Mechanisms Of G Protein-coupled Receptors Signaling Research Articles

Posttranslational modifications in GPCR internalization.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors that serve as the most important drug targets. Classically, GPCR internalization has been considered to lead to receptor desensitization. However, many studies over the past decade have reported that internalized membrane receptors can trigger distinct signal activation. The "internalized activation" provides a completely new understanding for the receptor internalization, the mechanism of physiology/pathology and novel drug targets for precision medicine. GPCR internalization undergoes a series of strict regulations, especially by posttranslational modifications (PTMs). Here, this review summarizes different PTMs in GPCR internalization and analyzes their significance in GPCR internalization dynamics, internalization routes, postinternalization fates, and related diseases, which will offer new insights into the regulatory mechanism of GPCR signaling and novel drug targets for precision medicine.

G protein-coupled receptor-G protein interactions: a single-molecule perspective.

G protein-coupled receptors (GPCRs) regulate many cellular and physiological processes, responding to a diverse range of extracellular stimuli including hormones, neurotransmitters, odorants, and light. Decades of biochemical and pharmacological studies have provided fundamental insights into the mechanisms of GPCR signaling. Thanks to recent advances in structural biology, we now possess an atomistic understanding of receptor activation and G protein coupling. However, how GPCRs and G proteins interact in living cells to confer signaling efficiency and specificity remains insufficiently understood. The development of advanced optical methods, including single-molecule microscopy, has provided the means to study receptors and G proteins in living cells with unprecedented spatio-temporal resolution. The results of these studies reveal an unexpected level of complexity, whereby GPCRs undergo transient interactions among themselves as well as with G proteins and structural elements of the plasma membrane to form short-lived signaling nanodomains that likely confer both rapidity and specificity to GPCR signaling. These findings may provide new strategies to pharmaceutically modulate GPCR function, which might eventually pave the way to innovative drugs for common diseases such as diabetes or heart failure.

Recent advances in computational studies of GPCR-G protein interactions.

Protein-protein interactions are key in cellular signaling. G protein-coupled receptors (GPCRs), the largest superfamily of human membrane proteins, are able to transduce extracellular signals (e.g., hormones and neurotransmitters) to intracellular proteins, in particular the G proteins. Since GPCRs serve as primary targets of ~1/3 of currently marketed drugs, it is important to understand mechanisms of GPCR signaling in order to design selective and potent drug molecules. This chapter focuses on recent advances in computational studies of the GPCR-G protein interactions using bioinformatics, protein-protein docking and molecular dynamics simulation approaches.

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.

Current Concepts and Treatments of Schizophrenia.

Schizophrenia is a debilitating mental illness which involves three groups of symptoms, i.e., positive, negative and cognitive, and has major public health implications. According to various sources, it affects up to 1% of the population. The pathomechanism of schizophrenia is not fully understood and current antipsychotics are characterized by severe limitations. Firstly, these treatments are efficient for about half of patients only. Secondly, they ameliorate mainly positive symptoms (e.g., hallucinations and thought disorders which are the core of the disease) but negative (e.g., flat affect and social withdrawal) and cognitive (e.g., learning and attention disorders) symptoms remain untreated. Thirdly, they involve severe neurological and metabolic side effects and may lead to sexual dysfunction or agranulocytosis (clozapine). It is generally agreed that the interactions of antipsychotics with various neurotransmitter receptors are responsible for their effects to treat schizophrenia symptoms. In particular, several G protein-coupled receptors (GPCRs), mainly dopamine, serotonin and adrenaline receptors, are traditional molecular targets for antipsychotics. Comprehensive research on GPCRs resulted in the exploration of novel important signaling mechanisms of GPCRs which are crucial for drug discovery: intentionally non-selective multi-target compounds, allosteric modulators, functionally selective compounds and receptor oligomerization. In this review, we cover current hypotheses of schizophrenia, involving different neurotransmitter systems, discuss available treatments and present novel concepts in schizophrenia and its treatment, involving mainly novel mechanisms of GPCRs signaling.

Making useful gadgets with miniaturized G proteins

G protein-coupled receptors (GPCRs) relay information from extracellular stimuli to intracellular responses in a wide range of physiological and pathological processes, but understanding their complex effects in live cells is a daunting task. In this issue of JBC, Wan et al repurpose "mini G proteins"-previously used as affinity tools for structural studies-to develop a suite of probes to visualize GPCR activation in live cells. The approach is expected to revolutionize our understanding of the spatiotemporal control and mechanisms of GPCR signaling.

Regulation of G-protein coupled receptor signalling underpinning neurobiology of mood disorders and depression

G-protein coupled receptors (GPCRs) have long been at the center of investigations of the neurobiology of depression and mood disorders. Different facets of GPCR signalling pathways, including those controlling monoaminergic and neuropeptidergic hormonal systems are believed to be dysregulated in major depressive and bipolar disorders. Although these receptors are key molecular targets for a variety of therapeutic agents and continue to be the focus of intense pharmaceutical development, the molecular mechanisms activated by these GPCRs and underpin the pathological basis of mood disorders remain poorly understood. This review will discuss some of the emerging regulatory mechanisms of GPCR signaling in the central nervous system (CNS) involving protein-protein interactions, downstream effectors and cross-talk with other signaling molecules and their potential involvement in the neurobiology of psychiatric disease.

NF1 Is a Direct G Protein Effector Essential for Opioid Signaling to Ras in the Striatum

It is well recognized that G-protein-coupled receptors (GPCRs) can activate Ras-regulated kinase pathways to produce lasting changes in neuronal function. Mechanisms by which GPCRs transduce these signals and their relevance to brain disorders are not well understood. Here, we identify a major Ras regulator, neurofibromin 1 (NF1), as a direct effector of GPCR signaling via Gβγ subunits in the striatum. We find that binding of Gβγ to NF1 inhibits its ability to inactivate Ras. Deletion of NF1 in striatal neurons prevents the opioid-receptor-induced activation of Ras and eliminates its coupling to Akt-mTOR-signaling pathway. By acting in the striatal medium spiny neurons of the direct pathway, NF1regulates opioid-induced changes in Ras activity, thereby sensitizing mice to psychomotor and rewarding effects of morphine. These results delineate a novel mechanism of GPCR signaling to Ras pathways and establish a critical role of NF1 in opioid addiction.

Effective Application of Bicelles for Conformational Analysis of G Protein-Coupled Receptors by Hydrogen/Deuterium Exchange Mass Spectrometry

G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein–protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic receptor [β2AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.

Structural Life Science towards the Regulation of Selective GPCR Signaling

G protein-coupled receptors (GPCRs) are the largest family of receptors in the human genome. They are involved in many diseases and also the target of approximately 30% of all modern medicinal drugs. GPCRs respond to a broad spectrum of chemical entities, ranging from photons, protons, and calcium ions to small organic molecules (including odorants and neurotransmitters), peptides, and glycoproteins. Many GPCRs are members of closely related subfamilies that respond to the same hormone or neurotransmitter. However, they have different physiologic functions based on the cells in which they are expressed and the different signaling pathways that they exploit (e.g., coupling through heterotrimeric G-proteins such as Gs, Gi, and Gq, as well as β-arrestins). Antibody fragments including Fab and Fv can effectively stabilize and crystallize membrane proteins. However, using the mouse hybridoma technology it has been difficult to develop monoclonal antibodies that can recognize conformational epitopes of native GPCRs. We have recently succeeded in developing antibodies against native GPCRs using this technology in combination with our improved immunization and screening methods. In this symposium review, I present a successful example of prostaglandin E2 receptor (one of the GPCRs) crystallization using antibody fragments. To avoid several adverse effects of current therapeutics, it is essential to understand the molecular mechanism of GPCR signaling in a monomeric, dimeric, or oligomeric state. Also, we are interested in selectively regulating GPCR signaling via functional antibodies developed using our methods and/or the designed small organic molecules depending on the GPCR structure.

Conformational analysis of g protein-coupled receptor signaling by hydrogen/deuterium exchange mass spectrometry.

Conformational change and protein-protein interactions are two major mechanisms of membrane protein signal transduction, including G protein-coupled receptors (GPCRs). Upon agonist binding, GPCRs change conformation, resulting in interaction with downstream signaling molecules such as G proteins. To understand the precise signaling mechanism, studies have investigated the structural mechanism of GPCR signaling using X-ray crystallography, nuclear magnetic resonance (NMR), or electron paramagnetic resonance. In addition to these techniques, hydrogen/deuterium exchange mass spectrometry (HDX-MS) has recently been used in GPCR studies. HDX-MS measures the rate at which peptide amide hydrogens exchange with deuterium in the solvent. Exposed or flexible regions have higher exchange rates and excluded or ordered regions have lower exchange rates. Therefore, HDX-MS is a useful tool for studying protein-protein interfaces and conformational changes after protein activation or protein-protein interactions. Although HDX-MS does not give high-resolution structures, it analyzes protein conformations that are difficult to study with X-ray crystallography or NMR. Furthermore, conformational information from HDX-MS can help in the crystallization of X-ray crystallography by suggesting highly flexible regions. Interactions between GPCRs and downstream signaling molecules are not easily analyzed by X-ray crystallography or NMR because of the large size of the GPCR-signaling molecule complexes, hydrophobicity, and flexibility of GPCRs. HDX-MS could be useful for analyzing the conformational mechanism of GPCR signaling. In this chapter, we discuss details of HDX-MS for analyzing GPCRs using the β2AR-G protein complex as a model system.

The Activation Dynamics of Class C Gpcrs Revealed by Single Molecule FRET

G-protein-coupled receptors (GPCRs) are the largest and most diverse family of membrane signaling proteins. Binding of an extracellular signal to a GPCR activates G protein, which in turn modulates the activity of different downstream cellular effectors. Recent biochemical, pharmacological and biophysical studies suggest that GPCRs do not simply act as on-off switches but instead exist in multiple conformational and functional states where they may signal to distinct G proteins and other partners. However, despite recent advances, many aspects of signal transduction through GPCRs is still not well understood. Here we report a novel functional assay based on single molecule FRET to probe real-time conformational dynamics of isolated receptors. Using our assay, we studied metabotropic glutamate receptors (mGluRs), dimeric members of class C GPCR family, which are involved in modulation of synaptic transmission and excitability of nerve cells. Experiments on mGluR2 and mGluR3 homodimers reveal multiple stable conformational states. Strikingly, in spite of more than 70% sequence identity between mGluR2 and mGluR3, we observe substantial differences in their inherent dynamics and basal activity in absence of ligand. Our approach should make it possible to directly quantify how drugs stabilize conformational states of receptors and provide new insights into the molecular mechanisms of GPCR signaling.

Adenosine A1 Receptor Signaling Unraveled By Particle Image Correlation Spectroscopy (PICS)

The adenosine A1 receptor is a typical example of a G protein coupled receptor (GPCR). Despite a wealth of biochemical data the general mechanism of GPCR signaling has not been fully clarified. Whether GPCR signaling takes place in membrane microdomains, and whether the respective G proteins are precoupled, is still heavily debated. Both mechanisms would explain the fast receptor G protein interaction that is observed in experiments. Using single-molecule microscopy in live CHO cells and our recently developed analysis technique (PICS, Semrau, Schmidt., Biophys. J., 2007) we unraveled the first steps of the A1 receptor signaling. We found that at least 7% of the receptors are precoupled to the G protein already before stimulation with an agonist. Furthermore, 9% of the receptors translocate to membrane microdomains upon agonist stimulation. These domains, which are about 150 nm in size, are related to the cytoskeleton. We believe that this knowledge about the molecular mechanisms of GPCR signaling will open up new ways to manipulate GPCRs and develop new, potent drugs.

Reviews in Molecular Biology and Biotechnology: Transmembrane Signaling by G Protein-Coupled Receptors

As the most diverse type of cell surface receptor, the importance heptahelical G protein-coupled receptors (GPCRs) to clinical medicine cannot be overestimated. Visual, olfactory and gustatory sensation, intermediary metabolism, cell growth and differentiation are all influenced by GPCR signals. The basic receptor-G protein-effector mechanism of GPCR signaling is tuned by a complex interplay of positive and negative regulatory events that amplify the effect of a hormone binding the receptor or that dampen cellular responsiveness. The association of heptahelical receptors with a variety of intracellular partners other than G proteins has led to the discovery of potential mechanisms of GPCR signaling that extend beyond the classical paradigms. While the physiologic relevance of many of these novel mechanisms of GPCR signaling remains to be established, their existence suggests that the mechanisms of GPCR signaling are even more diverse than previously imagined.

The Long and the Short of Relaxin Signaling

The hormone relaxin functions in the reproductive system and derived its name from its role in relaxation of the pubic symphysis during parturition. Relaxin and the related hormone insulin-like peptide 3 (INSL3) act through heterotrimeric guanine nucleotide­-binding protein (G protein)-coupled receptors (GPCRs). Scott et al . report studies of human splice variants of these proteins that reveal an unusual mode of signaling by these receptors. The receptors for relaxin and INSL3, called LGR7 (leucine-rich repeat-containing GPCR 7) and LGR8, respectively [or, in an alternative nomenclature, relaxin family peptide receptors 1 and 2 (RXFP1 and RXFP2)] differ from other GPCRs in that they contain N-terminal domains with similarity to the ligand-binding domains of low-density lipoprotein class A (LDLa). Splice variants exist in which this LDLa module is missing or in which a secreted protein mainly encoding just the LDLa module is made. The authors examined the signaling properties of natural or artificial variant receptors in transfected cells. Although they bound relaxin normally, LGR7 variants without the LDLa module failed to produce their normal G protein-coupled signaling effects on accumulation of cyclic AMP. The truncated excreted form inhibited signaling from the normal receptor. It is not yet clear precisely how the LDLa modules influence signaling. In LDL receptors, these modules are binding sites for ligands or for protein-protein interactions. The truncated, excreted form of LDL7 was detected in human uterus and brain and was most abundant in uteri of pregnant mice. Thus, the authors propose that LGR7 and LGR8 have an unusual mechanism of GPCR signaling and that the alternative splicing of the receptors may produce an inhibitory factor that provides an additional regulatory mechanism for controlling relaxin action during reproduction. D. J. Scott, S. Layfield, Y. Yan, S. Sudo, A. J. W. Hsueh, G. W. Tregear, R. A. D. Bathgate, Characterization of novel splice variants of LGR7 and LGR8 reveals that receptor signaling is mediated by their unique low density lipoprotein class A modules. J. Biol. Chem. 281 , 34942-34954 (2006). [Abstract] [Full Text]