PXD056561

PXD056561 is an original dataset announced via ProteomeXchange.

Title	GAIN domain cleavage of aGPCRs requires multiple domain-extrinsic factors coordinated by the 7TM region
Description	The G protein-coupled receptor (GPCR) autoproteolysis-inducing (GAIN) domain is a structurally conserved and tell-tale fold of class B2/adhesion GPCRs (aGPCRs). Self-cleavage of the GAIN domain is central for mechanobiological and signaling properties of aGPCRs. Biochemical analyses and atomistic details of several GAIN domain structures have recently provided a basic understanding on the molecular mechanism of the autoproteolytic reaction. However, if and how self-cleavage may be impacted by factors outside of the GAIN domain has not been systematically investigated. Here we used ADGRE2/EMR2 as a model receptor to explore processes contributing to the autoproteolytic cleavage of its GAIN domain. Retention Upon Selective Hook (RUSH), a quantitative pulse-chase assay to temporally follow newly synthesised receptor protein, showed that receptor self-cleavage serves a checkpoint function before exit of newly synthesised receptor protein from the ER, but not for plasma membrane trafficking. We established that autoproteolysis occured before or during synthesis of the first transmembrane helix, and that the extent of cleavage increases with the translation of the complete heptahelical (7TM) region of the receptor. We show that this effect is caused by tethering the nascent receptor to the lumenal side of the ER membrane during receptor biogenesis. ER membrane tethering facilitates GAIN domain processing by positioning the receptor near components of the N-glycosylation pathway, as demonstrated by genetic code expansion combined with biorthogonal labeling of the GAIN domain, photo-crosslinking and mass- spectrometric analysis. Collectively, these findings provide evidence that the extent of GAIN domain cleavage depends not only on intra-domain parameters, but can be modulated by the subcellular location of the domain, which aids its post-translational glycosylation. We anticipate that our findings will be valuable for understanding the GAIN domain self-cleavage of other aGPCRs, and will inform pharmacological strategies to modulate their activation and signaling.
HostingRepository	PRIDE
AnnounceDate	2025-10-06
AnnouncementXML	Submission_2025-10-06_12:20:51.482.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Christian Ihling
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	iodoacetamide derivatized residue
Instrument	timsTOF Pro

Revision	Datetime	Status	ChangeLog Entry
0	2024-10-07 11:42:05	ID requested
⏵ 1	2025-10-06 12:20:51	announced

10.1038/S41467-025-64589-3;

submitter keyword: Adhesion GPCR, photo-crosslinking, GAIN domain

Tobias Langenhan
contact affiliation	Rudolf-Schoenheimer Institute of Biochemistry, Division of General Biochemistry, Medical Faculty, Leipzig University, Johannisallee 30, 04103 Leipzig, Germany
contact email	tobias.langenhan@gmail.com
lab head
Christian Ihling
contact affiliation	MLU Halle, Inst. f. Pharmacy
contact email	christian.ihling@pharmazie.uni-halle.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD056561
     1. Label: PRIDE project
     2. Name: GAIN domain cleavage of aGPCRs requires multiple domain-extrinsic factors coordinated by the 7TM region