Dataset Information

XL-MS Analysis of Recombinant GPR158:RGS7:Gb5 Complex in Detergent Micelles

ABSTRACT: GPR158 is widely expressed in the brain and have been implicated in depression, cognition, mood and several cancers. GPR158 is also involved in retinal photoreceptor signaling along with it involvement in synaptic organization. GPR158 interact with G-protein regulator-RGS7 and brings it to plasma membrane to allosterically modulate GTPase activity of G-protein alpha subunit. However, the signaling mechanism and it structural organization is not known. The mnuscript associated with this deposition describes a structural analysis of GPR158 in complex with downstream effectors RGS7 and Gb5. This deposition pertains to the crosslinking mass spectrometry experiments conducted to complement these efforts.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Timothy Strutzenberg

LAB HEAD: Patrick R. Griffin

PROVIDER: PXD026603 | Pride | 2023-03-11

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20210129_GPR158-Complex_DMSO_TRY_REP1.raw	Raw
	20210129_GPR158-Complex_DMSO_TRY_REP2.raw	Raw
	20210129_GPR158-Complex_DMSO_TRY_REP3.raw	Raw
	20210129_GPR158-Complex_DSSO_TRY_REP1.raw	Raw
	20210129_GPR158-Complex_DSSO_TRY_REP2.raw	Raw

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Publications

Cryo-EM structure of human GPR158 receptor coupled to the RGS7-Gβ5 signaling complex.

Patil Dipak N DN Singh Shikha S Laboute Thibaut T Strutzenberg Timothy S TS Qiu Xingyu X Wu Di D Novick Scott J SJ Robinson Carol V CV Griffin Patrick R PR Hunt John F JF Izard Tina T Singh Appu K AK Martemyanov Kirill A KA

Science (New York, N.Y.) 20211118 6576

GPR158 is an orphan G protein–coupled receptor (GPCR) highly expressed in the brain, where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanisms of GPR158 are largely unknown. We used single-particle cryo–electron microscopy (cryo-EM) to determine the structures of human GPR158 alone and bound to an RGS signaling complex. The structures reveal a homodimeric organizati ...[more]

PMID: 34793198

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Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.