scholarly journals A luciferase complementation assay system using transferable mouse artificial chromosomes to monitor protein–protein interactions mediated by G protein-coupled receptors

2018 ◽  
Vol 70 (6) ◽  
pp. 1499-1508
Author(s):  
Narumi Uno ◽  
Tomohito Fujimoto ◽  
Shinya Komoto ◽  
Gene Kurosawa ◽  
Masaaki Sawa ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
G Protein Coupled Receptors ◽  
Assay System ◽  
Protein Protein Interactions ◽  
Complementation Assay ◽  
Artificial Chromosomes ◽  
G Protein Coupled

scholarly journals Intraflagellar transport-A complex mediates ciliary entry and retrograde trafficking of ciliary G protein–coupled receptors

2017 ◽  
Vol 28 (3) ◽  
pp. 429-439 ◽  
Author(s):  
Tomoaki Hirano ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Protein Trafficking ◽  
Intraflagellar Transport ◽  
G Protein Coupled Receptors ◽  
Protein Protein Interactions ◽  
Retrograde Trafficking ◽  
The Core ◽  
Ciliary Protein ◽  
G Protein Coupled

Cilia serve as cellular antennae where proteins involved in sensory and developmental signaling, including G protein–coupled receptors (GPCRs), are specifically localized. Intraflagellar transport (IFT)-A and -B complexes mediate retrograde and anterograde ciliary protein trafficking, respectively. Using a visible immunoprecipitation assay to detect protein–protein interactions, we show that the IFT-A complex is divided into a core subcomplex, composed of IFT122/IFT140/IFT144, which is associated with TULP3, and a peripheral subcomplex, composed of IFT43/IFT121/IFT139, where IFT139 is most distally located. IFT139-knockout (KO) and IFT144-KO cells demonstrated distinct phenotypes: IFT139-KO cells showed the accumulation of IFT-A, IFT-B, and GPCRs, including Smoothened and GPR161, at the bulged ciliary tips; IFT144-KO cells showed failed ciliary entry of IFT-A and GPCRs and IFT-B accumulation at the bulged tips. These observations demonstrate the distinct roles of the core and peripheral IFT-A subunits: IFT139 is dispensable for IFT-A assembly but essential for retrograde trafficking of IFT-A, IFT-B, and GPCRs; in contrast, IFT144 is essential for functional IFT-A assembly and ciliary entry of GPCRs but dispensable for anterograde IFT-B trafficking. Thus the data presented here demonstrate that the IFT-A complex mediates not only retrograde trafficking but also entry into cilia of GPCRs.

scholarly journals Heterodimerization Between the Lutropin and Follitropin Receptors is Associated With an Attenuation of Hormone-Dependent Signaling

Endocrinology ◽  
2013 ◽  
Vol 154 (10) ◽  
pp. 3925-3930 ◽  
Author(s):  
Xiuyan Feng ◽  
Meilin Zhang ◽  
Rongbin Guan ◽  
Deborah L. Segaloff
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Fsh Receptor ◽  
Bioluminescence Resonance Energy Transfer ◽  
Protein Protein Interactions ◽  
Lh Receptor ◽  
G Protein Coupled ◽  
Stimulation Of

The LH receptor (LHR) and FSH receptor (FSHR) are each G protein-coupled receptors that play critical roles in reproductive endocrinology. Each of these receptors has previously been shown to self-associate into homodimers and oligomers shortly after their biosynthesis. As shown herein using bioluminescence resonance energy transfer to detect protein-protein interactions, our data show that the LHR and FSHR, when coexpressed in the same cells, specifically heterodimerize with each other. Further experiments confirm that at least a portion of the cellular LHR/FSHR heterodimers are present on the cell surface and are functional. We then sought to ascertain what effects, if any, heterodimerization between the LHR and FSHR might have on signaling. It was observed that when the LHR was expressed under conditions promoting the heterodimerization with FSHR, LH or human chorionic gonadotropin (hCG) stimulation of Gs was attenuated. Conversely, when the FSHR was expressed under conditions promoting heterodimerization with the LHR, FSH-stimulated Gs activation was attenuated. These results demonstrate that the coexpression of the LHR and FSHR enables heterodimerizaton between the 2 gonadotropin receptors and results in an attenuation of signaling through each receptor.

scholarly journals Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor

2018 ◽  
Vol 115 (12) ◽  
pp. 3036-3041 ◽  
Author(s):  
Yinglong Miao ◽  
J. Andrew McCammon
Keyword(s):  
Protein Binding ◽  
G Protein ◽  
Protein Interactions ◽  
Intracellular Signaling ◽  
Md Simulations ◽  
Binding Pocket ◽  
Signaling Proteins ◽  
Protein Protein Interactions ◽  
Intracellular Loops ◽  
G Protein Coupled

Protein–protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein–protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR–nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein–protein interactions.

scholarly journals Lipid Protein Interactions Of G Protein Coupled Receptors

2016 ◽  
Vol 110 (3) ◽  
pp. 423a
Author(s):  
Besian Sejdiu ◽  
Christine DeGagne ◽  
Valentina Corradi ◽  
Peter Tieleman
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
G Protein Coupled Receptors ◽  
Lipid Protein Interactions ◽  
G Protein Coupled

scholarly journals Moonlighting Proteins and Protein–Protein Interactions as Neurotherapeutic Targets in the G Protein-Coupled Receptor Field

2013 ◽  
Vol 39 (1) ◽  
pp. 131-155 ◽  
Author(s):  
Kjell Fuxe ◽  
Dasiel O Borroto-Escuela ◽  
Wilber Romero-Fernandez ◽  
Miklós Palkovits ◽  
Alexander O Tarakanov ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
G Protein Coupled Receptor ◽  
Receptor Field ◽  
Moonlighting Proteins ◽  
G Protein Coupled
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