Employing Rhodobacter sphaeroides to functionally express and purify human G protein-coupled receptors

2008 ◽  
Vol 389 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Ankita Roy ◽  
Arun Kumar Shukla ◽  
Winfried Haase ◽  
Hartmut Michel
Keyword(s):  
G Protein ◽  
Rhodobacter Sphaeroides ◽  
Structural Information ◽  
Membrane Surface ◽  
Expression System ◽  
Gene Promoter ◽  
Photosynthetic Bacterium ◽  
G Protein Coupled Receptors ◽  
Recombinant Receptors ◽  
G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors and play crucial roles in many cellular and physiological processes. Functional production of recombinant GPCRs is one of the main bottlenecks to obtaining structural information. Here, we report the use of a novel bacterial expression system based on the photosynthetic bacteriumRhodobacter sphaeroidesfor the production of human recombinant GPCRs. The advantage of employingR. sphaeroidesas a host lies in the fact that it provides much more membrane surface per cell compared to other typical expression hosts. The system was tailored to overexpress recombinant receptors under the control of the moderately strong and highly regulated superoperonic photosynthetic promoterpufQ. We tested this system for the expression of some class A GPCRs, namely, the human adenosine A2a receptor (A2aR), the human angiotensin AT1a receptor (AT1aR) and the human bradykinin B2 receptor (B2R). Several different constructs were examined and functional production of the recombinant receptors was achieved. The best-expressed receptor, AT1aR, was solubilized and affinity-purified. To the best of our knowledge, this is the first report of successful use of a bacterial host –R. sphaeroides– to produce functional recombinant GPCRs under the control of a photosynthetic gene promoter.

scholarly journals Applications of molecular replacement to G protein-coupled receptors

2013 ◽  
Vol 69 (11) ◽  
pp. 2287-2292 ◽  
Author(s):  
Andrew C. Kruse ◽  
Aashish Manglik ◽  
Brian K. Kobilka ◽  
William I. Weis
Keyword(s):  
G Protein ◽  
Structural Biology ◽  
Structural Information ◽  
G Protein Coupled Receptors ◽  
Integral Membrane Proteins ◽  
Molecular Replacement ◽  
Gpcr Activation ◽  
Health And Disease ◽  
Almost All ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed.

scholarly journals Melatonin promotes sleep by activating the BK channel in C. elegans

2020 ◽  
Vol 117 (40) ◽  
pp. 25128-25137
Author(s):  
Longgang Niu ◽  
Yan Li ◽  
Pengyu Zong ◽  
Ping Liu ◽  
Yuan Shui ◽  
...  
Keyword(s):  
G Protein ◽  
Neurotransmitter Release ◽  
Expression System ◽  
Molecular Target ◽  
Bk Channel ◽  
G Protein Coupled Receptors ◽  
Wild Type ◽  
Heterologous Expression System ◽  
C Elegans ◽  
G Protein Coupled

Melatonin (Mel) promotes sleep through G protein-coupled receptors. However, the downstream molecular target(s) is unknown. We identified the Caenorhabditis elegans BK channel SLO-1 as a molecular target of the Mel receptor PCDR-1-. Knockout of pcdr-1, slo-1, or homt-1 (a gene required for Mel synthesis) causes substantially increased neurotransmitter release and shortened sleep duration, and these effects are nonadditive in double knockouts. Exogenous Mel inhibits neurotransmitter release and promotes sleep in wild-type (WT) but not pcdr-1 and slo-1 mutants. In a heterologous expression system, Mel activates the human BK channel (hSlo1) in a membrane-delimited manner in the presence of the Mel receptor MT1 but not MT2. A peptide acting to release free Gβγ also activates hSlo1 in a MT1-dependent and membrane-delimited manner, whereas a Gβλ inhibitor abolishes the stimulating effect of Mel. Our results suggest that Mel promotes sleep by activating the BK channel through a specific Mel receptor and Gβλ.

High-Level Expression of G Protein-Coupled Receptors with the Aid of the Semliki Forest Virus Expression System

1995 ◽  
Vol 15 (1-4) ◽  
pp. 23-32 ◽  
Author(s):  
Kenneth Lundstrom ◽  
Ann Mills ◽  
Elisabeth Allet ◽  
Karin Ceszkowski ◽  
Georges Agudo ◽  
...  
Keyword(s):  
G Protein ◽  
Semliki Forest Virus ◽  
Expression System ◽  
G Protein Coupled Receptors ◽  
High Level Expression ◽  
Virus Expression ◽  
High Level ◽  
G Protein Coupled

scholarly journals Structural conservation among the rhodopsin-like and other G protein-coupled receptors

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Mikitaka Kinoshita ◽  
Tetsuji Okada
Keyword(s):  
G Protein ◽  
Structural Information ◽  
G Protein Coupled Receptors ◽  
Extracellular Region ◽  
Wide Range ◽  
Class B ◽  
Frizzled Receptors ◽  
Unknown Structure ◽  
Structural Conservation ◽  
G Protein Coupled

Abstract Intramolecular remote coupling within the polypeptide backbones of membrane proteins is difficult to analyze owing to the limited structural information available at the atomic level. Nonetheless, recent progress in the crystallographic study of G protein-coupled receptors (GPCRs) has provided an unprecedented opportunity for understanding the sophisticated architecture of heptahelical transmembrane (7TM) bundles. These 7TM bundles can respond to a wide range of extracellular stimuli while retaining the common function of binding trimeric G proteins. Here we have systematically analyzed select sets of inactive-like 7TM bundles to highlight the structural conservation of the receptors, in terms of intramolecular Cα-Cα distances. Distances with the highest scores were found to be dominated by the intrahelical distances of helix III, regardless of the choice of bundles in the set, indicating that the intracellular half of this helix is highly conserved. Unexpectedly, the distances between the cytoplasmic side of helix I and the extracellular region of helix VI provided the largest contribution to the high score populations among the interhelical pairs in most of the selected sets, including class B, C and frizzled receptors. These findings are expected to be valuable in further studies of GPCRs with unknown structure and of other protein families.

scholarly journals β-Barrel scaffolds for the grafting of extracellular loops from G-protein-coupled receptors

2012 ◽  
Vol 393 (11) ◽  
pp. 1341-1355 ◽  
Author(s):  
Reto Walser ◽  
Jörg H. Kleinschmidt ◽  
Arne Skerra ◽  
Oliver Zerbe
Keyword(s):  
G Protein ◽  
Structural Information ◽  
Binding Mode ◽  
G Protein Coupled Receptors ◽  
Peptide Ligand ◽  
Receptor Ligands ◽  
Natural Systems ◽  
Comprehensive Information ◽  
Chimeric Construct ◽  
G Protein Coupled

Abstract Owing to the difficulties in production and purification of G-protein-coupled receptors (GPCRs), relatively little structural information is available about this class of receptors. Here we aim at developing small chimeric proteins, displaying the extracellular ligand-binding motifs of a human GPCR, the Y receptor. This allows the study of ligand-receptor interactions in simplified systems. We present comprehensive information on the use of transmembrane (OmpA) and soluble (Blc) β-barrel scaffolds. Whereas Blc appeared to be not fully compatible with our approach, owing to problems with refolding of the hybrid constructs, loop-grafted versions of OmpA delivered encouraging results. Previously, we described a chimeric construct based on OmpA displaying all three extracellular Y1 receptor loops in different topologies and showing moderate affinity to one of the natural ligands. Now, we present detailed data on the interaction of these constructs with several Y receptor ligands along with data on new constructs. Our findings suggest a common binding mode for all ligands, which is mediated through the C-terminal residues of the peptide ligand, supporting the functional validity of these hybrid receptors. The observed binding affinities, however, are well below those observed for the natural receptors, clearly indicating limitations in mimicking the natural systems.

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