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

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.

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Mutation of an aspartate residue highly conserved among G-protein-coupled receptors results in nonreciprocal disruption of alpha 2-adrenergic receptor-G-protein interactions. A negative charge at amino acid residue 79 forecasts alpha 2A-adrenergic receptor sensitivity to allosteric modulation by monovalent cations and fully effective receptor/G-protein coupling.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)43916-6 ◽

1994 ◽

Vol 269 (47) ◽

pp. 29557-29564

Author(s):

B P Ceresa ◽

L E Limbird

Keyword(s):

G Protein ◽

Protein Interactions ◽

Adrenergic Receptor ◽

Negative Charge ◽

G Protein Coupled Receptors ◽

Monovalent Cations ◽

Receptor Sensitivity ◽

G Protein Coupling ◽

G Protein Coupled ◽

Aspartate Residue

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

Endocrinology ◽

10.1210/en.2013-1407 ◽

2013 ◽

Vol 154 (10) ◽

pp. 3925-3930 ◽

Cited By ~ 32

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1800756115 ◽

2018 ◽

Vol 115 (12) ◽

pp. 3036-3041 ◽

Cited By ~ 43

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.

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