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

Protein-protein interactions between G protein-coupled receptors (GPCRs) can augment their functionality and increase the repertoire of signaling pathways they regulate. New therapeutics designed to modulate such interactions may allow for targeting of a specific GPCR activity, thus reducing potential for side effects. Dopamine receptor (DR) heteromers are promising candidates for targeted therapy of neurological conditions such as Parkinson’s disease since current treatments can have severe side effects. To facilitate development of such therapies, it is necessary to identify the various DR binding partners. We report here a new interaction partner for DRD2 and DRD3, the orphan receptor G protein-coupled receptor 143 (GPR143), an atypical GPCR that plays multiple roles in pigment cells and is expressed in several regions of the brain. We previously demonstrated that the DRD2/ DRD3 antagonist pimozide also modulates GPR143 activity. Using confocal microscopy and two FRET methods, we observed that the DRs and GPR143 colocalize and interact at intracellular membranes. Furthermore, co-expression of wildtype GPR143 resulted in a 57% and 67% decrease in DRD2 and DRD3 activity, respectively, as determined by β-Arrestin recruitment assay. GPR143-DR dimerization may negatively modulate DR activity by changing affinity for dopamine or delaying delivery of the DRs to the plasma membrane.

Download Full-text

Structural Characterization of Receptor–Receptor Interactions in the Allosteric Modulation of G Protein-Coupled Receptor (GPCR) Dimers

International Journal of Molecular Sciences ◽

10.3390/ijms22063241 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3241

Author(s):

Raudah Lazim ◽

Donghyuk Suh ◽

Jai Woo Lee ◽

Thi Ngoc Lan Vu ◽

Sanghee Yoon ◽

...

Keyword(s):

G Protein ◽

Protein Interactions ◽

Metabotropic Glutamate Receptor ◽

Three Dimensional ◽

Allosteric Modulation ◽

Experimental Investigations ◽

G Protein Coupled Receptor ◽

Metabotropic Glutamate ◽

Gpcr Activation ◽

G Protein Coupled

G protein-coupled receptor (GPCR) oligomerization, while contentious, continues to attract the attention of researchers. Numerous experimental investigations have validated the presence of GPCR dimers, and the relevance of dimerization in the effectuation of physiological functions intensifies the attractiveness of this concept as a potential therapeutic target. GPCRs, as a single entity, have been the main source of scrutiny for drug design objectives for multiple diseases such as cancer, inflammation, cardiac, and respiratory diseases. The existence of dimers broadens the research scope of GPCR functions, revealing new signaling pathways that can be targeted for disease pathogenesis that have not previously been reported when GPCRs were only viewed in their monomeric form. This review will highlight several aspects of GPCR dimerization, which include a summary of the structural elucidation of the allosteric modulation of class C GPCR activation offered through recent solutions to the three-dimensional, full-length structures of metabotropic glutamate receptor and γ-aminobutyric acid B receptor as well as the role of dimerization in the modification of GPCR function and allostery. With the growing influence of computational methods in the study of GPCRs, we will also be reviewing recent computational tools that have been utilized to map protein–protein interactions (PPI).

Download Full-text

Interprotein interactions are responsible for the confined diffusion of a G-protein-coupled receptor at the cell surface

Biochemical Society Transactions ◽

10.1042/bst0311001 ◽

2003 ◽

Vol 31 (5) ◽

pp. 1001-1005 ◽

Cited By ~ 10

Author(s):

F. Daumas ◽

N. Destainville ◽

C. Millot ◽

A. Lopez ◽

D. Dean ◽

...

Keyword(s):

G Protein ◽

Protein Interactions ◽

Domain Size ◽

Single Particle Tracking ◽

G Protein Coupled Receptor ◽

Short Term ◽

Detailed Statistical Analysis ◽

G Protein Coupled ◽

Dynamic Organization

The monitoring of the movements of membrane proteins (or lipids) by single-particle tracking enables one to obtain reliable insights into the complex dynamic organization of the plasma membrane constituents. Using this technique, we investigated the diffusional behaviour of a G-protein-coupled receptor. The trajectories of the receptors revealed a diffusion mode combining a short-term rapid confined diffusion with a long-term slow diffusion. A detailed statistical analysis shows that the receptors have a diffusion confined to a domain which itself diffuses, the confinement being due to long-range attractive inter-protein interactions. The existing models of the dynamic organization of the cell membrane cannot explain our results. We propose a theoretical Brownian model of interacting proteins that is consistent with the experimental observations and accounts for the variations found as a function of the domain size of the short-term and long-term diffusion coefficients.

Download Full-text

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.

Download Full-text

Protein–protein interactions at G-protein-coupled receptors

Trends in Pharmacological Sciences ◽

10.1016/s0165-6147(00)01801-0 ◽

2001 ◽

Vol 22 (10) ◽

pp. 513-518 ◽

Cited By ~ 94

Author(s):

Graeme Milligan ◽

Julia H White

Keyword(s):

G Protein ◽

Protein Interactions ◽

G Protein Coupled Receptors ◽

Protein Protein Interactions ◽

G Protein Coupled

Download Full-text

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

Cytotechnology ◽

10.1007/s10616-018-0231-7 ◽

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

Download Full-text

Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1711543114 ◽

2017 ◽

Vol 114 (51) ◽

pp. 13453-13458 ◽

Cited By ~ 20

Author(s):

John J. Skinner ◽

Sheng Wang ◽

Jiyoung Lee ◽

Colin Ong ◽

Ruth Sommese ◽

...

Keyword(s):

G Protein ◽

Protein Interactions ◽

Salt Bridge ◽

G Protein Coupled Receptor ◽

Inhibitory Protein ◽

Raf Kinase ◽

Protein Interfaces ◽

Partial Unfolding ◽

Direct Recognition ◽

G Protein Coupled

Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or “theft” mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein–coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein–Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.

Download Full-text