N-terminal fragment shedding contributes to signaling of the full-length adhesion receptor ADGRL3

Nicole A Perry-Hauser; Jonathan R Du Rand; Kuo Hao Lee; Lei Shi; Jonathan A Javitch

doi:10.1016/j.jbc.2025.108174

N-terminal fragment shedding contributes to signaling of the full-length adhesion receptor ADGRL3

J Biol Chem. 2025 Jan 9:108174. doi: 10.1016/j.jbc.2025.108174. Online ahead of print.

Authors

Nicole A Perry-Hauser¹, Jonathan R Du Rand¹, Kuo Hao Lee², Lei Shi², Jonathan A Javitch³

Affiliations

¹ Department of Psychiatry, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA; Department of Molecular Pharmacology and Therapeutics, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA; Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA.
² Computational Chemistry and Molecular Biophysics Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, Baltimore, MD, USA.
³ Department of Psychiatry, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA; Department of Molecular Pharmacology and Therapeutics, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA; Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA. Electronic address: jaj2@cumc.columbia.edu.

PMID: 39798870
DOI: 10.1016/j.jbc.2025.108174

Abstract

Most adhesion GPCRs undergo autoproteolytic cleavage during receptor biosynthesis, resulting in non-covalently bound N- and C-terminal fragments (NTF and CTF) that remain associated during receptor trafficking to the plasma membrane. While substantial evidence supports increased G protein signaling when just the CTF is expressed, there is an ongoing debate about whether NTF removal is required to initiate signaling in the context of the wild-type receptor. Here, we use adhesion GPCR latrophilin-3 (ADGRL3) as a model receptor to investigate tethered agonist-mediated activation. First, we show that extending the N terminus of the tethered agonist in ADGRL3 CTF disrupts G protein signaling. This suggests that if the tethered agonist is not fully exposed it is unlikely to interact with the orthosteric pocket in an optimal manner for G protein activation. Second, we show that when full-length ADGRL3 is expressed in heterologous cells, approximately ∼5% of the receptor population spontaneously sheds its NTF. We hypothesized that the signaling activity observed for full-length ADGRL3 is largely due to this shedding, which exposes the native tethered agonist. To test this hypothesis, we used a full-length, cleavage-deficient ADGRL3 mutant. Compared to wild-type receptor, this mutant lost ∼80% of its signaling through Gα_{13 and} showed a much lower level of spontaneous NTF shedding, approximately 20% of that observed for wild-type receptor. This loss of spontaneous NTF shedding likely explains its diminished signaling activity. These findings suggest that TA-mediated signal transduction by full-length ADGRL3 requires removal of its NTF.

Keywords: G protein; adhesion G protein-coupled receptor latrophilin (ADGRL); adhesion G protein-coupled receptors (aGPCRs); autoproteolysis; cell signaling; membrane protein; receptor reserve; tethered agonist.