPCR trafficking is essential for understanding GPCR signaling. We used real time imaging to show that somatostatin receptor 3 (SSTR3) traffics through Rab4-, Rab21-, and Rab11-containing endosomes, but largely bypasses Rab5 and Rab7 endosomes. We show that SSTR3 rapidly traffics through Rab4 endosomes but moves slower through Rab21 and Rab11 endosomes. SSTR3 passage through each endosomal compartment is regulated by the cognate Rab since expression of the inactive Rab4/S22N, Rab21/T33N, and Rab11/S25N inhibits SSTR3 trafficking. Thus, Rab4, Rab21, and Rab11 may represent therapeutic targets to modulate surface availability of SSTR3 for agonist binding. Our novel finding that Rab21 regulates SSTR3 trafficking suggests that Rab21 may play a role in trafficking of other GPCRs.     

Structural biology of G protein-coupled receptors

More than 60% of the current drugs are based on G protein-coupled receptors. Paradoxically, high-resolution structures are not available to facilitate rational drug design. Difficulties in expression, purification, and crystallization of these transmembrane receptors are the reasons for the low success rate. Recent individual and network-based technology development has significantly improved our knowledge of structural biology and might soon bring a major breakthrough in this area.     

Mechanisms of G protein-coupled receptors regulation

Within the last two decades of studies in the ever-expanding field of GPCR signaling, challenging insights were adopted. Growing evidence now asists the shift from classical linear model of signaling towards a considerably complex network of signaling pathways with many shared proteins and cross-talks. Considering the extensive and intriguing network of pathways activated by these receptors, it is apparent that multi-level system of regulation must exist to rigorously modulate the amplitude, duration and spatial aspects of the GPCR signaling. This review summarizes the principal mechanisms of GPCR regulation and gives the overview of recent advances in this field of research.     

Splice variants of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are encoded by a vast gene superfamily, reflecting the large number of ligands that must be specifically recognized at any given cell surface. The discovery that the variety of GPCRs is further expanded through the generation of splice variants was therefore somewhat surprising. Studies of the functional consequences of alternative splicing have focused on ligand binding, signaling, constitutive activity, and downregulation. However, GPCRs also appear to interact directly with many other intracellular proteins in addition to G proteins. Intriguingly, the domains involved in these interactions are the predominant sites of variation arising through splicing.     

G蛋白偶联受体的结构生物学研究

G蛋白偶联受体(G protein-coupled receptor,GPCR)在细胞信号转导过程中发挥关键的生理学功能,是极其重要的药物靶标,其三维结构信息对功能研究以及新药研发具有十分重要的意义。近年来,新技术的发展和应用使GPCR的结构生物学研究发生了跨越式的发展,本文简要回顾这些新的技术和方法以及已解析的GPCR三维结构,并以CCR5和P2Y12R两种受体的结构为例来具体阐明现阶段GPCR结构生物学研究的内容和意义。     

Desensitization of herpesvirus-encoded G protein-coupled receptors

Members of the herpesvirus family, including human cytomegalovirus (HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), encode G protein-coupled receptor (GPCR) homologs, which strongly activate classical G protein signal transduction networks within the cell. In animal models of herpesvirus infection, the viral GPCRs appear to play physiologically important roles by enabling viral replication within tropic tissues and by promoting reactivation from latency. While a number of studies have defined intracellular signaling pathways activated by herpesviral GPCRs, it remains unclear if their physiological function is subjected to the process of desensitization as observed for cellular GPCRs. G protein-coupled receptor kinases (GRK) and arrestin proteins have been recently implicated in regulating viral GPCR signaling; however, the role that these desensitization proteins play in viral GPCR function in vivo remains unknown. Here, we review what is currently known regarding viral GPCR desensitization and discuss potential biological ramifications of viral GPCR regulation by the host cell desensitization machinery.