Dimerization of G protein-coupled receptors: New avenues for somatostatin receptor signalling, control and functioning

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between β-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (~4 kDa), optimized α fragment peptide (termed ProLink™) derived from β-galactosidase, and β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the β-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active β-galactosidase enzyme, and thus GPCR activation can be determined by quantifying β-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Gαi-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified. ( Journal of Biomolecular Screening 2008:737-747)

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

Biochemical Journal ◽

10.1042/bj20031648 ◽

2004 ◽

Vol 379 (1) ◽

pp. 1-9 ◽

Cited By ~ 78

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.

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G-protein-coupled receptor signalling through protein networks

Biochemical Society Transactions ◽

10.1042/bst0350023 ◽

2007 ◽

Vol 35 (1) ◽

pp. 23-27 ◽

Cited By ~ 8

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.

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Analysis of ciliary status via G-protein-coupled receptors localized on primary cilia

Microscopy ◽

10.1093/jmicro/dfaa035 ◽

2020 ◽

Vol 69 (5) ◽

pp. 277-285

Author(s):

Yuki Kobayashi ◽

Akie Hamamoto ◽

Yumiko Saito

Keyword(s):

G Protein ◽

Primary Cilium ◽

Somatostatin Receptor ◽

G Protein Coupled Receptors ◽

The Body ◽

Cell Surface Receptor ◽

Specific Cell ◽

Gpcr Signaling ◽

Sequence Of Events ◽

G Protein Coupled

Abstract G-protein-coupled receptors (GPCRs) comprise the largest and most diverse cell surface receptor family, with more than 800 known GPCRs identified in the human genome. Binding of an extracellular cue to a GPCR results in intracellular G protein activation, after which a sequence of events, can be amplified and optimized by selective binding partners and downstream effectors in spatially discrete cellular environments. Because GPCRs are widely expressed in the body, they help to regulate an incredible range of physiological processes from sensation to growth to hormone responses. Indeed, it is estimated that ∼ 30% of all clinically approved drugs act by binding to GPCRs. The primary cilium is a sensory organelle composed of a microtubule axoneme that extends from the basal body. The ciliary membrane is highly enriched in specific signaling components, allowing the primary cilium to efficiently convey signaling cascades in a highly ordered microenvironment. Recent data demonstrated that a limited number of non-olfactory GPCRs, including somatostatin receptor 3 and melanin-concentrating hormone receptor 1 (MCHR1), are selectively localized to cilia on several mammalian cell types including neuronal cells. Utilizing cilia-specific cell biological and molecular biological approaches, evidence has accumulated to support the biological importance of ciliary GPCR signaling followed by cilia structural changes. Thus, cilia are now considered a unique sensory platform for integration of GPCR signaling toward juxtaposed cytoplasmic structures. Herein, we review ciliary GPCRs and focus on a novel role of MCHR1 in ciliary length control that will impact ciliary signaling capacity and neuronal function.

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Lysine63-linked ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

10.1101/2020.03.04.977090 ◽

2020 ◽

Cited By ~ 1

Author(s):

Swapnil Rohidas Shinde ◽

Andrew R. Nager ◽

Maxence V. Nachury

Keyword(s):

G Protein ◽

Somatostatin Receptor ◽

G Protein Coupled Receptors ◽

Dependent Manner ◽

Bardet Biedl Syndrome ◽

Ubiquitinated Proteins ◽

Molecular Sensor ◽

Regulated Trafficking ◽

G Protein Coupled ◽

Orphan Gpcr

ABSTRACTRegulated trafficking of G-protein coupled receptors (GPCRs) controls cilium-based signaling pathways. β-arrestin, a molecular sensor of activated GPCRs, and the BBSome, a complex of Bardet-Biedl Syndrome (BBS) proteins, are required for the signal-dependent exit of ciliary GPCRs but the functional interplay between β-arrestin and the BBSome remains elusive. Here we find that, upon activation, ciliary GPCRs become tagged with K63-linked ubiquitin (K63Ub) chains in a β-arrestin-dependent manner prior to BBSome-mediated exit. Removal of ubiquitin acceptor residues from the somatostatin receptor 3 (SSTR3) and from the orphan GPCR GPR161 demonstrates that ubiquitination of ciliary GPCRs is required for their regulated exit from cilia. Furthermore, targeting a K63Ub-specific deubiquitinase to cilia blocks the exit of GPR161, SSTR3 and Smoothened (SMO) from cilia. Finally, ubiquitinated proteins accumulate in cilia of mammalian photoreceptors and Chlamydomonas cells when BBSome function is compromised. We conclude that K63Ub chains mark GPCRs and other unwanted ciliary proteins for recognition by the ciliary exit machinery.

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Kinetic diversity in G-protein-coupled receptor signalling

Biochemical Journal ◽

10.1042/bj20060517 ◽

2006 ◽

Vol 401 (2) ◽

pp. 485-495 ◽

Cited By ~ 19

Author(s):

Vladimir L. Katanaev ◽

Matey Chornomorets

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Receptor Signalling ◽

Stable Production ◽

G Protein Signalling ◽

Simple Kinetic Model ◽

Signal Specificity ◽

Higher Eukaryotes ◽

G Protein Coupled ◽

Gpcr Signal

The majority of intracellular signalling cascades in higher eukaryotes are initiated by GPCRs (G-protein-coupled receptors). Hundreds of GPCRs signal through a handful of trimeric G-proteins, raising the issue of signal specificity. In the present paper, we illustrate a simple kinetic model of G-protein signalling. This model shows that stable production of significant amounts of free GαGTP (GTP-bound Gα subunit) and βγ is only one of multiple modes of behaviour of the G-protein system upon activation. Other modes, previously uncharacterized, are sustained production of βγ without significant levels of GαGTP and transient production of GαGTP with sustained βγ. The system can flip between different modes upon changes in conditions. This model demonstrates further that the negative feedback of receptor uncoupling or internalization, when combined with a positive feedback within the G-protein cycle, under a broad range of conditions results not in termination of the response but in relaxed oscillations in GPCR signalling. This variety of G-protein responses may serve to encode signal specificity in GPCR signal transduction.

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

FEMS Yeast Research ◽

10.1093/femsyr/foz087 ◽

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.

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Ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

The Journal of Cell Biology ◽

10.1083/jcb.202003020 ◽

2020 ◽

Vol 219 (12) ◽

Cited By ~ 2

Author(s):

Swapnil Rohidas Shinde ◽

Andrew R. Nager ◽

Maxence V. Nachury

Keyword(s):

G Protein ◽

Somatostatin Receptor ◽

G Protein Coupled Receptors ◽

Dependent Manner ◽

Bardet Biedl Syndrome ◽

Ubiquitinated Proteins ◽

Molecular Sensor ◽

Regulated Trafficking ◽

G Protein Coupled ◽

Orphan Gpcr

Regulated trafficking of G protein–coupled receptors (GPCRs) controls cilium-based signaling pathways. β-Arrestin, a molecular sensor of activated GPCRs, and the BBSome, a complex of Bardet–Biedl syndrome (BBS) proteins, are required for the signal-dependent exit of ciliary GPCRs, but the functional interplay between β-arrestin and the BBSome remains elusive. Here we find that, upon activation, ciliary GPCRs become tagged with ubiquitin chains comprising K63 linkages (UbK63) in a β-arrestin–dependent manner before BBSome-mediated exit. Removal of ubiquitin acceptor residues from the somatostatin receptor 3 (SSTR3) and from the orphan GPCR GPR161 demonstrates that ubiquitination of ciliary GPCRs is required for their regulated exit from cilia. Furthermore, targeting a UbK63-specific deubiquitinase to cilia blocks the exit of GPR161, SSTR3, and Smoothened (SMO) from cilia. Finally, ubiquitinated proteins accumulate in cilia of mammalian photoreceptors and Chlamydomonas cells when BBSome function is compromised. We conclude that Ub chains mark GPCRs and other unwanted ciliary proteins for recognition by the ciliary exit machinery.

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N-Terminal Fusion Tags for Effective Production of G-Protein-Coupled Receptors in Bacterial Cell-Free Systems

Acta Naturae ◽

10.32607/20758251-2012-4-4-58-64 ◽

2012 ◽

Vol 4 (4) ◽

pp. 58-64 ◽

Cited By ~ 9

Author(s):

E. N. Lyukmanova ◽

Z. O. Shenkarev ◽

N. F. Khabibullina ◽

D. S. Kulbatskiy ◽

M. A. Shulepko ◽

...

Keyword(s):

Membrane Proteins ◽

G Protein ◽

Bacterial Cell ◽

Somatostatin Receptor ◽

Rnase A ◽

G Protein Coupled Receptors ◽

Functional Studies ◽

Terminal Fragment ◽

Low Efficiency ◽

G Protein Coupled

G-protein-coupled receptors (GPCR) constitute one of the biggest families of membrane proteins. In spite of the fact that they are highly relevant to pharmacy, they have remained poorly explored. One of the main bottlenecks encountered in structural-functional studies of GPCRs is the difficulty to produce sufficient amounts of the proteins. Cell-free systems based on bacterial extracts from E. coli cells attract much attention as an effective tool for recombinant production of membrane proteins. GPCR production in bacterial cell-free expression systems is often inefficient because of the problems associated with the low efficiency of the translation initiation process. This problem could be resolved if GPCRs were expressed in the form of hybrid proteins with N-terminal polypeptide fusion tags. In the present work, three new N-terminal fusion tags are proposed for cellfree production of the human 2-adrenergic receptor, human M1 muscarinic acetylcholine receptor, and human somatostatin receptor type 5. It is demonstrated that the application of an N-terminal fragment (6 a.a.) of bacteriorhodopsin from Exiguobacterium sibiricum (ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein from B. subtilis allows to increase the CF synthesis of the target GPCRs by 538 times, resulting in yields of 0.63.8 mg from 1 ml of the reaction mixture, which is sufficient for structural-functional studies.

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Functional coupling of a mammalian somatostatin receptor to the yeast pheromone response pathway.

Molecular and Cellular Biology ◽

10.1128/mcb.15.11.6188 ◽

1995 ◽

Vol 15 (11) ◽

pp. 6188-6195 ◽

Cited By ~ 105

Author(s):

L A Price ◽

E M Kajkowski ◽

J R Hadcock ◽

B A Ozenberger ◽

M H Pausch

Keyword(s):

G Protein ◽

Growth Response ◽

Somatostatin Receptor ◽

G Protein Coupled Receptors ◽

Functional Coupling ◽

Pheromone Response ◽

Pheromone Response Pathway ◽

Yeast Strains ◽

Dependent Growth ◽

G Protein Coupled

A detailed analysis of structural and functional aspects of G-protein-coupled receptors, as well as discovery of novel pharmacophores that exert their effects on members of this class of receptors, will be facilitated by development of a yeast-based bioassay. To that end, yeast strains that functionally express the rat somatostatin receptor subtype 2 (SSTR2) were constructed. High-affinity binding sites for somatostatin ([125I-Tyr-11]S-14) comparable to those in native tissues were detected in yeast membrane extracts at levels equivalent to the alpha-mating pheromone receptor (Ste2p). Somatostatin-dependent growth of strains modified by deletion of genes encoding components of the pheromone response pathway was detected through induction of a pheromone-responsive HIS3 reporter gene, enabling cells to grow on medium lacking histidine. Dose-dependent growth responses to S-14 and related SSTR2 subtype-selective agonists that were proportional to the affinity of the ligands for SSTR2 were observed. The growth response required SSTR2, G alpha proteins, and an intact signal transduction pathway. The sensitivity of the bioassay was affected by intracellular levels of the G alpha protein. A mutation in the SST2 gene, which confers supersensitivity to pheromone, was found to significantly enhance the growth response to S-14. In sst2 delta cells, SSTR2 functionally interacted with both a chimeric yeast/mammalian G alpha protein and the yeast G alpha protein, Gpa1p; to promote growth. These yeast strains should serve as a useful in vivo reconstitution system for examination of molecular interactions of the G-protein-coupled receptors and G proteins.

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