The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Ga chaperone and GEF Ric-8A
- Institute of Physical-Chemistry Blas Cabrera, Spain
- University of Montana, United States
- University of Zaragoza, Spain
- Hospital Universitario Ramón y Cajal, Spain
- Software 4 Science Developments, Spain
- Institute of Physical-Chemistry, Spain
- Institute of Physical Chemistry Blas Cabrera, Spain
Abstract
The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga 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 Ga 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 Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a 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 Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.
Data availability
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, https://www.pdb.org/. PDB with codes: Structure 1 (8ALH), Structure 2 (8AHY), Structure 3 (8ALM).All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figure 2, Figure 3, Figure 4, Figure 5
Article and author information
Author details
Maria Jose Sanchez-Barrena
Funding
Spanish National Plan for Scientific and Technical Research and Innovation (PID2019-111737RB-I00)
- Maria Jose Sanchez-Barrena
Spanish National Plan for Scientific and Technical Research and Innovation (PID2019-106608RB-I00)
- Alicia Mansilla
Spanish National Plan for Scientific and Technical Research and Innovation (PDC2022-133775-I00)
- Alicia Mansilla
Spanish National Plan for Scientific and Technical Research and Innovation (RTI2018-099985-B-I00)
- Margarita Menendez
Spanish National Plan for Scientific and Technical Research and Innovation (PID2020-113359GA-I00)
- Javier García-Nafría
Spanish National Plan for Scientific and Technical Research and Innovation (RYC-2017-22392)
- Alicia Mansilla
Spanish National Plan for Scientific and Technical Research and Innovation (RYC2018-025731-I)
- Javier García-Nafría
National Institutes of Health (P30GM140963)
- Stephen Sprang
National Institutes of Health (R01GM105993)
- Stephen Sprang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Randy B Stockbridge, University of Michigan, United States
Version history
- Preprint posted: December 10, 2022 (view preprint)
- Received: January 12, 2023
- Accepted: November 24, 2023
- Accepted Manuscript published: November 29, 2023 (version 1)
- Version of Record published: December 20, 2023 (version 2)
Copyright
© 2023, Muñoz-Reyes et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 746
- views
-
- 157
- downloads
-
- 0
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Ga chaperone and GEF Ric-8A
eLife 12:e86151.
https://doi.org/10.7554/eLife.86151
Further reading
-
- Biochemistry and Chemical Biology
- Neuroscience
In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health, and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex/strain-selective ‘raw’ chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animal’s sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary ‘secretome’, both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1’s helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.
