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 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.

scholarly journals Development of OPLS-AA/M Parameters for Simulations of G Protein-Coupled Receptors and Other Membrane Proteins

2022 ◽  
Author(s):  
Michael J. Robertson ◽  
Georgios Skiniotis
Keyword(s):  
Membrane Proteins ◽  
Force Field ◽  
G Protein ◽  
Drug Targets ◽  
G Protein Coupled Receptors ◽  
Dynamic Nature ◽  
Post Translational Modifications ◽  
Dynamics Simulations ◽  
High Level ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) and other membrane proteins are valuable drug targets, and their dynamic nature makes them attractive systems for study with molecular dynamics simulations and free energy approaches. Here, we report the development, implementation, and validation of OPLS-AA/M force field parameters to enable simulations of these systems. These efforts include the introduction of post-translational modifications including lipidations and phosphorylation. We also modify previously reported parameters for lipids to be more consistent with the OPLS-AA force field standard and extend their coverage. These new parameters are validated on a variety of test systems, with the results compared to high-level quantum mechanics calculations, experimental data, and simulations with CHARMM36m where relevant. The results demonstrate that the new parameters reliably reproduce the behavior of membrane protein systems.

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.

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