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Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3
Adhesion G protein-coupled receptor latrophilin 3 (ADGRL3), a cell adhesion molecule highly expressed in the central nervous system, acts in synapse formation through trans interactions with its ligands. It is largely unknown if these interactions serve a purely adhesive function or can modulate G p...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9674912/ https://www.ncbi.nlm.nih.gov/pubmed/36244455 http://dx.doi.org/10.1016/j.jbc.2022.102594 |
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author | Perry-Hauser, Nicole A. VanDyck, Max W. Lee, Kuo Hao Shi, Lei Javitch, Jonathan A. |
author_facet | Perry-Hauser, Nicole A. VanDyck, Max W. Lee, Kuo Hao Shi, Lei Javitch, Jonathan A. |
author_sort | Perry-Hauser, Nicole A. |
collection | PubMed |
description | Adhesion G protein-coupled receptor latrophilin 3 (ADGRL3), a cell adhesion molecule highly expressed in the central nervous system, acts in synapse formation through trans interactions with its ligands. It is largely unknown if these interactions serve a purely adhesive function or can modulate G protein signaling. To assess how different structural elements of ADGRL3 (e.g., the adhesive domains, autoproteolytic cleavage site, or tethered agonist (TA)) impact receptor function, we require constructs that disrupt specific receptor features without impacting others. While we showed previously that mutating conserved Phe and Met residues in the TA of ADGRL3–C-terminal fragment (CTF), a CTF truncated to the G protein-coupled receptor proteolysis site, abolishes receptor-mediated G protein activation, we now find that autoproteolytic cleavage is disrupted in the full-length version of this construct. To identify a construct that disrupts TA-dependent activity without impacting proteolysis, we explored other mutations in the TA. We found that mutating the sixth and seventh residues of the TA, Leu and Met, to Ala impaired activity in a serum response element activity assay for both full-length and CTF constructs. We confirmed this activity loss results from impaired G protein coupling using an assay that acutely exposes the TA through controlled proteolysis. The ADGRL3 mutant expresses normally at the cell surface, and immunoblotting shows that it undergoes normal autoproteolysis. Thus, we found a construct that disrupts tethered agonism while retaining autoproteolytic cleavage, providing a tool to disentangle these functions in vivo. Our approach and specific findings are likely to be broadly applicable to other adhesion receptors. |
format | Online Article Text |
id | pubmed-9674912 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-96749122022-11-21 Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3 Perry-Hauser, Nicole A. VanDyck, Max W. Lee, Kuo Hao Shi, Lei Javitch, Jonathan A. J Biol Chem Research Article Adhesion G protein-coupled receptor latrophilin 3 (ADGRL3), a cell adhesion molecule highly expressed in the central nervous system, acts in synapse formation through trans interactions with its ligands. It is largely unknown if these interactions serve a purely adhesive function or can modulate G protein signaling. To assess how different structural elements of ADGRL3 (e.g., the adhesive domains, autoproteolytic cleavage site, or tethered agonist (TA)) impact receptor function, we require constructs that disrupt specific receptor features without impacting others. While we showed previously that mutating conserved Phe and Met residues in the TA of ADGRL3–C-terminal fragment (CTF), a CTF truncated to the G protein-coupled receptor proteolysis site, abolishes receptor-mediated G protein activation, we now find that autoproteolytic cleavage is disrupted in the full-length version of this construct. To identify a construct that disrupts TA-dependent activity without impacting proteolysis, we explored other mutations in the TA. We found that mutating the sixth and seventh residues of the TA, Leu and Met, to Ala impaired activity in a serum response element activity assay for both full-length and CTF constructs. We confirmed this activity loss results from impaired G protein coupling using an assay that acutely exposes the TA through controlled proteolysis. The ADGRL3 mutant expresses normally at the cell surface, and immunoblotting shows that it undergoes normal autoproteolysis. Thus, we found a construct that disrupts tethered agonism while retaining autoproteolytic cleavage, providing a tool to disentangle these functions in vivo. Our approach and specific findings are likely to be broadly applicable to other adhesion receptors. American Society for Biochemistry and Molecular Biology 2022-10-14 /pmc/articles/PMC9674912/ /pubmed/36244455 http://dx.doi.org/10.1016/j.jbc.2022.102594 Text en © 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Research Article Perry-Hauser, Nicole A. VanDyck, Max W. Lee, Kuo Hao Shi, Lei Javitch, Jonathan A. Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3 |
title | Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3 |
title_full | Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3 |
title_fullStr | Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3 |
title_full_unstemmed | Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3 |
title_short | Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3 |
title_sort | disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor adgrl3 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9674912/ https://www.ncbi.nlm.nih.gov/pubmed/36244455 http://dx.doi.org/10.1016/j.jbc.2022.102594 |
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