Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250317_Report_PTM.setup.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/Phosphosites_quantity.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_GLP_1_100nM_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_GLP_1_100nM_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_Semaglutide_100nM_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_Buffer_10mM_glucose_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_Buffer_10mM_glucose_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_Semaglutide_100nM_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_GLP_1_100nM_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_GLP_1_100nM_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_Semaglutide_100nM_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_Buffer_10mM_glucose_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_Semaglutide_100nM_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063931/20250228_EVO5_AST5_DIA_IO5cm_80SPD_ML_Collab_Phospho_islet_CPH_Donor3_Buffer_10mM_glucose_1.rawprimaryOK200jesper.olsen@cpr.ku.dkPierre SabatierMass SpectrometryData-independent acquisitionPhosphoproteomicsPancreatic isletsGlp-1rhttps://www.ebi.ac.uk/pride/archive/projects/PXD063931Pancreatic IsletPhosphopeptides enrichment Pancreatic islets were lysed in 50 µL of lysis buffer consisting of 5% sodium dodecyl sulfate, 100 mM Tris pH 8.5, and boiled for 10 minutes at 95°C. TCEP (tris(2-carboxyethyl)phosphine) and 2-chloroacetamide were added to a final concentration of 5 mM and 10 mM respectively, after which the samples were snap frozen. After thawing, 40U of Benzonase was added to each sample and incubated in a ThermoShaker for 15 min at 37°C. Proteins were digested using a protein aggregation capture-based digestion performed on the OpenTron2 robot. Briefly, the volume of each sample was adjusted to 40 µL using 50 mM TEAB (tetraethylammonium bromide). 5 µL of hydroxyl magnetic beads (ReSyn Bioscience) were added followed by 60 µL ACN (acetonitrile) to induce protein aggregation. After protein aggregation the beads were washed with 70% ethanol and 30 µL of digestion solution was added (Trypsin (1:25 enzyme:protein) and LysC (1:100) in 50mM TEAB). The samples were incubated for 4h at 37°C. The samples were then used for on-beads phosphopeptide enrichment on the KingFisher Flex robot using 5 µL of Zr-IMAC HP magnetic beads (ReSyn Bioscience). The phosphopeptide enrichment was performed a second time on the flow-through of the first enrichment. The enriched samples were acidified using 30 µL of 10% TFA (trifluoroacetic acid) and loaded on Evotips according to manufacturer’s protocol. LC-MS/MS analysis Samples were injected into an EvosepOne liquid chromatography (Evosep Biosystem) coupled to an Orbitrap Astral mass spectrometer (ThermoFisher Scientific). The separation was carried out on the whisper-zoom 80 SPD gradient (16 min) with an Aurora Rapid column (IonOpticks, 5cm x 75 µm x 1.9 µm). The phosphoproteomes were acquired in positive mode using a narrow window data independent strategy (nDIA) with a MS1 resolution set at 240,000 and analyzed on a mass range from 480 to 1080 m/z using 4 Th isolation windows with a maximum injection time of 6 ms.PrideNot availableMS data analysis The MS raw files were analyzed using Spectronaut v19 (Biognosys) with a directDIA + approach using the human protein reference database (Uniprot 2022 release, 20,588 sequences) complemented with common contaminants (246 sequences). Cysteine carbamylation was set as a fixed modification, and methionine oxidation, protein N-termini acetylation and phosphorylation of serine, threonine and tyrosine were set as variable modifications. The maximum number of variable modifications per peptide was limited to 3. The PTM localization cutoff was set as 0.75 and cross run normalization was not selected.ProteomicsJesper OlsenNovo Nordisk Foundation Center for Protein Research, University of CopenhagenPARTIALHomo Sapiens (human)pierre63.sabatier@gmail.comNot availableDepartment of Surgical Sciences, Uppsala University. Novo Nordisk Foundation Center for Protein Research, University of Copenhagen.SwedenfalseGLP-1R and GIPR heterodimerization and expression determine downstream signaling and metabolic phenotypesAdvancements in unimolecular drugs co-targeting the glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) have demonstrated remarkable clinical efficacy in treating type 2 diabetes (T2D) and obesity. However, the paradoxical ability of both GIPR agonism and antagonism to complement GLP-1R agonism underscores the need to elucidate molecular mechanisms underlying incretin receptor pharmacology and crosstalk. Here, we show that GLP-1 promotes heterodimerization via interactions between TM4 of GLP-1R and TM1/2 of GIPR. Phosphoproteomics profiling and molecular dynamics simulations reveal that dimerizing and non-dimerizing GLP-1R agonists differentially engage with GLP-1R and elicit distinct regulation of downstream signaling networks in human islets. Co-expression of GLP-1R and GIPR amplifies GIPR-mediated signaling in a β-arrestin-dependent manner, while GLP-1R-mediated signaling is suppressed by GIPR expression. Additionally, upregulation or downregulation of GIPR expression was associated with adiposity and diabetic traits, respectively. Collectively, these findings uncover mechanisms underlying the interplay between GLP-1R and GIPR and highlight heterodimerization as an innovative strategy for incretin-based therapies and personalized treatment.2026-05-132025-05-14PXD063931NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2102NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:5722NEWT:1247NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:2649997NEWT:272563NEWT:224326NCBITaxon:79857NEWT:1096976NEWT:82688NEWT:95648NEWT:3885NEWT:3888NEWT:5821NEWT:1589NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:626528NEWT:3347NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:197NEWT:1390363NEWT:288705NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:1270NEWT:374990NEWT:498217NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:286NEWT:391619NEWT:360104NEWT:287NEWT:246197NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:5508NEWT:294128NEWT:11676NEWT:55571NEWT:35500NEWT:1140NEWT:100226NEWT:4530NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:1094343NEWT:1336795NEWT:296543NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:105023NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:243277NEWT:990119NEWT:408172NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:1436733NEWT:460519NEWT:1187947NEWT:1432138NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:9598NEWT:8030NEWT:9483NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:3635NEWT:5811NEWT:235443NEWT:108458NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:225117NEWT:499175NEWT:109779NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:410289NEWT:373153NEWT:352472NEWT:357NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:38293NEWT:327159NEWT:178876NEWT:327160NEWT:573NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:7237NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1249668NEWT:7108NEWT:317NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:588858NEWT:9639NEWT:242231NEWT:7574NEWT:1715256NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:986NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:1194669NEWT:243230NEWT:8355NEWT:633NEWT:9685NEWT:7029NEWT:1080772NEWT:8479NEWT:1283300NEWT:632NEWT:6183NEWT:6063NEWT:630NEWT:334747NEWT:61235NEWT:15368NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:469008NEWT:260707NEWT:256318NCBITaxon:6191NEWT:1836NEWT:185431NEWT:29760NEWT:260704NEWT:1719NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:105231NEWT:1216981NEWT:52638NEWT:4097NCBITaxon:6073NEWT:884204NEWT:6279NEWT:1123869NEWT:9544NEWT:1329349NEWT:7370NEWT:83906NEWT:607699NCBITaxon:1502NEWT:6282NEWT:208963NEWT:208964NEWT:1134506NEWT:575584NEWT:38783NEWT:8727NEWT:4006NEWT:8726NEWT:6426NEWT:6669NEWT:10090NEWT:4120NEWT:51515NEWT:5693NEWT:2186NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:5334NEWT:37334NEWT:382352NCBITaxon:10359NEWT:242619NEWT:632957NEWT:34073NEWT:373995NEWT:544404NEWT:9925NEWT:8839NEWT:4232NEWT:2758385NEWT:37349NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:714NEWT:36015NEWT:421932NEWT:312017NEWT:196627NEWT:5691NEWT:48479NEWT:627025NEWT:1097677NEWT:61674NEWT:1117957NEWT:9913NEWT:8709NE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