{"paper_id":"84a811a4-ee12-4bfa-bf00-e8f0569dc405","body_text":"P-Rex1 Limits the Agonist-Induced Internalisation of GPCRs Independently of \nits Rac-GEF Activity \n \nMartin J. Baker1,2,3, Elizabeth Hampson1,2, Priota Islam2, Ruben Pelaez Moral2, Eve A. Maunders2, Kirsti \nHornigold2, Elpida Tsonou 2,4, David C. Hornigold4, Roderick E. Hubbard5, Andrew J. Massey5, Heidi C. \nE. Welch2,6 \n \n1 These authors contributed equally to the project and share first authorship \n \n2 Signalling Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK \n3 Present address: Cell Signalling Group, Cancer Research UK Manchester Institute, University of \nManchester, Wilmslow Road, Manchester, UK \n4 Bioscience Metabolism, Research and Early Development, Cardiovascular, Renal and Metabolism \n(CVRM), BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK \n5 Vernalis (R&D) Ltd., Granta Park, Cambridge, UK \n \n6 Correspondence: \nHeidi Welch \nSignalling Programme \nThe Babraham Institute \nBabraham Research Campus \nCambridge CB22 3AT, UK \n+44 (0)1223 406 596 \nheidi.welch@babraham.ac.uk \n \nKeywords: PREX1, PREX2, guanine -nucleotide exchange factor (GEF), G pr otein-coupled receptor \n(GPCR), agonist-induced internalisation, trafficking, GRK2 \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nSummary \nThe Rac-GEF P-Rex1 mediates GPCR signalling by activating the small GTPase Rac. We show here that \nP-Rex1 also controls GPCR trafficking. P-Rex1 inhibits the agonist-stimulated internalisation of the \nGPCR S1PR1 independently of its Rac-GEF activity, through its PDZ, DEP and IP4P domains. P-Rex1 also \nlimits the agonist-induced trafficking of CXCR4, PAR4, and GLP1R , but does not control steady-state \nGPCR levels, nor the agonist-induced internalisation of the RTKs PDGFR and EGFR. P-Rex1 blocks the \nphosphorylation required for GPCR internalisation. P-Rex1 binds Grk2, both in vitro and in cells, but \ndoes not appear to regulate Grk2 activity. We propose that P -Rex1 limits the agonist -induced \ninternalisation of GPCRs through its interaction with Grk2 to maintain high levels of active GPCR at the \nplasma membrane. Therefore, P-Rex1 plays a dual role in promoting GPCR responses, by controlling \nGPCR trafficking through an adaptor function as well as by mediating GPCR signalling through its Rac-\nGEF activity. \n \nHighlights  \n• P-Rex1 controls GPCR trafficking, independently of its Rac-GEF activity \n• P-Rex1 limits the agonist-induced internalisation of S1PR1, CXCR4, PAR4 and GLP1R \n• P-Rex1 does not control steady-state GPCR levels, or PDGFR and EGFR trafficking \n• P-Rex1 binds Grk2 and inhibits the phosphorylation required for GPCR internalisation \n \neTOC blurb  \nP-Rex1 activates Rac downstream of  GPCRs to regulate processes ranging from innate immunity to \nneuronal plasticity, its deregulation contributing to cancer. Here, Baker et al. show that P-Rex1 also \ncontrols GPCR trafficking, limiting agonist-induced GPCR internalisation through an adaptor function. \nThus, P-Rex1 promotes GPCR responses in a dual manner.   \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nIntroduction \nP-Rex1 is a guanine-nucleotide exchange factor (GEF) for Rac -type small GTPases which is widely \nexpressed and plays important roles in the immune and nervous systems 1-4. In the immune system, \nP-Rex1 is required for a range of pro-inflammatory and immune functions, including the recruitment \nof leukocytes to sites of inflammation and infection, and for the clearance of bacteria 3,5-8. In the \nnervous system, P-Rex1 is required for synaptic plasticity, fine motor skills, and social recognition 9-11. \nFurthermore, P -Rex1 is required for  melanoblast migration during development, controlling skin \npigmentation 12. De regulation of P -Rex1 levels contributes to a range of diseases. P -Rex1 \noverexpression is seen in many types of cancers, including breast cancer and melanoma, promoting \ntumour initiation, growth and/or invasiveness 12-19. Loss of P-Rex1 is associated with autism spectrum \ndisorders 11. Furthermore, in mice, P -Rex1 promotes  pulmonary fibrosis 20 and diet -induced non -\nalcoholic fatty liver disease 21. \nAll physiological and pathophysiological functions of P-Rex1 are either known or assumed to \nbe mediated through its catalytic Rac-GEF activity. P-Rex1 activates all isoforms of Rac (Rac1, Rac2, \nRac3, RhoG) upon itself being activated synergistically by the phosphoinositide 3 -kinase (PI3K) -\ngenerated lipid second messenger phosphoinositide 3,4,5 -trisphosphate (PIP 3) and by the Gβγ \nsubunits of heterotrimeric G proteins which are released upon activation of  G protein -coupled \nreceptors (GPCRs) 1. P-Rex1 has an N-terminal catalytic DH domain which serves to activate Rac, a PH \ndomain, two DEP and two PDZ domains , and a C-terminal half similar to inositol polyphosphate 4 -\nphosphatase (IP4P) but without phosphatase activity 1,3. Residues E56 and N238 in the DH domain are \nrequired for the interaction with Rac1 and for catalytic activity  4,22. PIP3 binds to the PH domain and \nGβγ bind to the DH domain and to C-terminal domains, whereas the DEP, PDZ and IP4P domains serve \nto keep the protein in an autoinhibited confirmation prior to cell stimulation 22-30. \nIn addition to P -Rex1, the P -Rex family also comprises P -Rex2, a Rac -GEF with the same domain \nstructure and regulated in the same manner by  PIP3 and Gβγ 9,31,32. However, unlike P -Rex1, P-Rex2 \nhas a known adaptor function, binding and inhibiting the tumour suppressor PTEN which converts PIP3 \nto PI(4,5)P2, and thus indirectly stimulating PI3K pathway activity, independently of its catalytic Rac -\nGEF activity 33-35. This adaptor function is specific to P-Rex2, as P-Rex1 cannot bind PTEN 33. \nGPCRs are the largest family of cell surface receptors, characterised by their seven transmembrane \nspanning regions 36. They signal in response to a vast array of stimuli, ranging from photons to proteins, \npeptides and lipids. Ligand binding induces a conformational change, which activates the receptor -\ncoupled heterotrimeric G protein, consisting of Gα and  Gβγ 37. The Gα subunit is a GTPase , and the \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nligand-bound GPCR acts as its GEF, leading to GTP -loading of Gα and release of the Gβγ dimer. Gα -\nGTP and Gβγ then interact with their respective effector proteins to elicit downstream signalling 37.   \nGPCRs can be classified according to the type of Gα  they couple to, Gαs Gαi, Gαq or Gα12/13. Very \ngenerally, Gαs signals to increase adenyl yl cyclase activity and thus cellular cAMP levels, Gαi inhibits \nadenylyl cyclase, Gαq activates phospholipase -C-β (PLCβ)  to stimulate Ca2+ signalling, and  Gα12/13 \nactivates Rho-GEFs to regulate cytoskeletal organisation 38. However, all Gα proteins signal through \nmultiple pathways. Like Gα, Gβγ proteins also regulate a variety of signalling pathways, including the \nP-Rex Rac-GEF, PLCβ and PI3Kγ pathways 39. \nThe desensitisation of activated GPCRs occurs through the agonist-induced internalisation of the \nreceptors 40,41. G protein-coupled receptor kinases (GRKs) recognise active GPCRs and phosphorylate \nserine or threonine residues in the ir cytoplasmic C -terminus. β-arrestin is recruited to the \nphosphorylated receptor, which sterically hinders the coupling between GPCR and heterotrimeric G \nprotein and recruits clathrin adaptor AP2, Arf-GTPase Arf6 and clathrin, leading to clathrin-mediated \nendocytosis of the GPCR. This usually terminates the GPCR signal, although some internalised GPCRs \nmay continue to signal intracellularly. The internalised GPCRs are then either recycled back to the cell \nmembrane following dephosphorylation, or they are transported to lysosomes for degradation.  \nThe GRK family, which comprises seven  members, serve to  phosphorylate active GPCRs for \ndesensitisation 42. Grk2, the prototype of the family, is a ubiquitously expressed kinase which  carries \nan N -terminal regulator of G proteins signalling homology (RH) domain, central catalytic kinase \ndomain and C-terminal PH domain 43,44. Binding of Gβγ to the PH domain upon GPCR activation recruits \nGrk2 to the plasma membrane, enabling the catalytic domain to phosphorylate the GPCR 42. \nHere, we investigated roles of P -Rex1 in GPCR trafficking, and show that P-Rex1 binds directly to \nGRK2 and limits the agonist-induced internalisation of GPCRs through an adaptor function. \n \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nResults \nP-Rex1 limits the agonist -induced internalisation of the GPCR S1PR1 independently of its catalytic \nRac-GEF activity \nSphingosine 1-phosphate receptor 1 (S1PR1) is a widely expressed GPCR for sphingosine 1-phosphate \n(S1P), with essential roles in vascular and neuronal development and pleiotropic other functions \nincluding immune cell migration 45. Upon S1P stimulation, S1PR1 signals through to Gα i and is then \ninternalised by clathrin-mediated endocytosis to switch off its signalling 46. To study S1PR1 trafficking, \nwe generated  HEK293 cells which stably express  S1PR1-GFP (HEK293-S1PR1 cells), similar to a \nneuronal PC12 -S1PR1 cell line we previously established 47. Widefield fluorescence microscopy \nshowed that S1PR1-GFP is localised at the plasma membrane of serum-starved cells, as judged by the \nsheet-like staining typically seen in widefield imaging, and i s internalised into vesicles upon  S1P \nstimulation in a dose- and time dependent manner, with an EC 50 of 5 nM S1P after 30 min and with \n50% internalisation after 17 min in response to 10 nM S1P (Supplemental Figure 1A-D). We assessed \nthe localisation of S1PR1-GFP by two image analysis methods, a semi-quantitative method comparing \nimages to a panel of standard images (Supplemental Figure 1A, B, E), and a quantitative method using \nVolocity image analysis 48 (Supplemental Figure 2A-C), which gave similar results.  \nTo investigate whether P -Rex1 plays a role in S1PR1  trafficking, we expressed EE-tagged P-Rex1 \ntransiently in HEK293-S1PR1 cells, stimulated the cells with 10 nM S1P, fixed, and imaged them for EE-\nP-Rex1 and for the localisation of S1PR1-GFP. EE -P-Rex1 express ion limited the S1P -stimulated \ninternalisation of  the receptor (Figure 1A) . The same results were obtained by  live-cell imaging. \nHEK293-S1PR1 cells showed the expected robust  internalisation of S1PR1 -GFP from the plasma \nmembrane into intracellular vesicles in response to S1P (Supplemental Movie 1), whereas mCherry-\nP-Rex1 reduced this receptor internalisation (Supplemental Movie 2). Therefore, P-Rex1 limits the \nagonist-induced internalisation of the GPCR S1PR1. In contrast, S1PR1 localisation was normal without \nS1P stimulation  (Figure 1A  and Supplemental Figure 2B ), suggesting that P -Rex1 inhibits agonist -\ndependent but not steady -state receptor trafficking.  Furthermore, P -Rex1 did not affect the total \nexpression level of the GPCR (Supplemental Figure 2D).  \nTo determine if suppression of GPCR trafficking depends on the catalytic Rac-GEF activity of P-Rex1, \nwe expressed GEF-dead P-Rex1, which contains point mutations E56A and N238A in the DH domain \nwhich abolish GEF-activity 22,25. As previously observed for wild type P-Rex1, S1PR1-GFP was retained \nat the plasma membrane of GEF-dead EE-P-Rex1 expressing cells stimulated with S1P (Figure 1B and \nSupplemental Figure 2C). Hence, P-Rex1-dependent control of GPCR trafficking is independent of its \ncatalytic Rac-GEF activity. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nTo test a different cell system, we used MDCK cells with doxycycline (dox)-inducible expression of \nwild type or GEF-dead P-Rex1 22,49 (Figure 1C), together with transient expression of S1PR1-GFP, and \nperformed confocal fluorescence microscopy. Without dox-induction, S1PR1-GFP was seen as a ring \nat the cell periphery, as typically seen for plasma membrane -localised proteins by confocal \nmicroscopy, and the receptor was internalised into intracellular vesicles upon S1P stimulation in a \ndose-dependent manner as expected. Expression of wild type or GEF -dead P -Rex1 inhibited this \nreceptor internalisation, as previously seen in HEK293-S1PR1 cells (Figure 1C).  \nTherefore, P-Rex1 inhibits the agonist -induced internalisation of S1PR1-GFP independently of its \ncatalytic Rac-GEF activity. This is the first Rac-GEF activity independent function of P-Rex1. \nP-Rex1 deficiency increases the agonist-induced internalisation of S1PR1  \nTo investigate whether endogenous P-Rex1 plays a role in the S1P-stimulated internalisation of S1PR1-\nGFP, we used wild type ( Prex1+/+) and two clones of P-Rex1-deficient (Prex1–/–) PC12-S1PR1 cells 47. \nThe cells were serum-starved, stimulated with S1P, and the localisation of PC12-S1PR1 was assessed \nby confocal fluorescence microscopy (Figure 2A-C and Supplemental Figure 1E). P-Rex1 deficiency \nincreased S1P-induced receptor internalisation, whereas the steady state -cell surface level of the \nreceptor was normal (Figure 2B, C). Hence, P-Rex1 expression limits and P-Rex1 deficiency promotes \nthe agonist-induced internalisation of S1PR1-GFP. \nTo assess whether P-Rex1 controls the trafficking of endogenous S1PR1, and for an alternative \nmethod of quantifying receptor internalisation, we adapted a previously described  cell fractionation \nmethod to separate endosomes from the plasma membrane in PC12 cells 50, using ultracentrifugation \nof detergent -free PC12 -S1PR1 cell lysates on an OptiPrep density -gradient followed by western \nblotting. This provided a K-Ras-enriched plasma-membrane fraction (fraction 1), EEA1-enriched early \nendosome fractions (2, 3) , and a Rab5 -enriched endosome fraction (5) (Supplemental Figure 3A-C). \nFractionation of serum-starved Prex1+/+ and Prex1–/– PC12-S1PR1 cells showed that endogenous S1PR1 \nwas mainly localised in the plasma membrane and early endosome fractions of both genotypes. Upon \nstimulation with a low concentration of S1P (5 nM), 22% of the total cellular S1PR1 translocated from \nthe plasma  membrane into the early endosome fraction in Prex1–/– PC12-S1PR1 cells, whereas \nreceptor localisation remained unchanged in Prex1+/+ PC12-S1PR1 cells (Figure 2D). This extent of \ninternalisation of endogenous S1PR1 measured by fractionation was identical t o that of S1PR1 -GFP \nassessed by imaging at that concentration of S1P. To test the specificity of S1PR1 internalisation, we \nalso determined the localisation of the receptor tyrosine kinase (RTK) EGFR. Endogenous EGFR was \nlocalised throughout plasma membrane and endosomal fractions in both Prex1+/+ and Prex1–/– PC12-\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nS1PR1 cells, and S1P stimulation did not affect its localisation (Supplemental Figure 3D). Therefore, P-\nRex1 limits the agonist-induced internalisation of endogenous S1PR1. \nLarge portions of the P-Rex1 protein are required for its control of GPCR trafficking \nTo identify which P-Rex1 domains are  required for the control of GCPR trafficking, we expressed \nvarious P-Rex1 mutants 22,51. EE-P-Rex1 ΔPH inhibited the S1P-induced internalisation of S1PR1 -GFP \nsimilar to wild type P-Rex1 (Figure 3A and Supplemental Figure 4A), suggesting that the PH domain is \ndispensable. In contrast, EE-P-Rex1 ΔPDZ, ΔDEP, and ΔIP4P mutants had no effect on the trafficking of \nS1PR1-GFP (Figure 3B -D and Supplemental Figure 4B -D). Therefore, large portions of the P -Rex1 \nprotein, including the DEP, PDZ and IP4P domains, are required for its ability to inhibit GPCR trafficking.  \nFurthermore, it was previously suggested that the isolated PD Z domains of P-Rex1 can interact with \nS1PR1 52. To investigate, we expressed myc-P-Rex1 iPDZ in HEK293-S1PR1 cells. This had no effect on \nthe S1P-stimulated trafficking of S1PR1-GFP (Supplemental Figure 5A), suggesting that isolated PDZ \ndomain tandem is not sufficient for the control of S1PR1 trafficking.  \nTo investigate whether the P-Rex1 homologue P-Rex2 plays a similar role in GPCR trafficking to P-\nRex1, we expressed myc -P-Rex2 in HEK293 -S1PR1 cells. P-Rex2 had a similar effect on S1P -induced \nS1PR1-GFP trafficking as P -Rex1 (Supplemental Figure 5B ). Therefore, both P -Rex family members \nlimit agonist-induced GPCR internalisation. \nP-Rex1 limits the agonist-induced internalisation of a range of GPCRs but not that of RTKs  \nTo investigate if other GPCRs  are affected by P -Rex1, in addition to S1PR1, we selected c andidate \nGPCRs to cover a range of classes that couple to different ty pes of heterotrimeric G protein  and \nexpressed them in MDCK cells with dox-inducible expression of wild type or GEF -dead P-Rex1. The \nGPCRs included CXCR4 which, like S1PR1, is Gα i-coupled 53, Gαs-coupled Glucagon-Like Peptide -1 \nReceptor (GLP1R) 54, and Gαq/12/13-coupled Protease-Activated Receptor 4 (PAR4) 55. Each GPCR was \nassessed by confocal fluorescence microscopy,  using stimulation with its own agonist , after pilot \nexperiments to determine appropriate agonist concentrations and timing.  \nMDCK cells expressing CXCR4-LSSmOrange were treated with dox to induce P-Rex1 expression, or \nmock-treated, serum-starved, and stimulated with 25 nM SDF1α for various periods of time . The \nlocalisation of CXCR4-LSSmOrange was more cytoplasmic than previously observed for S1PR1, and \neven partially nuclear, which has  been observed before 56, but SDF1α stimulation induced the \nexpected dose -dependent internalisation of CXCR4-LSSmOrange from the plasma membrane into \nintracellular vesicles. Both w ild type and GEF -dead P -Rex1 inhibited this  SDF1α-stimulated \ninternalisation of CXCR4-LSSmOrange (Figure 4A). Similarly, MDCK cells expressing GLP1R-mCherry \nwere stimulated for 10 min with various concentrations of Glucagon-Like Peptide-1 (GLP-1) 57. Again, \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nthe localisation of GLP1R-mCherry was more cytoplasmic than  previously observed for S1PR1, but \nGLP-1 stimulation caused the expected dose-dependent internalisation of the receptor, and both wild \ntype and GEF -dead P -Rex1 inhibited this  GLP-1 stimulated internalisation (Figure 4B ). MDCK cells \nexpressing PAR4-mCherry were stimulated with 500 µM AY-NH2 58 for various periods of time . AY-\nNH2 stimulation caused the expected internalisation of PAR4-mCherry, and again, both wild type and \nGEF-Dead P-Rex1 inhibited this internalisation (Figure 4C). Hence, P-Rex1 inhibits the agonist-induced \ninternalisation of all GPCRs tested, regardless of the type of heterotrimeric G protein the GPCRs couple \nto, and in a GEF-activity independent manner. \nTo determine if P -Rex1 also controls the trafficking of other classes of receptor, we tested the \nagonist-induced internalisation of the RTKs EGFR and PDGFRβ. MDCK cells expressing EGFR-GFP were \nserum-starved and stimulated with 100 ng/ml EGF 59 for various periods of time. EGFR-GFP was largely \nlocalised at the plasma membrane of serum -starved cells, and EGF stimulation caused the expected \ninternalisation of the receptor , but the agonist-induced internalisation of EGFR was not affected by \nwild type or GEF -dead P -Rex1 (Supplemental Figure 6A) . Similarly, PDGFRβ -GFP was also largely \nlocalised at the plasma membrane, and stimulation with 50 ng/ml PDGF 60 induced the internalisation \nof the receptor. Again, this internalisation was not affected by the expression of wild type or GEF-dead \nP-Rex1 (Supplemental Figure 6B). Hence, P-Rex1 controls the trafficking of a range of GPCRs, in a GEF-\nactivity independent manner, but  does not control the trafficking of RTKs.  Of note, we previously \nmeasured the cell surface levels of the L-selectin and of the β2 integrins LFA1 and Mac1 in Prex1–/– \nmouse neutrophils, which were also normal 7,61. Hence, P-Rex1 regulates the trafficking of GPCRs but \nnot of a range of other receptor classes.  \nP-Rex1 inhibits the phosphorylation which is required for GPCR internalisation \nThe first step in the agonist-induced internalisation of GPCRs is the phosphorylation of the GPCR at its \nC-terminal tail , mainly  by GRKs 42. To investigate the  effects of P -Rex1 on the phosphorylation of \nS1PR1, we used mass spectrometry. HEK293-S1PR1 cells expressing EE-P-Rex1 were serum -starved \nand stimulated with 10 nM S1P , or mock-stimulated, and S1PR1-GFP was immunoprecipitated from \ntotal lysates. Phosphorylated and non-phosphorylated peptides of the C-terminal tail, in particular a \npeptide encompassing S351, a residue critical for S1PR1 internalisation, were identified via LC-MS. In \ncontrol cells, S1P stimulation increased the phosphorylation of S1PR1-GFP on S351. This S1P-induced \nphosphorylation was inhibited by the expression of P-Rex1 (Figure 5A). Hence, P-Rex1 inhibits the S1P-\ndependent phosphorylation of S1PR1  on S351,  the first step in the process of clathrin -mediated \nendocytosis of the receptor.  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nTo investigate a different GPCR and test if limiting GPCR phosphorylation is a GEF -activity \ndependent role of P -Rex1, we assess the phosphorylation of endogenous  CXCR4 in dox -inducible \nMDCK cells . The agonist -induced phosphorylation of residues S324 and S 325 is required for the \ninternalisation of CXCR4 62. MDCK cells were induced with dox, or mock -treated, serum -starved, \nstimulated with 25 nM SDF1α for 10 min , or mock-stimulated, and total lysates western blotted for \nphospho-S324/S325 and total CXCR4.  In co ntrol cells, SDF1α stimulation increased the \nphosphorylation of S324/S325, whereas the expression of wild type or GEF-dead P-Rex1 reduced this \nphosphorylation (Figure 5B ). Hence, P -Rex1 blocks the phosphorylation required for the agonist -\ninduced internalisation of CXCR4, in a GEF-activity independent manner.  \nGβγ proteins, which are released from Gα upon activation of GPCRs, can bind and activate P-Rex1. \nTo investigate if Gβγ binding is involved in the effects of P-Rex1 on GPCR trafficking, we used gallein, \na small molecule inhibitor of Gβγ interactions with effectors 63 . We saw no effects of g allein on the \nS1P-dependent internalisation of S1PR1-GFP in pilot experiments, neither in the presence nor absence \nof P-Rex1 (data not shown) , suggesting that the interaction of P-Rex1 with Gβγ plays no role in its \nability to regulate GPCR trafficking. \nTo test if P -Rex1 interacts with GPCRs, HEK293-S1PR1 cells expressing myc -P-Rex1, or mock -\ntransfected, were serum-starved, stimulated with 100 nM S1P, or mock-stimulated, and total lysates \nwere subjected to immunoprecipitation with GFP or myc antibodies using various conditions of \nstringency. No interaction between P-Rex1 and S1PR1-GFP could be detected by western blotting of \nthe IP samples under any of the conditions tested (Supplemental Figure 7A, and data not shown). \nP-Rex1 interacts with Grk2 in cells and in vitro \nTo investigate if P-Rex1 interacts with Grks, which carry out the C-terminal phosphorylation of GPCRs \nupon agonist -stimulation, we selected Grk2, the best -understood and arguably most important of \nthese kinases. To test if P-Rex1 interacts with Grk2 in vivo, HEK293-S1PR1 cells expressing myc-P-Rex1 \nand/or flag-Grk2 were serum-starved, and Grk2 isolated from total lysates by immunoprecipitation \nwith flag antibody. Myc-P-Rex1 co-immunoprecipitated with flag-Grk2 under these conditions (Figure \n6A), which shows that P-Rex1 constitutively associates with Grk2 in cells. \nTo test if P -Rex1 binds directly to Grk2  in vitro, and to determine if the interaction requires \nthe GEF activity of P -Rex1, we incubated purified recombinant wild type and GEF -dead EE -P-Rex1 \nproteins with purified recombinant GST or GST -Grk2, isolated the GST -containing proteins by pull \ndown with GSH bea ds, and analysed P -Rex1 binding by western  blotting. EE-P-Rex1 bound to GST -\nGrk2 but not to GST (Figure 6B and Supplemental Figure 7B). Hence, the constitutive binding between \nP-Rex1 and Grk2 is direct. The GEF activity was not required for this direct binding, as GEF -dead P-\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nRex1 also bound to flag -Grk2 (Figure 6C). To test which domains of P -Rex1 are required for binding, \nwe used purified recombinant P -Rex1 deletion mutants 22,51. The PH and PDZ domains were \ndispensable for Grk2 binding, but the DEP domain tandem was required (Figure 6C). Hence, P-Rex1 \nbinds Grk2 directly in vitro through its DEP domains, independently of its GEF activity. \nWe showed in Supplemental Figure 5B that P-Rex2 can regulate GPCR trafficking similar to P-\nRex1. To test if P -Rex2 also binds Grk2, we generated human recombinant wild type and GEF -dead \nHis-P-Rex2 proteins, the latter by introducing alanine mutations at Glu30 and Asn212 in the catalytic \nDH domain of P -Rex2, the equivalent residues to those in GEF -dead P -Rex1 22,51, and purified the \nproteins from Sf9 cells (Supplemental Figure 7C) . For quality control, w e used an in vitro Rac-GEF \nactivity assay in the presence of the signalling lipid PIP3 to stimulate P-Rex2 activity, which confirmed \nthat the purified wild type His-P-Rex2 is an active Rac-GEF and that P-Rex2E30A,N212A is indeed GEF-dead \n(Supplemental Figure 7C). We tested the binding of these proteins to GST -Grk2 in the same way as \nfor P-Rex1. Both wild type and GEF -dead P-Rex2 bound to GST -Grk2 but not to GST (Supplemental \nFigure 7D). Hence, like P-Rex1, P-Rex2 binds Grk2 directly in vitro, independently of its GEF activity. \nFinally, to test if P-Rex1 affects Grk2 activity, we measured the kinase activity of GST-Grk2 in vitro \nwith tubulin as the substrate, in the presence and absence of EE -P-Rex1. GST-Grk2 was able to \nphosphorylate tubulin, but EE-P-Rex1 had no effect on this kinase activity (Figure 6D). Thus, while P-\nRex1 constitutively interacts with Grk2, it does not seem to control Grk2 kinase activity. \nTogether our data show that P-Rex1 limits the agonist-induced internalisation of GPCRs, but not \nother types of receptors, interacts constitutively with the kinase Grk2 which is required for GPCR \ninternalisation, and inhibits the C-terminal phosphorylation of GPCRs which is carried out by Grk2, all \nindependently of its catalytic Rac -GEF activity and without obviously affecting the kinase activity of \nGrk2. We propose that P-Rex1 inhibits GPCR trafficking likely by physically hindering the access of Grk2 \nto the GPCR. \n \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nDiscussion \nOur study shows that P-Rex1 inhibits the agonist-stimulated internalisation of GPCRs that switches-\noff GPCR signalling. This affects all GPCRs we tested, irrespective of the type of heterotrimeric G \nprotein the receptors couple to. P -Rex1 inhibits the first step of the GPCR trafficking process, Grk -\nmediated receptor phosphorylation, which implies that all subsequent steps are also abrogated, as -\narrestin cannot be recruited to non -phosphorylated GPCRs, and the rest of the clathrin -mediated \nendocytosis machinery depends on -arrestin recruitment 40,41. P-Rex1 did not affect the steady-state \ncell surface levels of GPCRs, nor total cellular GPCR levels, suggesting it plays no role in the constitutive \ntrafficking or degradation of GPCRs. P-Rex1 is also unlikely to affect GPCR recycling back to the plasma \nmembrane, as pilot experiments revealed normal localisation of the recycling endosome marker \nRab11 in HEK293 -S1PR1 cells (not shown).  Furthermore, the localisation of EGFR was unaffected in \nS1P-stimulated Prex1–/– PC12-S1PR1 cell s, and the agonist -stimulated internalisation of EGFR and \nPDGFR was normal. Therefore, P-Rex1 specifically limits the agonist-induced internalisation of GPCRs. \nP-Rex1 controls GPCR trafficking independently of its Rac-GEF activity. This is the first description \nof a GEF -activity independent role of P-Rex1, although other adaptor roles have previously been \nsuggested. P-Rex1 binding to the actin remodelling protein FLII enhances FLII interaction with Rac1 to \ncontrol cell/cell contacts, migration and contraction 49. As P-Rex1 binds FLII independently of its Rac-\nGEF activity, this was proposed to be a scaffolding role. However, the Rac-GEF activity of P-Rex1 was \nrequired for the downstream effects of FLII, so  P-Rex1 still functions as a Rac -GEF in this context. \nSimilarly, P -Rex1 deletion in mice causes a melanoblast migration defect which affects skin \npigmentation 12. Combined deletion of P-Rex1 and Rac1 brings about a more pronounced phenotype, \nwhich suggested that P-Rex1 may have a GEF-independent role in this context 64. However, use of GEF-\ndead P -Rex1 showed that melanoblast migration requires  the Rac -GEF activity 12, so P -Rex1 likely \nactivates other Rac -type GTPases in addition to Rac1  in this scenario , for example RhoG. It is \nunsurprising that a protein as large as P -Rex1 should have adaptor functions. Some other Rac -GEFs \nare also known to play important adaptor roles. For example , Vav family Rac -GEFs have adaptor \nfunctions in the NFAT-dependent transcription and integrin-mediated spreading of lymphocytes 65, \nand the Rac-GEF Tiam1 controls dendrite morphology of somatosensory neurons independently of its \nRac-GEF activity 66. \nOur study is not the first to describe a role  for P-Rex1 in vesicle trafficking processes. P-Rex1 was \nshown to be required for the insulin -stimulated upregulation of glucose transporter 4 (GLUT4) from \nsecretory vesicles to the plasma membrane in 3T3-L1 adipocytes, regulating glucose uptake. This could \nbe abolished by the expression of dominant -negative Rac1, suggesting the role of  P-Rex1 in GLUT4 \ntrafficking requires its Rac-GEF activity 67. Platelets from Prex1–/– mice have defective dense granule \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nsecretion in response to thromboxane stimulation 68, and P-Rex1 knockdown in endothelial cells \nimpairs the epinephrine-stimulated secretion of Weibel-Palade bodies 69. Further research is needed \nto elucidate the underlying mechanisms. \nWe previously identified a direct interaction of P-Rex1 with the GPCR adaptor protein Norbin 70, \nwhich is mediated through the PH domain  and occurs in vitro and in cells. Norbin binding increases \nthe catalytic activity of P-Rex1, and both proteins promote each other’s plasma membrane localisation \n71. Norbin binds the C -terminal tails of many GPCRs to regulate GPCR signalling and trafficking 70. \nNorbin also controls S1PR1 trafficking in PC12-S1PR1 cells 48, similar to P-Rex1. However, Norbin is not \njust a positive regulator of P-Rex1, and the roles of P-Rex1 and Norbin in GPCR trafficking appear not \nto be linked. We showed in mouse neutrophils that Norbin can also function independently of P-Rex1 \nor even oppose P -Rex1 functions, acting as a suppressor of neutrophil -mediated innate immunity, \nwhereas P-Rex1 is required for this immunity 8. We found here that P -Rex1 only affects the agonist -\ninduced internalisation of GPCRs, whereas Norbin largely controls steady-state GPCR trafficking 70. \nP-Rex1 inhibits the phosphorylation of the C-terminal tails of GPCR which is required for receptor \ninternalisation. P-Rex1 binds Grk2, a major kinase responsible for GPCR phosphorylation, both in cells \nand in vitro, although it does not appear to regulate Grk2 activity. P-Rex1 binding to Grk2 requires the \nDEP domains, which are central to P -Rex1 regulation and to the downstream transmission of P -Rex1 \nsignals 3,4. In addition to Grk2, these domains are also involved in P -Rex1 binding to G and mTOR, \nand they harbour S436, a residue phosphorylated by PKA to prevent P -Rex1 activation. However, \nbinding of the DEP domains to Grk2 was not sufficient for the regulation of GPCR trafficking, as the \nPDZ and IP4P domains of P-Rex1 were also required, whereas the PH domain was dispensable. Others \npreviously suggested that the isolated PDZ domain s of P-Rex1 can bind  S1PR1 52, but we show that \nthis is not sufficient to affect the trafficking of the receptor . As P-Rex1 does not affect Grk2 activity, \nwe propose that its direct interaction with Grk2 limits the agonist -induced internalisation of GPCRs \nsomehow sterically, for example by preventing access of the Grk2 to the receptor. P -Rex2, the other \nP-Rex family member, inhibits GPCR trafficking in a similar manner to P-Rex1, and also binds Grk2 in a \nGEF-activity independent manner. The effect of P-Rex2 on GPCR trafficking appeared less pronounced \nthan with P-Rex1, but this likely reflects lower expression of P-Rex2 in our transient transfections.  \nThe synergistic mode of P -Rex1 activation by PIP 3 and Gβγ make it an ideal transducer of GPCR \nsignals, and there are numerous examples. Prex1 is required for the fMLP - or C5a -stimulated \nactivation of Rac2, ROS production, actin polymerisation, chemokinesis and chemotaxis in neutrophils \n1,2, for C5a-and MCP1-stimulated Rac1 activity and chemotaxis in macrophages 72, thromboxane A2- \nand thrombin-dependent secretion of dense granules in platelets 68, and SDF1-stimulated Rac1 activity \nand chemotaxis in endothelial cells 73. Furthermore, we previously showed that P-Rex1 is required for \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nthe S1P-stimulated Rac1 and Akt activities, spreading and neurite outgrowth in PC12 -S1PR1 cells 47. \nThe fact that large parts of P -Rex1 are required to control GPCR trafficking hinders the design of \nmutants which abolish its receptor trafficking role while retaining its signalling capacity. Therefore, it \nis difficult to dissect how much P -Rex1 mediates GPCR responses through GPCR signalling compared \nto GPCR trafficking. In light of our findings, we propose that at least in part this will occur through the \ncontrol of GPCR trafficking , with  P-Rex1 preventing the Grk2 -depedent desensitisation resulting in \nretaining high levels of active GPCR at the plasma membrane and therefore prolonging  GPCR \nsignalling. Thus, P-Rex1 plays a dual role in promoting GPCR functions. \nFinally, our findings have implications for the clinical relevance of P -Rex1 in disease, in particular \ncancer. P-Rex1 promotes many types of cancer, but like most GEFs, it is difficult to target P -Rex1 \ndirectly 3,4. In comparison, GPCRs are straightforward targets. Many GPCRs, including S1PR1 and \nCXCR4, promote cancer growth and metastasis 74-78. From our findings, one would predict that cancers \nwith overexpression of P -Rex1 also show high plasma membrane levels of GPCRs, which could be \nexploited, and therapeutics for these GPCRs may already be approved in the clinic or in development. \n \nAcknowledgements: We thank Prof. Angeliki Malliri (CRUK Manchester Institute) for the gift of \ninducible MDCK cells, Prof. Timothy Hla (Harv ard University) and  Prof. Graham Ladds (University of \nCambridge) for GPCR constructs, and  Prof. Julie Pitcher (University College London)  for Grk2 \nconstructs. We thank the staff of the Babraham imaging facility for their expert help.  MB received a \nPhD studentship from UK Biotechnology and Biological Sciences Research Council (BBSRC) . EH \nreceived a BBSRC iCASE PhD studentship in collaboration with Vernalis. PI receives a PhD studentship \nfrom the Cambridge Trust. EM was awarded a Summer Vacation Studentship from the British Society \nfor Cell Biology. ET received a  BBSRC iCASE PhD studentship in collaboration with AstraZeneca.  The \nproject was funded by Institute Strategic Programme Grant BB/P013384/1 from the BBSRC to the \nBabraham Institute Signalling Programme. \nAuthor contributions: MB, EH, PI, RPM, EM, KH and ET designed, performed and analysed \nexperiments. DH, RH, AM and HW planned and supervised the project and procured funding. MB, EH \nand HW wrote the manuscript. \nDeclaration of interests: The authors declare no competing interests. \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nFigure Legends \nFigure 1. P-Rex1 limits the S1P -dependent internalisation of S1PR1  independently of its catalytic \nRac-GEF activity. (A, B) Wild type and GEF-dead P-Rex1 limit the S1P-induced internalisation of S1PR1. \nHEK293-S1PR1 cells, which express S1PR1 -GFP, were transfected with wild type (A) or GEF -dead (B) \nEE-P-Rex1 (blue symbols), or mock transfected (grey symbols), serum-starved, stimulated with 10 nM \nS1P for the indicated periods of time, fixed, and stained with EE antibody. Representative images show \ncells stimulated for 0 and 30 min, respectively. S1PR1 -GFP localisation at the plasma membrane was \nquantified by comparison to standard images (see Supplemental Figure 1). Alternative quantification \nof the same data by Volocity image analysis is shown in Supplemental Figure 2B-C. Data are mean ± \nSEM of 3 independent experiments. Statistics are two-way ANOVA with Sidak’s multiple comparisons \ncorrection; stars denote differences between genotypes for each time point. (C) Inducible expression \nof wild type or GEF-dead P-Rex1 inhibits the S1P-stimulated internalisation of S1PR1. MDCK cells with \ndoxycycline (dox)-inducible expression of wild type or GEF-dead P-Rex1 were treated with (blue) or \nwithout (grey) 1 µg/ml dox for 24 h, serum-starved, and stimulated with the indicated concentrations \nof S1P for 10 min, fixed, stained with Hoechst 33342, and imaged by confocal fluorescence \nmicroscopy. Representative confocal images are shown. Quantification was done as in (A, B). Data are \nmean ± SE M of 66 -95 cells per condition. S tatistics are two-way ANOVA  with Sidak’s multiple \ncomparisons correction; stars denote differences between genotypes for each S1P concentration. The \nwestern blot shows the dox-induced expression of wild type or GEF -dead P-Rex1 under the same \nconditions. Recombinant EE-P-Rex1 was loaded as a control. Coomassie staining was used as a control \nfor protein loading.  \nFigure 2. P-Rex1 deficiency promotes the S1P-dependent internalisation of S1PR1. (A -C) Wild type \nPC12-S1PR1 (Prex1+/+, grey symbols) and Prex1-deficient PC12-S1PR1 cells clone 1 (blue) and clone 2 \n(purple) were serum-starved, stimulated with the indicated concentrations of S1P for 10 min , fixed, \nstained with Hoechst 33342, and imaged by confocal microscopy. (A) Representative confocal images. \nS1PR1 localisation at the plasma membrane was quantified by (B) comparison to standard images (see \nSupplemental Figure 1E) or by (C) CellProfiler analysis (see Supplemental Figure 2A). Data in (B) and \n(C) are mean ± SEM of ≥3 independent experiments; the same experiments were analysed by both \nmethods. Statistics are two-way ANOVA, with Sidak’s multiple comparisons correction; s tars denote \ndifferences between genotypes for each S1P concentration . (D) Fractionation of PC12 -S1PR1 cells. \nPrex1+/+ (grey) and Prex1–/– (blue) PC12-S1PR1 cells were serum-starved and stimulated with 5 nM S1P, \nor mock -stimulated, for 10 min. Detergent-free cell l ysates were fractionated by discontinuous  \nOptiPrep density gradient (see Supplemental Figure 3), and proteins from each fraction  analysed by \nwestern blotting with S1PR1 antibody. All of  fractions 1-5 and 50% of fraction 6 were loaded. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nRepresentative western blots are shown. Coomassie staining was used to show total protein. Lower \nleft: The amount of endogenous S1PR1 per fraction was quantified by Fiji densitometry . Lower right: \nchange in S1PR1 localisation in fractions 1 and 2 upon S1P stimulation. Data are mean ± SEM from 4-\n6 independent experiments. Statistics are two -way ANOVA with Sidak’s multiple comparisons \ncorrection; black p-values denote significant differences, grey p-values are not significant. \nFigure 3. The DEP, PDZ and IP4P domains of P -Rex1 are required for the inhibition of S1PR1 \ninternalisation. (A-D) HEK293-S1PR1 cells were transfected with (A) EE -P P-Rex1 PH, (B) EE -P P-\nRex1 DEP, (C) EE-P P-Rex1 PDZ,  or EE-P-Rex1 IP4P (D) (blue symbols), or mock-transfected (grey \nsymbols), serum -starved, stimulated with 10 nM S1P for the indicated periods of time, fixed and \nstained for EE. Representative images show cells stimulated with S1P for 30 min. S1PR1 -GFP \nlocalisation at the plasma memb rane was  quantified by comparison  to standard images (see \nSupplemental Figure 1). Alternative quantification of the same data by Volocity image analysis is \nshown in Supplemental Figure 4. Data are mean ± SEM of 3 independent experiments for each mutant; \nstatistics are two-way ANOVA with Sidak’s multiple comparisons correction; stars denote differences \nbetween cells with and without P-Rex1 mutant for each time point. \nFigure 4. Expression of P -Rex1 inhibits the agonist -induced internalisation of CXCR4, GLP1R and \nPAR1, independently of its catalytic Rac-GEF activity. (A) CXCR4. CXCR4-LSSmOrange was expressed \nin MDCK cells with dox-inducible wild type (circles) or GEF-dead (triangles) P-Rex1. Cells were treated \nwith 1 µg/ml dox (blue symbols) for 24 h, or mock-treated (grey symbols), serum starved, stimulated \nwith 25 nM SDF1α for the indicated periods of time, fixed, stained with Hoechst 33342, and imaged \nby confocal fluorescence microscopy. Representative confocal images are shown. CXCR4-LSSmOrange \nlocalisation at the plasma membrane was quantified by comparison to standard images (see \nSupplemental Figure 1). (B) GLP1R. MDCK cells were treated as in (A) except that GLP1R-mCherry was \nexpressed and cells were stimulated with the indicated concentrations of GLP -1 for 10 min. GLP1R-\nmCherry localisation was quantified as in (A) . (C) PAR4. MDCK cells were treated as in (A, B) except \nthat PAR4-mCherry was expressed and cells were stimulated with 500 µM AY -NH2 for the indicated \nperiods of time. PAR4-mCherry localisation was quantified as in (A). Data in (A-C) are mean ± SEM of \nthree independent experiments for each receptor. Statistics are two-way ANOVA with Sidak’s multiple \ncomparisons correction; stars denote differences between mock and dox conditions. \nFigure 5. P-Rex1 inhibits the phosphorylation required for internalisation of S1PR1 and CXCR4. (A) \nP-Rex1 inhibits the S1P-stimulated phosphorylation of S1PR1-GFP at S351. S1PR1 -GFP was \nimmunoprecipitated from lysates of HEK293 -S1PR1 cells that had been transfected with EE-P-Rex1 \n(blue symbols), or mock transfected (grey symbols), serum starved, and stimulated with 10 nM S1P \nfor 10 min (filled bars), or mock-stimulated (open bars). Samples were analysed by LC-MS, targeted to \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nphosphopeptides in the  S1PR1 C-terminus. Data show the  ratio of phosphorylated/non -\nphosphorylated S351 and are mean ±  SEM of 3 independent experiments. Statistics are two-way \nANOVA with Sidak’s multiple comparisons correction; black p-values denote significant differences, \ngrey p-values are not significan t. (B) Wild type and GEF-dead P-Rex1 inhibit the phosphorylation of \nCXCR4 at S3254/S325. MDCK cells were treated with 1 µg/ml dox for 24 h to induce the expression of \nwild type or GEF-dead P-Rex1 (blue), or were mock-treated (grey), serum-starved, and stimulated with \n25 nM SDF1α for 10 min  (filled symbols), or mock-stimulated (open symbols). Total cell lysates were \nwestern blotted with phospho-S324/S325-CXCR4 and total CXCR4 antibodies. Representative blots are \nshown. Blots were quantified by Fiji densitometry . Data show t he ratio of phosphorylated/non -\nphosphorylated CXCR4 and are mean ± SEM of 4 independent experiments; statistics two-way ANOVA \nwith Sidak’s multiple comparisons correction. \nFigure 6 . P -Rex1 interacts with Grk2 . (A) P-Rex1 interacts with Grk2  in vivo . HEK293-S1PR1 cells \nexpressing myc-P-Rex1 and/or flag-Grk2, were serum-starved, and total lysates were subjected to \nimmunoprecipitation (IP) with flag antibody and analysed by western blotting with myc and flag  \nantibodies. 1.5% of the total lysate ( TL) and IP supernatant (sup ) were loaded alongside all the IP \nsample (red boxes). Coomassie staining was used as a loading control. Representative western blots \nare shown. Blots were qua ntified by Fiji densitometry. Data are mean ± SEM of 3 independent \nexperiments. Statistics are one-way ANOVA with Tukey’s multiple comparisons correction ; black p-\nvalues denote significant differences, grey p-values are not significant. (B, C) P-Rex1 binds directly to \nGrk2 in vitro through its DEP domains. Wild type (B) or mutant (C) P -Rex1 proteins were incubated \nwith GST or GST-Grk2, isolated using GSH-beads, and western blotted with P-Rex1 and GST antibodies. \n10% of the reaction mix (RM) and, where indicated, pull down supernatant (sup) controls and all of \nthe pull down (PD) sample were loaded. Blots are representative of 3 (B) and 2 -3 (C) independent \nexperiments per P-Rex protein. The quantification for (B) shown in Supplemental Figure 7. (D) P-Rex1 \ndoes not affect the kinase activity of Grk2. The kinase activity of GST-Grk2 was measured in vitro, with \ntubulin as the substrate, in the presence and absence of EE -P-Rex1. Data are mean ± SEM of 6 \nindependent experiments . Statistics are one -way ANOVA with Tukey’s multiple comparisons \ncorrection; black p-values denote significant differences, grey p-values are not significant. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nSTAR★Methods \nKey resources table \n \nREAGENT or RESOURCE SOURCE IDENTIFIER \nAntibodies \nCXCR4 Novus Biologicals NB100-56437 \nEE  Babraham Bioscience \nTechnologies \nEE \nEEA1  BD Biosciences 610456 \nEGFR  Abcam ab52894 \nflag   Sigma F3165 \nflag agarose-conjugated Sigma M8823 \nGFP  Sigma G6539 \nGFP  Abcam ab290 \nGrk2  \n \nCell Signaling \nTechnology \n3982S \nGST  Merck Cytiva 27-4577-01 \nK-Ras  Sigma Aldrich WH0003845M1 \nmyc  Babraham Bioscience \nTechnologies \nmyc \nphospho-S324/S325 CXCR4  ECM Biosciences CP435 \nP-Rex1  Marcus Thelen 1 IRB, Bellinzona, \nSwitzerland \nP-Rex2  Welch lab 9 78 \nRab5  Abcam ab18211 \nS1PR1  Abcam ab11424 \nAF568 goat-anti-mouse IgG Invitrogen A-11031 \nHRP  donkey anti-goat IgG Santa Cruz sc-3851 \nHRP goat anti-mouse IgG Bio-Rad 1706516 \nHRP goat anti-rabbit IgG Bio-Rad 1706515 \nBacterial and virus strains  \n   \n   \n   \nBiological samples   \nTubulin purified from pig brain  Tetubio T240 \n   \n   \nChemicals, peptides, and recombinant proteins \nAqua-Poly/Mount  Polysciences,  18606-20 \nAY-NH2  Tocris 1487 \nChromoTek agarose Proteintech bmab-20 \nDulbecco’s Modified Eagle’s Medium  Gibco 41965-039 \nEGF Sigma 11376454001 \nEscort IV  Sigma-Aldrich L3287 \nG418 disulphate  Melford G0175 \nGLP-1  Tocris 5374 \nGST  Welch lab 71 Babraham Institute \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nHoechst  33342  Thermo Fisher \nScientific \n62249 \nJetPEI  Polyplus 101-10N \nMagnetic high-capacity glutathione agarose Merck G0924 \nOptiPrep solution  StemCell Technologies 07820 \nPDGF  Invitrogen ABC125 \nPenicillin/streptomycin  Gibco 15140-122 \nProtein-A Sepharose Sigma-Aldrich P3391 \nPhosphate Buffered Saline (PBS) Invitrogen 70011-036 \nProLongGold Antifade  Life Technologies P36934 \nRecombinant human wild type and mutant EE-P-Rex1  Welch lab1,22,51 Babraham Institute \nRecombinant human GST-Grk2 Abcam ab125620 \nS1P  Sigma S9666 \nSDF1  Sigma SRP3276 \nSepharose Sigma-Aldrich 4B-200 \nX-tremeGENE 9  Roche 06366511001 \nCritical commercial assays \nADP-Glo™ kinase assay Promega V6930 \nClarity western ECL substrate  Bio-Rad 170-5060 \nGateway Bac-to-Bac system  Invitrogen 11827-011 \nSite-directed mutagenesis kit  New England Biolabs E0554 \nExperimental models: Cell lines \nHuman Embryonic Kidney 293 (HEK293) cells  Stephens/Hawkins lab Babraham Institute \nMadin-Darby Canine Kidney (MDCK) cells with inducible \nexpression of wild type or GEF-dead myc-P-Rex1  \nAngeliki Malliri 49 CRUK Manchester \nInstitute \nPC12-S1PR1 cells, wild type or knock-out for Prex1  Welch lab 47 Babraham Institute \nSf9 insect cells  Stephens/Hawkins lab Babraham Institute \nOligonucleotides \nPrimers (listed below) Merck  \n   \n   \nRecombinant DNA \npcDNA3-flag-GRK2 Julie Pitcher University College \nLondon \npcDNA3-S1PR1-GFP  Timothy Hla  Harvard University \npcDNA3.1-SNAP-GLP1R-mCherry  Graham Ladds  University of \nCambridge \npcDNA3.1-PAR4-mCherry  Graham Ladds  University of \nCambridge \npcDNA5FRT-EF-PDGFR-eGFP  Addgene 66790 \npCMV3 EE-P-Rex1 and pCMV3 myc-P-Rex1 constructs  Welch lab 1,22,51,79 Babraham Institute \npDEST10  Invitrogen 11806-015 \npEGFP-N1-EGFR-GFP Addgene 32751 \npENTR3C Thermo Fisher \nScientific \nA10464 \npLSSmOrange-N1-hCXCR4-Orange Addgene 110197 \nSoftware and algorithms \n   \n   \n   \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nOther \n13 mm glass coverslips Thermo Scientific 12392128 \n35 mm glass bottom dish  World Precision \nInstruments \nFD35-100 \n16 × 102 mm ultra-clear ultracentrifuge tubes Beckman Instruments 344661 \nImmobilon-P PVDF Millipore IPVH00010 \nLoBind tubes Eppendorf 0030108132 \nNunc T175 Easy flasks Thermo Fisher \nScientific \n159920 \n \nResource availability \nLead contact \nFurther information and requests for resources and reagents should be directed to and will be \nfulfilled by the lead contact, Heidi Welch (Heidi.welch@babraham.ac.uk). \nMaterials availability \nNewly generated materials associated with the paper are available from the lead contact. \nData and code availability \n• All data reported in this paper will be shared by the lead contact upon request \n• This paper does not report original code. \n• Any additional information required to reanalyze the data reported in this work paper is \navailable from the lead contact upon request. \n \nExperimental model and study participant details \nCell lines. Please see Key resources table and Method details sections. \n \nMethod details \nExpression vectors: Human P-Rex1 cDNA constructs with N-terminal myc or EE epitope tags in pCMV3 \nwere described previously  1,22,51,79. mCherry-P-Rex1 w as subcloned by replacing the EE -tag of \npCMV3(EE)P-Rex1 wit h mCherry using Kpn1 and EcoR1.  pCDNA3-S1PR1-GFP was a gift from Prof . \nTimothy Hla  (Harvard University) . pcDNA3.1-SNAP-GLP1R-mCherry and pcDNA3.1 -PAR4-mCherry \nwere gifts from Prof. Graham Ladds ( University of Cambridge) . pLSSmOrange -N1-hCXCR4-Orange \n(110197), pEGFP -N1-EGFR-GFP ( 32751) and pcDNA5FRT-EF-PDGFR-eGFP (66790) were from \nAddgene. cDNA3-flag-GRK2 was a gift from Prof. J ulie Pitcher (University College London ). For the \nproduction of recombinant P-Rex2 proteins in Sf9 cells, human P-Rex2 31 was subcloned into pENTR3C. \nCatalytically inactive (GEF-dead) P-Rex2E30A,N212A was generated in pENTR3C using a site-directed \nmutagenesis kit (New England Biolabs, E0554) following the manufacturer’s instructions, with primers \nCGCGTGTGCGTGCTCAGCGCGCTCCAGAAGACCGAGCGG and \nGCTGTCTGTTCCAACATAGCCGAGGCCAAGAGACAGATG to introduce the E30A and N212A mutations, \nrespectively. The wild type and GEF-dead P-Rex2 clones were recombined with pDEST10 (Invitrogen, \n11806-015) to gain an N -terminal 6His tag and generate baculovirus using the Gateway Bac -to-Bac \nsystem (Invitrogen, 11827-011). \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nWestern blotting: Proteins were transferred onto Immobilon-P PVDF (Millipore, IPVH00010) following \nSDS-PAGE. Primary antibodies were CXCR4 (Novus Biologicals, NB100 -56437, 1:250), EE (clone Glu-\nGlu, Babraham Bioscience Technologies, 1:50 ), EEA1 (BD B iosciences 610456, 1:100), EGFR (Abcam, \nab52894, 1:1000), flag (clone M2, Sigma, F3165, 1:6000), GFP (Sigma, G6539, 1:2000), Grk2 (Cell \nSignaling Technology, 3982S, 1:250), GST (Merck, Cytiva 27-4577-01, 1:1000), K-Ras (clone 3B10-2F2, \nSigma Aldrich, WH0003845M1, 1:1000), myc (clone 9E10, Babraham Bioscience Technologies, 1:50), \nphospho-S324/S325 CXCR4 (ECM Biosciences, CP435, 1:250), P-Rex1 1 (clone 6F12, from Prof. Marcus \nThelen, IRB, Bellinzona, Switzerland, 1:50), P -Rex2 9 (affinity-purified ‘78’, 1:10000), Rab5 (Abcam, \nab18211, 1:1000), and S1PR1 (Abcam, Ab11424, 1:1000). Secondary  antibodies were horseradish \nperoxidase (HRP) -coupled goat anti -rabbit ( Bio-Rad, 1706515, 1:3000 ), goat anti -mouse ( Bio-Rad, \n1706516, 1:3000) or donkey anti -goat (Santa Cruz, sc -3851, 1:3000). Clarity Western ECL Substrate \n(Bio-Rad, 170-5060) was used. Where required, membranes were stripped in 25 mM glycine (pH 2.0), \n1% SDS for 5 min at RT and reprobed. Coomassie staining (0.1% Coomassie brilliant blue R-250, 50% \nmethanol, 10% acetic acid) of gels and membranes was used to control for protein loading. X-ray films \nwere scanned, and band intensities were quantified by densitometry using Fiji (ImageJ). \nCell culture: Mammalian cell lines were used between 1 and 12 weeks in culture.  Human Embryonic \nKidney 293 (HEK293) cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, 41965-\n039) supplemented with 10% foetal bovine serum  (FBS), 1 00 U/ml penicillin and 100 g/ml \nstreptomycin (Gibco, 15140 -122) at 37°C  in a humidified incubator at 5 % CO 2. To generate HEK293 \ncells with stable expression of S1PR1 -GFP (HEK293-S1PR1 cells), HEK293 cells were transfected with \npcDNA.3-S1PR1-GFP using JetPEI, and maintained in the same medium as HEK293 cells except with  \n500 μg/ml G418 disulphate (Melford, G0175) to select for resistance, and FACS sorted to choose cells \nwith moderate GFP signal. Madin-Darby Canine Kidney (MDCK) cell s with doxycline (dox) -inducible \nexpression of myc-tagged wild type or GEF -dead P-Rex1 49 were grown in DMEM with 10% FBS, 100 \nU/ml penicillin, 100 g/ml streptomycin, 1 µg/ml puromycin, and 500 µg/ml G418. Expression of wild \ntype or GEF -dead P-Rex1 was induced by adding 1 µg/ ml dox for 24 h. PC12 (rat adrenal  gland \nphaeochromocytoma) cells with stable expression of S1PR1-GFP (PC12-S1PR1 cells) which were either \nwild type or knock -out for Prex1 47, were grown in poly -D-lysine coated flasks,  in DMEM with 10% \nhorse serum, 5% FBS, 100 U/ml penicillin, 100 g/ml streptomycin, 1× glutamine, and 500 µg/ml G418. \nTransient transfections were done  using JetPEI (Polyplus , 101-10N) or X -tremeGENE 9 (Roche , \n06366511001) following the manufacturers’ protocols. Sf9 insect cells for the expression of \nrecombinant proteins were cultured, lipofected using Escort IV t ransfection reagent (Sigma-Aldrich, \nL3287), baculovirus particles generated, amplified, and viral titres optimised for protein production as \npreviously described 51. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nGPCR localisation (imaging ): To measure S1PR1 internalisation in HEK293-S1PR1 cells, cells were  \nseeded onto 13 mm coverslips (Thermo Scientific, 12392128) in 24-well plates (Nunc) and transfected \nthe following day  with EE-tagged or myc -tagged P-Rex constructs using JetPEI, incubated for 21 h , \nwashed, serum-starved for 6.5 h in DMEM, and then stimulated with 10 nM S1P for various periods of \ntime. Cells were fixed in  4% paraformaldehyde (PFA) in 50 mM Pipes (pH 6.5), 1 mM EGTA, 10 mM \nMgCl2 for 15 min at RT, washed, permeabilised with 0.1% Triton X-100 in PBS for 10 min and washed \nagain. Samples were blocked in PBS/0.5% BSA, incubated with EE antibody (clone Glu-Glu, Babraham \nBioscience Technologies, UK, 1:10) or myc antibody (clone 9E10, Babraham Bioscience Technologies, \nUK, 1:10), washed again, and incubated with goat-anti-mouse AF568-IgG (Invitrogen, A-11031, 1:200), \nwashed in PBS, rinsed in H2O, and mounted using ProLongGold Antifade (Life Technologies, P36934). \nCells were imaged using the 60× objective of a Zeiss AxioImager D2 widefield microscope with \nAxioCam HRm camera. Duplicate coverslips were imaged for each condition and 15 images acquired \nper coverslip. Images were blinded prior to analysis. To determine  the localisation of S1PR1-GFP at \nthe plasma membrane, images were either assessed semi-quantitatively by comparison to a panel of \nstandard images, or were quantified using Volocity or CellProfiler software essentially as described 48, \nby generating a mask covering the entire cell and a second mask shrunk inwards by 0.619 µm (3 pixels), \nand calculating the GFP signal at the cell edge (mask 1 minus mask 2) as % of the total GFP signal (see \nalso Supplemental Figures 1 and 2). \nTo measure GPCR internalisation in MDCK cell s with inducible expression of P -Rex1 49, cells \nwere seeded onto 13 mm coverslips and transfected the next day using JetPEI to transiently express \nS1PR1-GFP, GLP1R-mCherry, PAR4-mCherry, or CXCR4-LSSmOrange. Alternatively, EGFR -eGFP or \nPDGFR-eGFP were expressed. T he medium was changed  24 h after transfection , and 1 µg/ml dox \nwas added 6 h later to half the samples to induce the expression of wild type or GEF-dead P-Rex1. 24 \nh after dox treatment, cells were serum-starved for 18 h in DMEM with 100 U/ml penicillin, 100 g/ml \nstreptomycin, 1 µg/ml puromycin, 500 µg/ml G418, and 0.1% FAF-BSA. The cells were stimulated with \nthe appropriate  receptor agonists, namely S1P (Sigma, S9666)  for S1PR1,  GLP-1 (Tocris, 5374) for \nGLP1R, AY-NH2 (Tocris, 1487) for PAR4, SDF1 (Sigma, SRP3276) for CXCR4, EGF (Sigma, 11376454001) \nfor EGFR, or PDGF (Invitrogen, ABC125) for PDGFβ, at various concentrations and periods of time, or \nwere mock stimulated. The medium was aspirated, and cells were fixed in 4% PFA for 15 min, washed \nin PBS, stained with Hoechst  33342 (Thermo Fisher Scientific, 62249, 1:1000), washed again, mounted \nusing ProLongGold Antifade and imaged using the 60× objective of a Nikon AR1 confocal microscope. \nReceptor localisation was quantified as described here-above. \nTo measure S1PR1 internalisation in  wild type and P -Rex1 deficient PC12 -S1PR1 cells, cells \nwere seeded onto 13 mm glass coverslips, serum-starved the next day overnight in DMEM, 0.1% FAF-\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nBSA, and then stimulated with 10 nM S1P for various periods of time, or mock stimulated, and fixed \nin 4% PFA. Samples washed in PBS, stained with Hoechst 33342, and mounted using Aqua-Poly/Mount \n(Polysciences, 18606 -20). Cells were imaged using the 60 × objective of a Nikon AR1 confocal \nmicroscope. Duplicate coverslips were assessed per condition, and 5 images acquired per coverslip. \nReceptor localisation was quantified as described here-above. \nGPCR localisation (live cell-imaging): HEK293 S1PR1-GFP cells were seeded into 35 mm glass bottom, \ndishes (World Pr ecision Instruments, FD35 -100) and transfected the following day with mCherry -P-\nRex1 using JetPEI. After 21 h, the cells were serum -starved in DMEM for 6 h . Cells were live-imaged \nusing an Olympus CellR widefield imaging system  at 37°C,  5% CO 2, acquiring frames for GFP and \nmCherry every 30 s over 45 min. At the flash, an aspirator was used to gently replace the DMEM with \nDMEM containing 100 nM S1P, keeping a constant volume of 2 ml. Movies were processed using Fiji. \nGPCR localisation (cell fractionation): Wild type and P-Rex1 deficient PC12-S1PR1 cells were seeded \ninto poly -D-lysine coated T175 flasks, serum -starved overnight in DMEM,  0.1% FAF-BSA, and then \nstimulated with 5 nM S1P for 10 min, or mock -stimulated. The medium was aspirated, flasks  were \ntransferred onto metal trays on ice, rinsed with ice-cold PBS, and cells harvested by scraping into ice-\ncold PBS. Cells were centrifuged at 800 × g for 5 min at 4˚C, resuspended in 3 ml of ice-cold detergent-\nfree homogenisation buffer (25 mM sucrose, 20 mM Tricine-NaOH, 1 mM EDTA pH 7.8, 2 mM MgCl2, \n2 mM DTT, 100 μM PMSF, and 10 μg/ml  each of leupeptin, pepstatin-A, aprotinin and antipain) and \nhomogenised in a Teflon-coated homogeniser by douncing. Samples were centrifuged at 800 × g for \n10 min at 4˚C. 20% OptiPrep solution (StemCell Technologies, 07820) in homogenisation buffer was \nadded to the supernatant to give a concentration of 10% OptiPrep . The sample was loaded onto an \nOptiPrep step-gradient in 16 × 102 mm ultra-clear ultracentrifuge tubes (Beckman Instruments, Palo \nAlto, CA, 344661). The step gradient consisted of 5 layers of OptiPrep, 2.3 ml  per layer, at 13.3%, \n16.6%, 20%, 25% and 40% OptiPrep in homogenisation buffer from top to bottom . Samples were \nultracentrifuged for 18 h at 90 ,000 × g at 4˚C in a swinging bucket SW32.1Ti rotor with \nbreak/acceleration settings on minimum. 1 ml fractions were collected from each interphase. Proteins \nwere precipitated from the fractions by addition of an equal volume of 25% trichloroacetic acid (TCA) \nand incubation for 30 min on ice. Samples were centrifuged at 13 ,225 × g for 15 min at 4˚C, and the \nsupernatant was removed. 1 ml  of ice-cold acetone was added to each sample, and samples were \ncentrifuged again. The superna tant was removed and the pellet  left to air -dry for 20 min. Samples \nwere resuspended in SDS-PAGE sample buffer, with addition of NaOH where necessary to adjust pH, \nand were analysed by western blotting.  \nMass spectrometry of S1PR1 phosphorylation: HEK293 S1PR1-GFP cells were seeded into Nunc T175 \nEasy flasks (Thermo Fisher Scientific, 159920). Half were transfected with pCMV3-EE-P-Rex1 and half \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nwith non-expressing control DNA using JetPEI. 21 h later, cells were serum-starved in DMEM for 6.5 h \nand then stimulated with 10 nM S1P for 10 min , or mock -stimulated. Flasks were transferred onto \niced metal trays, washed in PBS, and cells were scraped into 1 ml ice-cold lysis buffer 1 (50 mM Hepes \npH 7.2, 150 mM NaCl, 1% Triton X -100, 5 mM EDTA, 0.1 mM PMSF, 1 mM DTT, 20 mM β -glycerol \nphosphate, 25 mM NaF, 1 mM Na 3VO4, 10 μg/ml each of leupeptin, aprotinin, pepstatin -A and \nantipain). Lysates were centrifuged at 110,000 × g for 30 min at 4°C and the supernatant incubated \nwith 100 μl Sepharose beads (Sigma -Aldrich, 4B-200, prewashed in lysis buffer  1) for 20 min at 4°C \nwith end -over-end rotation. The beads were sedimented at 18,000 × g for 30 s at 4°C and the \nsupernatant transferred into precooled 1.5 ml Eppendorf tubes. 150 μl of supernatant taken as a total \nlysate sample. 6 μl GFP antibody (Abcam, a b290) was added to the remaining supernatant, and \nsamples were incubated for 1.5 h at 4°C with end -over-end rot ation before  60 μl of protein -A \nSepharose (Sigma-Aldrich, P3391, prewashed in lysis buffer  1) was added, and samples were \nincubated for 1 h at 4°C with end-over-end rotation. The beads were sedimented at 18,000 × g for 30 \ns at 4°C, and the supernatant was removed. 150 μl of the supernatant was kept as a post -\nimmunoprecipitation control. The beads were washed 5 times in lysis buffer 1, protein was eluted by \n3 additions of 50 μl 0.1 M glycine, pH 2.5, and the pH was neutralised using 1 M Tris (pH 7.8 at 4°C).  \nThe eluates were centrifuged at 18,000  × g for 30 s at 4°C, and the supernatant was transferred to \nfresh precooled tubes. Boiling 4× SDS-PAGE buffer was added to final 1.3×, and samples were boiled \nfor 10 min and snap-frozen in liquid nitrogen. Samples were subjected to tryptic digest, treated with \ntitanium dioxide to enrich phosphopeptides, and analysed by targeted  liquid chromatography mass \nspectrometry LC -MS. The ratio of phosphorylated to non -phosphorylated peptide s was used to \nquantify the C-terminal phosphorylation of the GPCR. \nInteraction of P -Rex1 with Grk2 in HEK293 -S1PR1 cells: HEK-293-S1PR1-GFP cells were plated into \nT175 flasks, transfected with myc -P-Rex1 and/or flag -Grk2 using  jetPEI for 72 h, and then serum -\nstarved in DMEM for 14 h. Cells were washed in PBS (Invitrogen, 70011-036), scraped, centrifuged at \n10,000 × g for 30 s at 4˚C, and resuspended in ice-cold lysis buffer 2 (50 mM Hepes, pH 7.2 at 4˚C, 150 \nmM NaCl, 1% NP -40, 1 mM EDTA, 2 mM EGTA , 1 mM DTT, 0.1 mM PMSF, and 25 μg/ml each of \nleupeptin, pepst atin-A, aprotin in, and antipain). The lysate was incubated on ice for 10 min  with \nintermittent vortexing, cleared by centrifugation at 10,000 × g for 3 min at 4°C, and the supernatant \nrecovered. For a total lysate control, boiling 4× SDS-sample buffer was added to 75 l of cleared lysate, \nsamples boiled for 5 min , and frozen in liquid nitrogen . The rest of the lysate was transferred into \nprecleared 2 ml  LoBind tubes (Eppendorf,  0030108132)  and precleared with 10 µg prewashed \nmagnetic ChromoTek agarose (Proteintech, bmab-20) for 30 min at 4 °C with end-over-end rotation. \nThe beads were removed magnetically, and the supernatant was transferred into fresh tubes a nd \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nincubated with 10 µg prewashed magnetic anti-flag agarose beads (Sigma, M8823) for 60 min at 4°C \nwith end-over-end rotation. 75 µl of the supernatant was retained as a ‘supernatant’ control and \nprocessed like the total lysate control . The beads were washed 4 times and resuspended in 30 µl \nboiling 1.3× SDS-sample buffer, boiled for 5 min, and frozen in liquid nitrogen. Samples were analysed \nby western blotting with P-Rex1 and flag antibodies. \nCo-immunoprecipitation of P-Rex1 with S1PR1-GFP was performed the same way, except that \nHEK293-S1PR1 cells were transfected with myc -P-Rex1 alone, or mock -transfected, and stimulated \nwith 100 nM S1P for 10  min, or mock -stimulated, after the serum -starvation, S1PR1 -GFP was \nimmunoprecipitated using  magnetic GFP -trap agarose, and samples were analysed by western \nblotting with myc and S1PR1 antibodies.  \nRecombinant proteins: Recombinant human wild type and mutant EE-P-Rex1 proteins, purified from \nbaculovirus-infected Sf9 cells  using their EE tag, were as previously described 1,22,51. Recombinant \nhuman wild type and GEF-dead His-P-Rex2 proteins were purified from baculovirus-infected Sf9 cells \nusing their His tag. Pellets from 400 ml Sf9 cell cultures infected with high titre baculovirus (see above) \nwere thawed  into 25 ml  ice-cold lysis buffer  3 (PBS, 1% Tr iton X -100, 25 mM NaF, 20 mM β -\nglycerophosphate, 1 mM DTT, 0.1 mM PMSF and 10 μg/ml each of antipain, pep statin A, leupeptin, \naprotinin), lysed on ice for 5 min, and ultracentrifuged at 200,000 × g for 1 h at 4°C. The supernatant \nwas incubated with  prewashed Ni-NTA agarose for 90  min at 4°C and with end-over-end rotation. \nBeads were washed 3  times in ice-cold 2× PBS, 1% Triton X -100 and 4 times in wash/elution buffer \n(PBS, 10% gly cerol, 1 mM DTT, 0.01% azide,  20 mM imidazole). For elution, wash/elution buffer \ncontaining 600 mM imidazole was added , and samples  were incubated for 10 min  on ice. Samples \nwere centrifuged at 800 × g for 1 min at 4°C and the supernatant recovered . A second elution was \nperformed with 300 mM imidazole  and pooled  with the first . To remove the imidazole, a PD -10 \ndesalting column (GE Healthcare Life Sciences, 52130800) was used according to the manufacturer’s \ninstructions. Desalted protein was concentrated using  a 100 kDa  Amicon Ultra f ilter (Merck, \nUCF210024). Once the sample volume was reduced to 100 μl, 2 ml of equilibration buffer (1 × PBS, \n10% glycerol, 1 mM DTT, 1 mM EGTA, 0.01% azide) were added, and the samples concentrated again. \nGlycerol was added to 50%, and protein for GEF activity assays was supplemented with 2 mg/ml FAF-\nBSA. To test the quality of the purified P -Rex2 proteins, Rac -GEF activity was measured using a \nliposome-based assay  as previously described  1,31,5. GST was purified from E. coli  as previously \ndescribed 71. Sf9 cell-derived recombinant human GST-Grk2 was from Abcam (Abcam, ab125620).  \nDirect binding of P-Rex proteins to Grk2: 5 pmol of P-Rex1 or P-Rex2 protein were incubated with 5 \npmol of either GST or GST-Grk2 in a volume of 20 µl in detergent-free buffer (50 mM Hepes, pH 7.2 at \n4°C, 150 mM NaCl, 1 mM EDTA, 2 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 25 μg/ml each of leupeptin, \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\npepstatin-A, aprotinin, and antipain) for 1 h on ice,  with frequent vortexing. For experiments with P-\nRex1 mutants, proteins were reduced to 2.5 pmol. A 2 µl aliquot was taken as the ‘reaction mix’ \ncontrol. Boiling 1.3x SDS-sample buffer was added, and the sample was boiled  for 5 min, and frozen \nin liquid nitrogen . The remaining reaction mix was added to 300 μl detergent -free buffer in LoBind \ntubes containing 5 μl prewashed magnetic high-capacity glutathione a garose (Merck, G0924)  and \nincubated for 45 min at 4°C with end-over-end rotation. The beads were sedimented using a magnet, \nand 75 μl  of the supernatant was retained as a ‘supernatant’ control, which was processed like the \n‘reaction mix’ control. The beads were washed four times in lysis b uffer 2, boiling 1.3 × SDS-sample \nbuffer was added, and samples were boiled  for 5 min, and frozen in liquid nitrogen . Samples were \nanalysed by western blotting using P-Rex1 or P-Rex2 and GST antibodies.  \nGrk2 kinase activity:  To measure the catalytic activity of G rk2, the ADP -Glo™ kinase assay k it \n(Promega, V6930) was used with tubulin as the substrate. 40 nM human recombinant GST-Grk2 \nand/or 40 nM EE -P-Rex1 proteins were incubated with 150  nM tubulin purified from pig brain \n(Tetubio, T240) and 400 µM ATP in k inase buffer (40 mM Tris, pH 7.5 (RT),  20 mM MgCl2, and 0.1% \nBSA), in a volume of 25 µl for 30 min at 30°C . Controls included samples without protein  and with \nkinase detection reagent only. To control for potential effects of the storage buffers of EE-P-Rex1 (PBS, \n1 mM EGTA, 1 mM DTT, 50% glycerol, 0.01% sodium azide) and GST-Grk2 (0.79% Tris HCl, 0.88% NaCl, \n0.31% glutathione, 0.002% PMSF, 0.004% DTT, 0.003% EDTA, 25% glycerol), the buffers were added \nto samples without protein at the equivalent dilution. After the incubation, 25 µl ADP-Glo reagent was \nadded for 40 min at RT to deplete any remaining ATP. 50 µl of kinase detection reagent was added for \na further 40 min, and luminescence was measured in a PHERAstar FS luminometer (BMG Labtech). \nQuantification and statistical analysis \nData were tested for normality of distribution to determine if parametric or non-parametric methods \nof analysis were appropriate. For comparison of two groups, unpaired Student’s t -test was used, \nwhereas for comparison of multiple groups, one -way or two-way ANOVA was used, as appropriate, \nwith repeated measures followed by post-hoc test with multiple comparisons correction. Parameters \nwith values of p ≤ 0.05 were considered to differ significantly. In the figures, * indicates p < 0.05, ** p \n< 0.01, *** p < 0.001, and **** p < 0.0001. Results are presented as mean ± standard error of the \nmean (SEM). The number of experimental repeats is indicated in the figure legends. 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It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nGraphical Abstract\n10’\n0’\nGrk2\nβ-arr\nα\n βγ\n α\nβγ\nβ-arr\nP P\nP\ndesensitisationsignalling\n0’\ndesensitisation\nendocytosis\nrecycling\ndegradation\n GPCR\nGrk2\nα\n βγ\nsignalling\nP-Rex1\nα\nβγ\nP-Rex1\n α\nβγ\nsignalling\nP-Rex1\nwithout P-Rex1\nwith P-Rex1\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nFigure 1\nmock\nP-Rex1\nC\n - +dox wild type P-Rex1\n300 nM S1P mock\nHoechst S1PR1-GFP\n10 µm\n- +dox GEF-dead P-Rex1\n300 nM S1P mock\n10 µm\nRecomb.\nEE-P-Rex1\ndox\nCoom.\n225\n150\n102\n225\n150\n102\nwild type\nP-Rex1\n- +\nGEF- dead\nP-Rex1\n- +\nP-Rex1\nwild type P-Rex1\n100\n60\n20\n80\n40\nPlasma membrane\nS1PR1-GFP (%)\n[S1P] (nM)\n0 100 3005 50\n \n    \n  ** **** ****\nGEF-dead P-Rex1\n \n    \n  \n     \n  \n*** **** *******100\n60\n20\n80\n40\nPlasma membrane\nS1PR1-GFP (%)\n[S1P] (nM)\n0 100 3005 50\nA\n \n    \nP-Rex1\n100\n50\n0\n75\n25\nPlasma membrane\nS1PR1-GFP (%)\n0 5030 4010 20\n* ** ***\n***\n20 µm\nEE-P-Rex1S1PR1-GFP\nDH PH DEP PDZ IP4P\nTime (min)\n0 min30 min\nB\n \n    \n \nGEF-dead P-Rex1\n100\n50\n0\n75\n25\nPlasma membrane\nS1PR1-GFP (%)\nTime (min)\n0 5030 4010 20\n**** **** ****\n****\n20 µm\nGEF-dead EE-P-Rex1S1PR1-GFP\n0 min30 min\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nA\nS1PR1-GFP Hoechst\n20 µm\nPrex1+/+\n5 nM S1P mock\nPrex1–/– clone 1 Prex1–/– clone 2\nPrex1+/+\nPrex1–/– clone 1\nPrex1–/– clone 2\nB C\n \n      \n \n80\n40\n0\n60\n20\nPlasma membrane\nS1PR1-GFP (%)\n[S1P] (nM)\n0 50 3005 20 100\n* * ****\n* * ****\n \n    \n \n \n100\n50\n0\n75\n25\nPlasma membrane\nS1PR1-GFP (%)\n[S1P] (nM)\n0 50 3005 20 100\n** ***\n**** ******\nD\n76\n52\n3831\nPrex1+/+ mock Prex1+/+ S1P Prex1–/– mock Prex1–/– S1P\n76\n52\n3831\n1 2 3 4 5 61 2 3 4 5 61 2 3 4 5 6fraction 1 2 3 4 5 6\nS1PR1\ncoom.\n      \n    \n \n  \n100\n50\n0\n75\n25\nS1PR1 (% of total)\nFraction 1 42 3 1 42 3 1 42 3 1 42 3\nPrex1+/+ mock Prex1+/+ S1P Prex1–/– mock Prex1–/– S1P\n    \n  \n \nChange in S1PR1 localisation\nupon S1P stimulation (%)\n60\n0\n-60\n40\n-20\n-40\n20\nPrex1+/+ Prex1–/–\n1 2 1 2fraction\n0.0318\n0.0214\n0.0254 <0.00010.0225\n0.0182\n0.9793\n0.9994\n1\n2, 3\n4, 5\n6\nplasma membrane\nearly endosomes\nendosomes\nother membranes\nFigure 2\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\n \n    \n \n \n    \n \n \n    \n \n \n    \n \nDH PH DEP PDZ\nDH PH DEP IP4P\nV\nA P-Rex1 ∆PH\n100\n50\n0\n75\n25\nPlasma membrane\nS1PR1-GFP (%)\nTime (min)\n0 5030 4010 20\n** **** ****\n****\n20 µm\nEE-P-Rex1 ∆-PHS1PR1-GFP\nB P-Rex1 1 ∆DEP\n100\n50\n0\n75\n25\nPlasma membrane\nS1PR1-GFP (%)\nTime (min)\n0 5030 4010 20\n*\n**\n20 µm\nEE-P-Rex1 ∆-DEPS1PR1-GFP\nC P-Rex1 ∆PDZ\n100\n50\n0\n75\n25\nPlasma membrane\nS1PR1-GFP (%)\nTime (min)\n0 5030 4010 20\n*\n20 µm\nEE-P-Rex1 ∆-PDZS1PR1-GFP\nD P-Rex1 ∆IP4P\n100\n50\n0\n75\n25\nPlasma membrane\nS1PR1-GFP (%)\nTime (min)\n0 5030 4010 20\n20 µm\nEE-P-Rex1 ∆-IP4PS1PR1-GFP\nDH DEP PDZ IP4P\nV\nDH PH PDZ IP4P\nV\nFigure 3\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nC\n   \n \n   \n80\n40\n0\n60\n20\nPlasma membrane\nPAR4-mCherry (%)\nTime (min)\n0 6030 4515\n**** **** *** **\n*** **** **** **\nPAR4\nwild type\n0 min AY-NH2\nwild type\n30 min AY-NH2\nGEF-dead\n30 min AY-NH2\nmockdoxycycline\nPAR4-mCherry Hoechst\n10 µm\n[GLP-1] µM\n40\n20\n0\n30\n10\nPlasma membrane\nGLP1R-mCherry (%)\n0 10.03 0.3O.01 0.1\n     \n \n    \n \n   ** **** ******\n**** **** ********\nB GLP1R\nwild type\n0 µM GLP-1\nwild type\n0.1 µM GLP-1\nGEF-dead\n0.1 µM GLP-1\nmockdoxyclycline\n10 µm\nGLP1R-mCherry Hoechst\n     \n  \n    \n \n   \n \n   \n60\n30\n0\n45\n15\nPlasma membrane\nCXCR4-mOrange (%)\nTime (min)\n0 2010 155\n** **** ******* **** *\nA CXCR4\nwild type\n0 min SDF1α\nwild type\n10 min  SDF1α\nGEF-dead\n10 min SDF1α\nmockdoxycycline\nCXCR4-mOrange Hoechst\n10 µm\nmock wild type\ndox wild type\nmock GEF-dead\ndox GEF-dead\nFigure 4\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nFigure 5\n  \n      \n3.2\n1.6\n0\n2.4\n0.8\nRatio\nphospho-S1PR1/S1PR1\nS1PS1P mockmock\nA S1PR1\n- P-Rex1\n+ P-Rex1\n0.0143\n0.8909\n0.0054 0.0303\n    \n    \n \n \n \n \nB CXCR4\n1.6\n0.8\n0\n1.2\n0.4\nRatio\nphospho-CXCR4/CXCR4\nGEF-dead P-Rex1wild type P-Rex1\nSDF1α - - ++ - - ++\n- dox\n+ dox\n<0.0001 <0.0001\n52\n38\n76\n52\n38\n76\nCXCR4\nphospho-\nCXCR4\nGEF-dead P-Rex1wild type P-Rex1\ndox\nSDF1α\n- + +-\n- - ++\n- + +-\n- - ++\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint \n\nA co-IP P-Rex1 + Grk2\nmyc-P-Rex1 \n(longer exposure)\ncoomassie\nflag-Grk2 \nmyc-P-Rex1 \n102\n76\n225\n150\n102\n76\n225\n150\n225\n150\nflag-Grk2 myc-P-Rex1\nflag-Grk2\nmyc-P-Rex1\nα-flag-IP:\n \n   \n  \n8\n4\n0\n6\n2\nIP signal (densitometry units x 103)\n0.9636\n0.0370 0.0300\nB recombinant P-Rex1 + Grk2\nGST-Grk2 \nEE-P-Rex1 \nGST \nRM PD RM PD\nGST-Grk2\nEE-P-Rex1\nGST\nEE-P-Rex1\nGSH PD:\n225\n150\n102\n76\n150\n102\n52\n38\n31\n24\n17\nC recombinant P-Rex1 mutants + Grk2\nRM PDsup RM PDsup\nGST-Grk2\nP-Rex1 mutant\nGST\nP-Rex1 mutant\nGSH PD:\nGEF-dead \n∆PDZ \n∆DEP\n∆PH\n225\n150\n225\n150\n102\n225\n150\n102\n225\n150\n102\nD Grk2 kinase activity\n \n  \n \n  \n \n60\n30\n0\n45\n15\nPhosphorylation of tubulin\n(luminometer units x 103)\n0.0582\n0.9244 0.0313\nFigure 6\nDH PH DEP PDZ IP4P\nV\nV\nV\nGEF-dead \n∆PDZ \n∆DEP\n∆PH\nwild type\nRH kinase PH\nGrk2\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.14.648762doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}