scholarly journals Engineering G protein-coupled receptor signalling in yeast for biotechnological and medical purposes

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Bettina Lengger ◽  
Michael K Jensen
Keyword(s):  
G Protein ◽  
Pathogen Detection ◽  
G Protein Coupled Receptors ◽  
Common Denominator ◽  
Orphan Receptors ◽  
Receptor Signalling ◽  
Functional Membrane ◽  
Membrane Bound ◽  
Engineering Parameters ◽  
G Protein Coupled

ABSTRACT G protein-coupled receptors (GPCRs) comprise the largest class of membrane proteins in the human genome, with a common denominator of seven-transmembrane domains largely conserved among eukaryotes. Yeast is naturally armoured with three different GPCRs for pheromone and sugar sensing, with the pheromone pathway being extensively hijacked for characterising heterologous GPCR signalling in a model eukaryote. This review focusses on functional GPCR studies performed in yeast and on the elucidated hotspots for engineering, and discusses both endogenous and heterologous GPCR signalling. Key emphasis will be devoted to studies describing important engineering parameters to consider for successful coupling of GPCRs to the yeast mating pathway. We also review the various means of applying yeast for studying GPCRs, including the use of yeast armed with heterologous GPCRs as a platform for (i) deorphanisation of orphan receptors, (ii) metabolic engineering of yeast for production of bioactive products and (iii) medical applications related to pathogen detection and drug discovery. Finally, this review summarises the current challenges related to expression of functional membrane-bound GPCRs in yeast and discusses the opportunities to continue capitalising on yeast as a model chassis for functional GPCR signalling studies.

G-Protein-Coupled Receptors in Drug Discovery: Nanosizing Using Cell-Free Technologies and Molecular Biology Approaches

2005 ◽  
Vol 10 (8) ◽  
pp. 765-779 ◽  
Author(s):  
Wayne R. Leifert ◽  
Amanda L. Aloia ◽  
Olgatina Bucco ◽  
Richard V. Glatz ◽  
Edward J. McMurchie
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
Drug Targets ◽  
G Protein Coupled Receptors ◽  
Orphan Receptors ◽  
Molecular Switching ◽  
Physiological Processes ◽  
Current Cell ◽  
G Protein Coupled ◽  
Signaling Components

Signal transduction by G-protein-coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to activation of a genericmolecular switch involving heterotrimeric G-proteins and guanine nucleotides. Signal transduction has been studied extensively with both cell-based systems and assays comprising isolated signaling components. Interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, highthroughput screening, biosensors, and so on will focus greater attention on assay development to allow for miniaturization, ultra-high throughput and, eventually, microarray/biochip assay formats. Although cell-based assays are adequate for many GPCRs, it is likely that these formatswill limit the development of higher density GPCRassay platforms mandatory for other applications. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR assay platforms adaptable for such applications as microarrays. The authors review current cell-free GPCR assay technologies and molecular biological approaches for construction of novel, functional GPCR assays.

Functional membrane diffusion of G-protein coupled receptors

2007 ◽  
Vol 36 (8) ◽  
pp. 849-860 ◽  
Author(s):  
Aurélie Baker ◽  
Aude Saulière ◽  
Fabrice Dumas ◽  
Claire Millot ◽  
Serge Mazères ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Membrane Diffusion ◽  
Functional Membrane ◽  
G Protein Coupled

scholarly journals AKAPs (A-kinase anchoring proteins) and molecules that compose their G-protein-coupled receptor signalling complexes

2004 ◽  
Vol 379 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Craig C. MALBON ◽  
Jiangchuan TAO ◽  
Hsien-yu WANG
Keyword(s):  
G Protein ◽  
Tyrosine Kinases ◽  
Cell Signalling ◽  
G Protein Coupled Receptors ◽  
Dynamic Regulation ◽  
Receptor Signalling ◽  
Anchoring Proteins ◽  
Β2 Adrenergic Receptor ◽  
Cytoskeletal Elements ◽  
G Protein Coupled

Cell signalling mediated via GPCRs (G-protein-coupled receptors) is a major paradigm in biology, involving the assembly of receptors, G-proteins, effectors and downstream elements into complexes that approach in design ‘solid-state’ signalling devices. Scaffold molecules, such as the AKAPs (A-kinase anchoring proteins), were discovered more than a decade ago and represent dynamic platforms, enabling multivalent signalling. AKAP79 and AKAP250 were the first to be shown to bind to membrane-embedded GPCRs, orchestrating the interactions of various protein kinases (including tyrosine kinases), protein phosphatases (e.g. calcineurin) and cytoskeletal elements with at least one member of the superfamily of GPCRs, the prototypical β2-adrenergic receptor. In this review, the multivalent interactions of AKAP250 with the cell membrane, receptor, cytoskeleton and constituent components are detailed, providing a working model for AKAP-based GPCR signalling complexes. Dynamic regulation of the AKAP–receptor complex is mediated by ordered protein phosphorylation.

G-protein-coupled receptor signalling through protein networks

2007 ◽  
Vol 35 (1) ◽  
pp. 23-27 ◽  
Author(s):  
A.D. Strosberg ◽  
C. Nahmias
Keyword(s):  
Structure Function ◽  
G Protein ◽  
Short Review ◽  
G Protein Coupled Receptors ◽  
Angiotensin Receptors ◽  
Receptor Signalling ◽  
Wide Range ◽  
Associated Proteins ◽  
Work Done ◽  
G Protein Coupled

This short review provides a broad, and therefore necessarily incomplete and personal, overview of G-protein-coupled receptors, which are often targets for a wide range of important drugs: I will discuss successively their structure, function and interactions with associated proteins. Examples will be drawn from work done over the last 30 years by scientists that worked at different times in my laboratories, mainly in the field of β-adrenoceptors, muscarinic acetylcholine, melatonin and angiotensin receptors.

scholarly journals Multiplexed analysis of the secretin-like GPCR-RAMP interactome

2019 ◽  
Vol 5 (9) ◽  
pp. eaaw2778 ◽  
Author(s):  
Emily Lorenzen ◽  
Tea Dodig-Crnković ◽  
Ilana B. Kotliar ◽  
Elisa Pin ◽  
Emilie Ceraudo ◽  
...  
Keyword(s):  
Membrane Protein ◽  
G Protein ◽  
Chemokine Receptors ◽  
G Protein Coupled Receptors ◽  
Orphan Receptors ◽  
Native Proteins ◽  
Receptor Activity Modifying Proteins ◽  
G Protein Coupled ◽  
Suspension Bead Array ◽  
Multiplexed Analysis

Receptor activity–modifying proteins (RAMPs) have been shown to modulate the functions of several G protein–coupled receptors (GPCRs), but potential direct interactions among the three known RAMPs and hundreds of GPCRs have never been investigated. Focusing mainly on the secretin-like family of GPCRs, we engineered epitope-tagged GPCRs and RAMPs, and developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized lysates. Using 64 antibodies raised against the native proteins and 4 antibodies targeting the epitope tags, we mapped the interactions among 23 GPCRs and 3 RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. The results provide a complete interactome of secretin-like GPCRs with RAMPs. The SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues.

Strategies to identify ligands for orphan G-protein-coupled receptors

2002 ◽  
Vol 30 (4) ◽  
pp. 789-793 ◽  
Author(s):  
G. Milligan
Keyword(s):  
Drug Design ◽  
G Protein ◽  
Small Molecule ◽  
Drug Targets ◽  
G Protein Coupled Receptors ◽  
Orphan Receptors ◽  
Protein Targets ◽  
Small Molecule Drug ◽  
Natural Ligands ◽  
G Protein Coupled

G-protein-coupled receptors are the most tractable class of protein targets for small molecule drug design. Sequencing of the human genome allied to bio-informatic analysis has identified a large number of putative receptors for which the natural ligands remain undefined. A range of currently employed and developing strategies to identify ligands that interact with these orphan receptors and to validate them as drug targets are described and discussed.

scholarly journals The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors

2016 ◽  
Vol 44 (2) ◽  
pp. 624-629 ◽  
Author(s):  
Laura E. Kilpatrick ◽  
Stephen J. Hill
Keyword(s):  
G Protein ◽  
Fluorescence Correlation Spectroscopy ◽  
Photon Counting ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
G Protein Coupled Receptors ◽  
Fluorescence Correlation ◽  
Membrane Bound ◽  
Molecular Brightness ◽  
G Protein Coupled

The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties.

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