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.     

Sensing and transduction of nutritional and chemical signals in filamentous fungi: Impact on cell development and secondary metabolites biosynthesis

Filamentous fungi respond to hundreds of nutritional, chemical and environmental signals that affect expression of primary metabolism and biosynthesis of secondary metabolites. These signals are sensed at the membrane level by G protein coupled receptors (GPCRs). GPCRs contain usually seven transmembrane domains, an external amino terminal fragment that interacts with the ligand, and an internal carboxy terminal end interacting with the intracellular G protein. There is a great variety of GPCRs in filamentous fungi involved in sensing of sugars, amino acids, cellulose, cell-wall components, sex pheromones, oxylipins, calcium ions and other ligands. Mechanisms of signal transduction at the membrane level by GPCRs are discussed, including the internalization and compartmentalisation of these sensor proteins. We have identified and analysed the GPCRs in the genome of Penicillium chrysogenum and compared them with GPCRs of several other filamentous fungi. We have found 66 GPCRs classified into 14 classes, depending on the ligand recognized by these proteins, including most previously proposed classes of GPCRs. We have found 66 putative GPCRs, representatives of twelve of the fourteen previously proposed classes of GPCRs, depending on the ligand recognized by these proteins. A staggering fortytwo putative members of the new GPCR class XIV, the so-called Pth11 sensors of cellulosic material as reported for Neurospora crassa and some other fungi, were identified. Several GPCRs sensing sex pheromones, known in yeast and in several fungi, were also identified in P. chrysogenum, confirming the recent unravelling of the hidden sexual capacity of this species. Other sensing mechanisms do not involve GPCRs, including the two-component systems (HKRR), the HOG signalling system and the PalH mediated pH transduction sensor. GPCR sensor proteins transmit their signals by interacting with intracellular heterotrimeric G proteins, that are well known in several fungi, including P. chrysogenum. These G proteins are inactive in the GDP containing heterotrimeric state, and become active by nucleotide exchange, allowing the separation of the heterotrimeric protein in active Gα and Gβγ dimer subunits. The conversion of GTP in GDP is mediated by the endogenous GTPase activity of the G proteins. Downstream of the ligand interaction, the activated Gα protein and also the Gβ/Gγ dimer, transduce the signals through at least three different cascades: adenylate cyclase/cAMP, MAPK kinase, and phospholipase C mediated pathways.     

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.     

The GAPs,GEFs, GDIs and…now,GEMs: New kids on the heterotrimeric G protein signaling block

The canonical process of activation of heterotrimeric G proteins by G protein coupled receptors (GPCRs) is well studied. Recently, a rapidly emerging paradigm has revealed the existence of a new, non-canonical set of cytosolic G protein modulators, guanine exchange modulators (GEMs). Among G proteins regulators, GEMs are uniquely capable of initiating pleiotropic signals: these bifunctional modulators can activate cAMP inhibitory (Gi) proteins and inhibit cAMP-stimulatory (Gs) proteins through a single short evolutionarily conserved module. A prototypical member of the GEM family, GIV/Girdin, integrates signals downstream of a myriad of cell surface receptors, e.g., growth factor RTKs, integrins, cytokine, GPCRs, etc., and translates these signals into G protein activation or inhibition. By their pleiotropic action, GIV and other GEMs modulate several key pathways within downstream signaling network. Unlike canonical G protein signaling that is finite and is triggered directly and exclusively by GPCRs, the temporal and spatial features of non-canonical activation of G protein via GIV-family of cytosolic GEMs are unusually relaxed. GIV uses this relaxed circuitry to integrate, reinforce and compartmentalize signals downstream of both growth factors and G proteins in a way that enables it to orchestrate cellular phenotypes in a sustained manner. Mounting evidence suggests the importance of GIV and other GEMs as disease modulators and their potential to serve as therapeutic targets; however, a lot remains unknown within the layers of the proverbial onion that must be systematically peeled. This perspective summarizes the key concepts of the GEM-dependent G protein signaling paradigm and discusses the multidisciplinary approaches that are likely to revolutionize our understanding of this paradigm from the atomic level to systems biology.     

Recognition in the face of diversity: interactions of heterotrimeric G proteins and G protein-coupled receptor (GPCR) kinases with activated GPCRs

G protein-coupled receptors (GPCRs) represent the largest class of integral membrane protein receptors in the human genome. Despite the great diversity of ligands that activate these GPCRs, they interact with a relatively small number of intracellular proteins to induce profound physiological change. Both heterotrimeric G proteins and GPCR kinases are well known for their ability to specifically recognize GPCRs in their active state. Recent structural studies now suggest that heterotrimeric G proteins and GPCR kinases identify activated receptors via a common molecular mechanism despite having completely different folds.     

Tuberin: a stimulus-regulated tumor suppressor protein controlled by a diverse array of receptor tyrosine kinases and G protein-coupled receptors

Tuberin, a tumor suppressor protein, is involved in various cellular functions including survival, proliferation, and growth. It has emerged as an important effector regulated by receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Regulation of tuberin by RTKs and GPCRs is highly complex and dependent on the type of receptors and their associated signaling molecules. Apart from Akt, the first kinase recognized to phosphorylate and inactivate tuberin upon growth factor stimulation, an increasing number of kinases upstream of tuberin have been identified. Furthermore, recruitment of different scaffolding adaptor components to the activated receptors appears to play an important role in the regulation of tuberin activity. More recently, the differential regulation of tuberin by various G protein family members have also been intensively studied, it appears that G proteins can both facilitate (e.g., G(i/o)) as well as inhibit (e.g., G(q)) tuberin phosphorylation. In the present review, we attempt to summarize our emerging understandings of the roles of RTKs, GPCRs, and their cross-talk on the regulation of tuberin.     

Identification of a novel family of G protein-coupled receptor associated sorting proteins

During the past few years several new interacting partners for G protein-coupled receptors (GPCRs) have been discovered, suggesting that the activity of these receptors is more complex than previously anticipated. Recently, candidate G protein-coupled receptor associated sorting protein (GASP-1) has been identified as a novel interacting partner for the delta opioid receptor and has been proposed to determine the degradative fate of this receptor. We show here that GASP-1 associates in vitro with other opioid receptors and that the interaction domain in these receptors is restricted to a small portion of the carboxyl-terminal tail, corresponding to helix 8 in the three-dimensional structure of rhodopsin. In addition, we show that GASP-1 interacts with COOH-terminus of several other GPCRs from subfamilies A and B and that two conserved residues within the putative helix 8 of these receptors are critical for the interaction with GASP-1. In situ hybridization and northern blot analysis indicate that GASP-1 mRNA is mainly distributed throughout the central nervous system, consistent with a potential interaction with numerous GPCRs in vivo. Finally, we show that GASP-1 is a member of a novel family comprising at least 10 members, whose genes are clustered on chromosome X. Another member of the family, GASP-2, also interacts with the carboxyl-terminal tail of several GPCRs. Therefore, GASP proteins may represent an important protein family regulating GPCR physiology.     

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.     

A reporter assay for G-protein-coupled receptors using a B-cell line suitable for stable episomal expression

We have established a cAMP response element (CRE)-mediated reporter assay system for G-protein-coupled receptors (GPCRs) using an oriP-based estrogen-inducible expression vector and the B-cell line (GBC53 or GBCC71) that expresses EBNA-1 and is adapted to serum-free culture. GBC53 harbors a GAL4-ER expression unit and a CRE-luciferase gene in the genome, and GBCC71 also harbors expression units for two chimeric Gαs proteins (Gs/q and Gs/i). Introduction of a GPCR expression plasmid into GBC53 or GBCC71 creates polyclonal stable transformants in 2 weeks, and these are easily expanded and used for assays after induction of the GPCR expression. Using GBC53, we detected ligand-dependent signals of Gs-coupled GPCRs such as glucagon-like peptide 1 receptor (GLP1R) and β2 adrenergic receptor (β2AR) with high sensitivity. Interestingly, we also detected constitutive activity of β2AR. Using GBCC71, we detected ligand-dependent signals of Gq- or Gi-coupled GPCRs such as H1 histamine receptor and CXCR1 chemokine receptor in addition to Gs-coupled GPCRs. An agonist, antagonist, or inverse agonist was successfully evaluated in this system. We succeeded in constructing a 384-well high-throughput screening (HTS) system for GLP1R. This system enabled us to easily and rapidly make a large number of efficient GPCR assay systems suitable for HTS as well as ligand hunting of orphan GPCRs.     

Reversible translocation of p115-RhoGEF by G(12/13)-coupled receptors

G protein-coupled receptors (GPCRs) are important targets for medicinal agents. Four different G protein families, G(s), G(i), G(q), and G(12), engage in their linkage to activation of receptor-specific signal transduction pathways. G(12) proteins were more recently studied, and upon activation by GPCRs they mediate activation of RhoGTPase guanine nucleotide exchange factors (RhoGEFs), which in turn activate the small GTPase RhoA. RhoA is involved in many cellular and physiological aspects, and a dysfunction of the G(12/13)-Rho pathway can lead to hypertension, cardiovascular diseases, stroke, impaired wound healing and immune cell functions, cancer progression and metastasis, or asthma. In this study, regulator of G protein signaling (RGS) domain-containing RhoGEFs were tagged with enhanced green fluorescent protein (EGFP) to detect their subcellular localization and translocation upon receptor activation. Constitutively active Galpha(12) and Galpha(13) mutants induced redistribution of these RhoGEFs from the cytosol to the plasma membrane. Furthermore, a pronounced and rapid translocation of p115-RhoGEF from the cytosol to the plasma membrane was observed upon activation of several G(12/13)-coupled GPCRs in a cell type-independent fashion. Plasma membrane translocation of p115-RhoGEF stimulated by a GPCR agonist could be completely and rapidly reversed by subsequent application of an antagonist for the respective GPCR, that is, p115-RhoGEF relocated back to the cytosol. The translocation of RhoGEF by G(12/13)-linked GPCRs can be quantified and therefore used for pharmacological studies of the pathway, and to discover active compounds in a G(12/13)-related disease context.     

Helix packing moments reveal diversity and conservation in membrane protein structure

Helical membrane proteins are more tightly packed and the packing interactions are more diverse than those found in helical soluble proteins. Based on a linear correlation between amino acid packing values and interhelical propensity, we propose the concept of a helix packing moment to predict the orientation of helices in helical membrane proteins and membrane protein complexes. We show that the helix packing moment correlates with the helix interfaces of helix dimers of single pass membrane proteins of known structure. Helix packing moments are also shown to help identify the packing interfaces in membrane proteins with multiple transmembrane helices, where a single helix can have multiple contact surfaces. Analyses are described on class A G protein-coupled receptors (GPCRs) with seven transmembrane helices. We show that the helix packing moments are conserved across the class A family of GPCRs and correspond to key structural contacts in rhodopsin. These contacts are distinct from the highly conserved signature motifs of GPCRs and have not previously been recognized. The specific amino acid types involved in these contacts, however, are not necessarily conserved between subfamilies of GPCRs, indicating that the same protein architecture can be supported by a diverse set of interactions. In GPCRs, as well as membrane channels and transporters, amino acid residues with small side-chains (Gly, Ala, Ser, Cys) allow tight helix packing by mediating strong van der Waals interactions between helices. Closely packed helices, in turn, facilitate interhelical hydrogen bonding of both weakly polar (Ser, Thr, Cys) and strongly polar (Asn, Gln, Glu, Asp, His, Arg, Lys) amino acid residues. We propose the use of the helix packing moment as a complementary tool to the helical hydrophobic moment in the analysis of transmembrane sequences.     

G protein-coupled receptor systems and their lipid environment in health disorders during aging

Cells, tissues and organs undergo phenotypic changes and deteriorate as they age. Cell growth arrest and hyporesponsiveness to extrinsic stimuli are all hallmarks of senescent cells. Most such external stimuli received by a cell are processed by two different cell membrane systems: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). GPCRs form the largest gene family in the human genome and they are involved in most relevant physiological functions. Given the changes observed in the expression and activity of GPCRs during aging, it is possible that these receptors are directly involved in aging and certain age-related pathologies. On the other hand, both GPCRs and G proteins are associated with the plasma membrane and since lipid-protein interactions regulate their activity, they can both be considered to be sensitive to the lipid environment. Changes in membrane lipid composition and structure have been described in aged cells and furthermore, these membrane changes have been associated with alterations in GPCR mediated signaling in some of the main health disorders in elderly subjects. Although senescence could be considered a physiologic process, not all aging humans develop the same health disorders. Here, we review the involvement of GPCRs and their lipid environment in the development of the major human pathologies associated with aging such as cancer, neurodegenerative disorders and cardiovascular pathologies.     

Signaling mechanisms and physiological functions of G‐protein Gα13 in blood vessel formation,bone homeostasis,and cancer

Heterotrimeric G‐proteins are cellular signal transducers. They mainly relay signals from G‐protein‐coupled receptors (GPCRs). GPCRs function as guanine nucleotide‐exchange factors to active these G‐proteins. Based on the sequence and functional similarities, these G‐proteins are grouped into four subfamilies: G_s, G_i, G_q, and G_12/13. The G_12/13 subfamily consists of two members: G₁₂ and G₁₃. G_12/13‐mediated signaling pathways play pivotal roles in a variety of physiological processes, while aberrant regulation of this pathway has been identified in various human diseases. Here we summarize the signaling mechanisms and physiological functions of Gα₁₃ in blood vessel formation and bone homeostasis. We further discuss the expanding roles of Gα₁₃ in cancers, serving as oncogenes as well as tumor suppressors.     

Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases

G protein-coupled receptors (GPCRs) are integral membrane proteins that, in response to activation by extracellular stimuli, regulate intracellular second messenger levels via their coupling to heterotrimeric G proteins. GPCR activation also initiates a series of molecular events that leads to G protein-coupled receptor kinase-mediated receptor phosphorylation and the binding of beta-arrestin proteins to the intracellular face of the receptor. beta-Arrestin binding not only contributes to the G protein-uncoupling of GPCRs, but also mediates the targeting of many GPCRs for endocytosis in clathrin-coated pits. Several GPCRs internalize as a stable complex with beta-arrestin and the stability of this complex appears to regulate, at least in part, whether the receptors are dephosphorylated in early endosomes and recycled back to the cell surface as fully functional receptors, retained in early endosomes or targeted for degradation in lysosomes. More recently, it has become appreciated that the movement of GPCRs through functionally distinct intracellular membrane compartments is regulated by a variety of Rab GTPases and that the activity of these Rab GTPases may influence GPCR function. Moreover, it appears that GPCRs are not simply passive cargo molecules, but that GPCR activation may directly influence Rab GTPase activity and as such, GPCRs may directly control their own targeting between intracellular compartments. This review provides a synopsis of the current knowledge regarding the role of beta-arrestins and Rab GTPases in regulating the intracellular trafficking and function of GPCRs.     

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.     

Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits

G protein-coupled receptors (GPCRs) constitute one of the largest families of genes in the human genome, and are the largest targets for drug development. Although a large number of GPCR genes have recently been identified, ligands have not yet been identified for many of them. Various assay systems have been employed to identify ligands for orphan GPCRs, but there is still no simple and general method to screen for ligands of such GPCRs, particularly of G(i)-coupled receptors. We have examined whether fusion proteins of GPCRs with G protein alpha subunit (Galpha) could be utilized for ligand screening and showed that the fusion proteins provide an effective method for the purpose. This article focuses on the followings: (1) characterization of GPCR genes and GPCRs, (2) identification of ligands for orphan GPCRs, (3) characterization of GPCR-Galpha fusion proteins, and (4) identification of ligands for orphan GPCRs using GPCR-Galpha fusion proteins.     

Lipids, lipid rafts and caveolae: their importance for GPCR signaling and their centrality to the endocannabinoid system

Scientific views of cell membrane organization are presently changing. Rather than serving only as the medium through which membrane proteins diffuse, lipid bilayers have now been shown to form compartmentalized domains with different biophysical properties (rafts/caveolae). For membrane proteins such as the G protein coupled receptors (GPCRs), a raft domain provides a platform for the assembly of signaling complexes and prevents cross-talk between pathways. Lipid composition also has a strong influence on the conformational activity of GPCRs. For certain GPCRs, such as the cannabinoid receptors, the lipid bilayer has additional significance. Endocannabinoids such as anandamide (AEA) are created in a lipid bilayer from lipid and act at the membrane embedded CB1 receptor. Endocannabinoids exiting the CB1 receptor are transported either by a carrier-mediated or a simple diffusion process to the membrane of the postsynaptic cell. Following cellular uptake, perhaps via caveolae/lipid raft-related endocytosis, AEA is rapidly metabolized by a membrane-associated enzyme, fatty acid amide hydrolase (FAAH) located in the endoplasmic reticulum. The entry point for AEA into FAAH appears to be from the lipid bilayer. This review explores the importance of lipid composition and lipid rafts to GPCR signaling and then focuses on the intimate relationship that exists between the lipid environment and the endocannabinoid system.     

G蛋白偶联受体与G蛋白相互作用的最新研究进展

传统观念认为,在激动剂作用下,G蛋白偶联受体(GPCRs)能够激活G蛋白的α亚基,从而使Gα亚基与Gβγ亚基分离,被激活的Gα亚基通过信号转导进一步参与细胞的生理过程。但是,最新研究发现GPCRs和G蛋白存在多种偶联关系,GPCRs不仅能够激活Gα亚基,还可以与Gβγ亚基相互靠近,甚至会使G蛋白亚基构象发生重排而不分离,这对于疾病发病机制的研究及新的药物靶点的发现具有重要意义。就GPCRs与G蛋白之间的相互作用以及最新研究技术作一简要综述。     

G‐protein coupled receptor array technologies: Site directed immobilisation of liposomes containing the H1‐histamine or M2‐muscarinic receptors

This paper describes a novel strategy to create a microarray of G‐protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H₁‐histamine receptor and the M₂‐muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol‐modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM‐D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the ZeptoREADER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self‐sorting liposome array of GPCRs which would underpin a variety of future novel applications.     

Soluble mimics of a chemokine receptor: chemokine binding by receptor elements juxtaposed on a soluble scaffold

Despite the broad biological importance of G protein-coupled receptors (GPCRs), ligand recognition by GPCRs remains poorly understood. To explore the roles of GPCR extracellular elements in ligand binding and to provide a tractable system for structural analyses of GPCR/ligand interactions, we have developed a soluble protein that mimics ligand recognition by a GPCR. This receptor analog, dubbed CROSS5, consists of the N-terminal and third extracellular loop regions of CC chemokine receptor 3 (CCR3) displayed on the surface of a small soluble protein, the B1 domain of Streptococcal protein G. CROSS5 binds to the CCR3 ligand eotaxin with a dissociation equilibrium constant of 2.9 +/- 0.8 microM and competes with CCR3 for eotaxin binding. Control proteins indicate that juxtaposition of both CCR3 elements is required for optimal binding to eotaxin. Moreover, the affinities of CROSS5 for a series of eotaxin mutants are highly correlated with the apparent affinities of CCR3 for the same mutants, demonstrating that CROSS5 uses many of the same interactions as does the native receptor. The strategy used to develop CROSS5 could be applied to many other GPCRs, with a variety of potential applications.