Biology subjects

Garcia-Nafria, J.

Publications and source records attributed to Garcia-Nafria, J..

4 recordsLinked to original sources

bioRxiv2025

FDA drug repurposing uncovers modulators of dopamine D2 receptor localization via disruption of the NCS-1 interaction

Dopamine D2 receptor (D2R) regulates key aspects of motor control, cognition, and reward. Its function depends not only on ligand binding and signaling efficacy, but also on the dynamic control of receptor localization at the cell surface. Neuronal Calcium Sensor 1 (NCS-1) is a calcium binding protein which directly interacts with D2R in a Ca2+-dependent manner. Here, we investigated the regulatory role of NCS-1 in D2R localization and function. We found that NCS-1 promotes the trafficking of D2R to the plasma membrane through a mechanism dependent on active exocytosis. Functional signaling assays confirmed that NCS-1 does not alter the canonical receptor pharmacology. Using a library of FDA-approved drugs, a structure-based drug-repurposing strategy was designed to find protein-protein interaction modulators that allowed the exploration of the NCS-1/D2R interface as a new pharmacological target. Azilsartan medoxomil, atorvastatin, and vilazodone disrupted its interaction with D2R, reducing receptor surface expression in cells. Crystallography and molecular dynamics simulations revealed their mechanism of action. These compounds target the NCS-1 hydrophobic crevice and overlap the D2R binding site, perturbing the dynamics of the regulatory helix H10 in NCS-1. These findings uncover a previously unexploited intracellular mechanism for modulating D2R function and highlight the potential of targeting protein-protein interactions for therapeutic purposes. Our results provide a framework for fine-tuning dopaminergic tone through receptor localization mechanisms, offering an alternative strategy to conventional approaches based on receptor blockade or direct agonism. SIGNIFICANCEFDA-approved drugs targeting the calcium sensor NCS-1 selectively disrupt its interaction with the dopamine D2 receptor, offering a new strategy to modulate receptor trafficking without altering receptor signaling.

biochemistry↗

bioRxiv2022

The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

The Neuronal Calcium Sensor 1 and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to G have revealed how Ric-8A phosphorylation promotes G recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with G subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with G. Our data show that the binding of NCS-1 and G to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a profound structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards G when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here that define the NCS-1/Ric-8A interface will allow the development of therapeutic synapse function regulators with improved activity and selectivity.

biochemistry↗

bioRxiv2020

Molecular determinants of β-arrestin coupling to formoterol-bound β1-adrenoceptor

The {beta}1-adrenoceptor ({beta}1AR) is a G protein-coupled receptor (GPCR) activated by the hormone noradrenaline, resulting in the coupling of the heterotrimeric G protein Gs1. G protein-mediated signalling is terminated by phosphorylation of the receptor C-terminus and coupling of {beta}-arrestin 1 ({beta}arr1, also known as arrestin-2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins (biased agonism)3 is important in drug development, as the therapeutic effect may arise from only one signalling cascade, whilst the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, we determined the electron cryo-microscopy (cryo-EM) structure of the {beta}1AR-{beta}arr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound {beta}1AR coupled to the G protein mimetic nanobody Nb806. {beta}arr1 couples to {beta}1AR in a distinct manner to how Gs couples to {beta}2AR7, with the finger loop of {beta}arr1 occupying a narrower cleft on the intracellular surface closer to transmembrane helix H7 than the C-terminal 5 helix of Gs. The conformation of the finger loop in {beta}arr1 is different from that adopted by the finger loop in visual arrestin when it couples to rhodopsin8, and its {beta}-turn configuration is reminiscent of the loop in Nb80 that inserts at the same position. {beta}1AR coupled to {beta}arr1 showed significant differences in structure compared to {beta}1AR coupled to Nb80, including an inward movement of extracellular loop 3 (ECL3) and the cytoplasmic ends of H5 and H6. In the orthosteric binding site there was also weakening of interactions between formoterol and the residues Ser2115.42 and Ser2155.46, and a reduction in affinity of formoterol for the {beta}1AR-{beta}arr1 complex compared to {beta}1AR coupled to mini-Gs. These differences provide a foundation for the development of small molecules that could bias signalling in the {beta}-adrenoceptors.

molecular biology↗

bioRxiv2018

Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein

The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here we determine the structure by electron cryo-microscopy (cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the {beta}{gamma} subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 [A] or better. Comparison with the 3.4 [A] resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the {beta} subunit of the G protein was observed.

biophysics↗