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.     

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.     

S-nitrosylation-regulated GPCR signaling

G protein-coupled receptors (GPCRs) are the most numerous and diverse type of cell surface receptors, accounting for about 1% of the entire human genome and relaying signals from a variety of extracellular stimuli that range from lipid and peptide growth factors to ions and sensory inputs. Activated GPCRs regulate a multitude of target cell functions, including intermediary metabolism, growth and differentiation, and migration and invasion. The GPCRs contain a characteristic 7-transmembrane domain topology and their activation promotes complex formation with a variety of intracellular partner proteins, which form basis for initiation of distinct signaling networks as well as dictate fate of the receptor itself. Both termination of active GPCR signaling and removal from the plasma membrane are controlled by protein post-translational modifications of the receptor itself and its interacting partners. Phosphorylation, acylation and ubiquitination are the most studied post-translational modifications involved in GPCR signal transduction, subcellular trafficking and overall expression. Emerging evidence demonstrates that protein S-nitrosylation, the covalent attachment of a nitric oxide moiety to specified cysteine thiol groups, of GPCRs and/or their associated effectors also participates in the fine-tuning of receptor signaling and expression. This newly appreciated mode of GPCR system modification adds another set of controls to more precisely regulate the many cellular functions elicited by this large group of receptors. This article is part of a Special Issue entitled: Regulation of cellular processes by S-nitrosylation.     

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.     

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.     

G protein-coupled receptors stimulation and the control of cell migration

Cell migration is a fundamental biological process involved in normal physiology. Altered motile phenotypes are however often associated with the development and progression of diseases such as cancer and atherosclerosis. Remodeling of the actin cytoskeleton is required for cell shape changes and is controlled by a broad variety of cellular proteins. Interestingly, several extracellular stimuli can promote actin reorganization and result in enhanced cell migration. Namely, G protein-coupled receptors (GPCRs), which are activated by factors ranging from small amines, to hormones, and chemokines, initiate signalling cascades resulting in cell shape changes, formation of a migrating front (leading edge) and altered adhesion. GPCRs are heptahelical membrane proteins, which classically transmit signal via the activation of heterotrimeric G proteins. Sustained stimulation leads to the activation of G protein-coupled receptor kinases (GRKs) and the recruitment of arrestin proteins, which engage alternative signalling pathways. In this review, we will discuss the role of GPCR mediated signal transduction and review their importance in the regulation of actin remodeling leading to cell migration.     

Theme and variations on kinetics of GPCR activation/deactivation

G protein-coupled receptors (GPCRs) initiate intracellular signaling pathways in response to physiologically and medically important extracellular ligands such as peptide and large glycoprotein hormones, neurotransmitters, sensory stimuli (odorant and taste molecules, light), calcium, l-amino acids, and are the target of many clinical drugs. The conversion of these extracellular stimuli into intracellular signals involves sequential and reversible reactions that initially take place at the plasma membrane. These reactions are mediated not only by dynamic interactions between ligands, receptors and heterotrimeric G proteins, but also by conformational changes associated with the activation/deactivation process of each protein. This review discusses the kinetic characteristics and rate-limiting reactions engaged in signal propagation that are involved in systems as diverse as neurotransmitter and hormonal signaling, and that have been recorded in live cells by Förster resonance energy transfer (FRET) approaches.     

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.     

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.     

Membranes: a meeting point for lipids, proteins and therapies

Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.     

In vitro expression and analysis of the 826 human G protein-coupled receptors

G protein-coupled receptors (GPCRs) are involved in all humanphysiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic targets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826humanGPCRs using two different fusion constructs. Expression characteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between different fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility.     

GPCR stabilization using the bicelle-like architecture of mixed sterol-detergent micelles

The biophysical characterization of purified membrane proteins typically requires detergent mediated extraction from native lipid membrane environments. In the case of human G protein-coupled receptors (GPCRs), this process has been complicated by their conformational heterogeneity and the general lack of understanding the composition and interactions within the diverse human cellular membrane environment. Several successful GPCR structure determination efforts have shown that the addition of cholesterol analogs is often critical for maintaining protein stability. We have identified sterols that substantially increase the stability of the NOP receptor (ORL-1), a member of the opioid GPCR family, in a mixed micelle environment. Using dynamic light scattering and small-angle X-ray scattering, we have determined that the most thermal stabilizing sterol, cholesteryl hemisuccinate, induces the formation of a bicelle-like micelle architecture when mixed with dodecyl maltoside detergent. Together with mutagenesis studies and recent GPCR structures, our results provide indications that stabilization is attained through a combination of specific sterol binding to GPCRs and modulation of micelle morphology.     

Structural insights into G-protein-coupled receptor activation

G-protein-coupled receptors (GPCRs) are the largest family of eukaryotic plasma membrane receptors, and are responsible for the majority of cellular responses to external signals. GPCRs share a common architecture comprising seven transmembrane (TM) helices. Binding of an activating ligand enables the receptor to catalyze the exchange of GTP for GDP in a heterotrimeric G protein. GPCRs are in a conformational equilibrium between inactive and activating states. Crystallographic and spectroscopic studies of the visual pigment rhodopsin and two beta-adrenergic receptors have defined some of the conformational changes associated with activation.     

Lipid-Protein Interactions Are a Unique Property and Defining Feature of G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are membrane-bound proteins that depend on their lipid environment to carry out their physiological function. Combined efforts from many theoretical and experimental studies on the lipid-protein interaction profile of several GPCRs hint at an intricate relationship of these receptors with their surrounding membrane environment, with several lipids emerging as particularly important. Using coarse-grained molecular dynamics simulations, we explore the lipid-protein interaction profiles of 28 different GPCRs, spanning different levels of classification and conformational states and totaling to 1 ms of simulation time. We find a close relationship with lipids for all GPCRs simulated, in particular, cholesterol and phosphatidylinositol phosphate (PIP) lipids, but the number, location, and estimated strength of these interactions is dependent on the specific GPCR as well as its conformational state. Although both cholesterol and PIP lipids bind specifically to GPCRs, they utilize distinct mechanisms. Interactions with PIP lipids are mediated by charge-charge interactions with intracellular loop residues and stabilized by one or both of the transmembrane helices linked by the loop. Interactions with cholesterol, on the other hand, are mediated by a hydrophobic environment, usually made up of residues from more than one helix, capable of accommodating its ring structure and stabilized by interactions with aromatic and charged/polar residues. Cholesterol binding to GPCRs occurs in a small number of sites, some of which (like the binding site on the extracellular side of transmembrane 6/7) are shared among many class A GPCRs. Combined with a thorough investigation of the local membrane structure, our results provide a detailed picture of GPCR-lipid interactions. Additionally, we provide an accompanying website to interactively explore the lipid-protein interaction profile of all GPCRs simulated to facilitate analysis and comparison of our data.     

Discovery of new G protein-coupled receptors for lipid mediators

Successful sequencing of the human genome has opened a new era in the life sciences and has greatly accelerated biomedical research. Among various research endeavors benefiting from established genomic information, one of the most fruitful areas is the research on orphan G protein-coupled receptors (GPCRs). Many intercellular mediators, including peptides, lipids, and other small molecules, have found their GPCRs in the plasma membrane, e.g., relaxin and tyramine. In the past 14 months, more than one dozen papers have been published reporting the finding of intercellular lipid mediators acting on rhodopsin family GPCRs. This review focuses primarily on intercellular lipid mediators and their recently discovered GPCRs.     

Intracellular α(2C)-adrenoceptors: storage depot, stunted development or signaling domain?

G-protein coupled receptors (GPCRs) are generally considered to function as cell surface signaling structures that respond to extracellular mediators, many of which do not readily access the cell's interior. Indeed, most GPCRs are preferentially targeted to the plasma membrane. However, some receptors, including α(2C)-Adrenoceptors, challenge conventional concepts of GPCR activity by being preferentially retained and localized within intracellular organelles. This review will address the issues associated with this unusual GPCR localization and discuss whether it represents a novel sub-cellular niche for GPCR signaling, whether these receptors are being stored for rapid deployment to the cell surface, or whether they represent immature or incomplete receptor systems.     

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.     

Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains

Supported cell-membrane sheets are promising in vitro systems to investigate the properties of membranes with native protein/lipid composition, in particular their sub-compartmentalization and the differential localization of proteins associated to them. While such studies are usually performed using static microscopy techniques, we demonstrate here the potential offered by dynamic diffusion measurements. Whereas the overall fluidity of the lipid bilayer was preserved, the preparation of the membrane sheets led to the selective immobilization of extracellular and transmembrane (TM) glycosylated proteins and the anchored proteins/lipids associated with them. Taking advantage of this, we investigated the association of the G protein Gq with TM proteins, in particular G-protein coupled receptors (GPCRs), by monitoring the changes in diffusion occurring after preparation of the supported membranes. Two fluorescently tagged Galphaq proteins were constructed, which remained either mostly monomeric in the plasma membrane or associated with Gbetagamma in heterotrimers. While both constructs diffused similarly in living cells, the preparation of the supported membranes led to the selective immobilization of the heterotrimers with minimal changes of the diffusion of the monomeric Galphaq. The diverse mobility of monomeric and heterotrimeric Galphaq was a result of their different lipid anchors as demonstrated by monitoring the diffusion of the corresponding anchors alone. We propose that the immobilization of the heterotrimer was caused by its partitioning inside membrane microdomains surrounding GPCRs.