{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/392494"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/392494","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Identification of the Role of P-Rex1 and its Adaptor Functions in Neutrophils","abstract":"P-Rex1 is a guanine-nucleotide exchange factor (Rac-GEF) for the small GTPase Rac, recognised for its involvement in neutrophil migration and ROS generation in response to GPCR activation—functions traditionally attributed to its Rac-GEF activity. Additionally, previous findings from our laboratory demonstrated a GEF-independent role for P-Rex1 in GPCR trafficking. This thesis investigates Rac-GEF-independent functions of P-Rex1 in neutrophil biology, as well as the molecular mechanisms by which it regulates GPCR trafficking. To dissect the necessity of P-Rex1 and of its catalytic function, we employed both P-Rex1 knockout (Prex1–/–) and catalytically inactive (Prex1GD) mouse models to examine neutrophil responses, including migration, ROS production, degranulation, phagocytosis, neutrophil extracellular trap formation, and bacterial clearance. Our results show that the Rac-GEF activity of P-Rex1 is essential for driving neutrophil migration, ROS production, and NET formation. In contrast, degranulation of azurophilic, specific, and gelatinase granules occurs independently of P-Rex1. Surprisingly, we discovered that P-Rex1 contributes to bacterial clearance in vivo during septic peritonitis, as well as to bacterial killing and phagocytosis by isolated neutrophils, through mechanisms that do not rely on its catalytic function. Specifically, P-Rex1 promotes the phagocytosis of IgG-opsonised zymosan particles and supports both integrin- and Fc receptor-dependent uptake independently of its Rac-GEF activity. Furthermore, P-Rex1 facilitates the Fc receptor-mediated activation of Rac and Syk. To investigate the underlying mechanism by which P-Rex1 mediates GPCR trafficking, we found that P-Rex1 does not associate with the GPCR S1PR1 in HEK293-S1PR1 cells to exert this effect. Instead, P-Rex1 binds GRK2, both in vitro and in cells, but does not appear to regulate GRK2 activity. Moreover, P-Rex1 binds GRK2 directly in vitro through its DEP domains, independently of its GEF activity, and the catalytic domain of GRK2 is not essential for this interaction. P-Rex2, a close homologue of P-Rex1, similarly binds GRK2 in a Rac-GEF-independent manner. These data suggest that P-Rex1 may regulate GPCR internalisation by sequestering GRK2, thereby maintaining receptor presence at the plasma membrane and enhancing GPCR signalling. In summary, this work identifies P-Rex1 as a key modulator of neutrophil effector functions such as NET formation and phagocytosis, with some distinct roles beyond its catalytic activity. It also defines a novel adaptor-like mechanism by which P-Rex1 regulates GPCR trafficking, reinforcing its dual role in modulating GPCR responses both through Rac activation and direct protein–protein interactions.","abstract_html":"P-Rex1 is a guanine-nucleotide exchange factor (Rac-GEF) for the small GTPase Rac, recognised for its involvement in neutrophil migration and ROS generation in response to GPCR activation—functions traditionally attributed to its Rac-GEF activity. Additionally, previous findings from our laboratory demonstrated a GEF-independent role for P-Rex1 in GPCR trafficking. This thesis investigates Rac-GEF-independent functions of P-Rex1 in neutrophil biology, as well as the molecular mechanisms by which it regulates GPCR trafficking. To dissect the necessity of P-Rex1 and of its catalytic function, we employed both P-Rex1 knockout (Prex1–/–) and catalytically inactive (Prex1GD) mouse models to examine neutrophil responses, including migration, ROS production, degranulation, phagocytosis, neutrophil extracellular trap formation, and bacterial clearance. Our results show that the Rac-GEF activity of P-Rex1 is essential for driving neutrophil migration, ROS production, and NET formation. In contrast, degranulation of azurophilic, specific, and gelatinase granules occurs independently of P-Rex1. Surprisingly, we discovered that P-Rex1 contributes to bacterial clearance in vivo during septic peritonitis, as well as to bacterial killing and phagocytosis by isolated neutrophils, through mechanisms that do not rely on its catalytic function. Specifically, P-Rex1 promotes the phagocytosis of IgG-opsonised zymosan particles and supports both integrin- and Fc receptor-dependent uptake independently of its Rac-GEF activity. Furthermore, P-Rex1 facilitates the Fc receptor-mediated activation of Rac and Syk. To investigate the underlying mechanism by which P-Rex1 mediates GPCR trafficking, we found that P-Rex1 does not associate with the GPCR S1PR1 in HEK293-S1PR1 cells to exert this effect. Instead, P-Rex1 binds GRK2, both in vitro and in cells, but does not appear to regulate GRK2 activity. Moreover, P-Rex1 binds GRK2 directly in vitro through its DEP domains, independently of its GEF activity, and the catalytic domain of GRK2 is not essential for this interaction. P-Rex2, a close homologue of P-Rex1, similarly binds GRK2 in a Rac-GEF-independent manner. These data suggest that P-Rex1 may regulate GPCR internalisation by sequestering GRK2, thereby maintaining receptor presence at the plasma membrane and enhancing GPCR signalling. In summary, this work identifies P-Rex1 as a key modulator of neutrophil effector functions such as NET formation and phagocytosis, with some distinct roles beyond its catalytic activity. It also defines a novel adaptor-like mechanism by which P-Rex1 regulates GPCR trafficking, reinforcing its dual role in modulating GPCR responses both through Rac activation and direct protein–protein interactions.","abstract_has_math":false,"creators":["Islam, Priota"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Christophorou, Maria A"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-08-04","date_published":"2025-08-04","updated_at":"2026-07-22T22:23:56Z","subjects":["Fc receptors","NETs","PREX1","ROS","Rac","Syk","neutrophils","phagocytosis"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/b33fb464-5f23-4cfa-95e5-e035e87a66d2/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.123192","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Christophorou, Maria A"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Jameel Education Foundation and Cambridge Trust."]},{"key":"dc:creator","label":"Author","values":["Islam, Priota"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2025-08-04"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/392494"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Fc receptors","NETs","PREX1","ROS","Rac","Syk","neutrophils","phagocytosis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/b33fb464-5f23-4cfa-95e5-e035e87a66d2/download","http://purl.org/NET/rdflicense/allrightsreserved"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.123192"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/8e0266d6-9710-40e9-8f9a-9e1b41038fa3/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["P-Rex1 is a guanine-nucleotide exchange factor (Rac-GEF) for the small GTPase Rac, recognised for its involvement in neutrophil migration and ROS generation in response to GPCR activation—functions traditionally attributed to its Rac-GEF activity. Additionally, previous findings from our laboratory demonstrated a GEF-independent role for P-Rex1 in GPCR trafficking. This thesis investigates Rac-GEF-independent functions of P-Rex1 in neutrophil biology, as well as the molecular mechanisms by which it regulates GPCR trafficking. To dissect the necessity of P-Rex1 and of its catalytic function, we employed both P-Rex1 knockout (Prex1–/–) and catalytically inactive (Prex1GD) mouse models to examine neutrophil responses, including migration, ROS production, degranulation, phagocytosis, neutrophil extracellular trap formation, and bacterial clearance. Our results show that the Rac-GEF activity of P-Rex1 is essential for driving neutrophil migration, ROS production, and NET formation. In contrast, degranulation of azurophilic, specific, and gelatinase granules occurs independently of P-Rex1. Surprisingly, we discovered that P-Rex1 contributes to bacterial clearance in vivo during septic peritonitis, as well as to bacterial killing and phagocytosis by isolated neutrophils, through mechanisms that do not rely on its catalytic function. Specifically, P-Rex1 promotes the phagocytosis of IgG-opsonised zymosan particles and supports both integrin- and Fc receptor-dependent uptake independently of its Rac-GEF activity. Furthermore, P-Rex1 facilitates the Fc receptor-mediated activation of Rac and Syk. To investigate the underlying mechanism by which P-Rex1 mediates GPCR trafficking, we found that P-Rex1 does not associate with the GPCR S1PR1 in HEK293-S1PR1 cells to exert this effect. Instead, P-Rex1 binds GRK2, both in vitro and in cells, but does not appear to regulate GRK2 activity. Moreover, P-Rex1 binds GRK2 directly in vitro through its DEP domains, independently of its GEF activity, and the catalytic domain of GRK2 is not essential for this interaction. P-Rex2, a close homologue of P-Rex1, similarly binds GRK2 in a Rac-GEF-independent manner. These data suggest that P-Rex1 may regulate GPCR internalisation by sequestering GRK2, thereby maintaining receptor presence at the plasma membrane and enhancing GPCR signalling. In summary, this work identifies P-Rex1 as a key modulator of neutrophil effector functions such as NET formation and phagocytosis, with some distinct roles beyond its catalytic activity. 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Surprisingly, we discovered that P-Rex1 contributes to bacterial clearance in vivo during septic peritonitis, as well as to bacterial killing and phagocytosis by isolated neutrophils, through mechanisms that do not rely on its catalytic function. Specifically, P-Rex1 promotes the phagocytosis of IgG-opsonised zymosan particles and supports both integrin- and Fc receptor-dependent uptake independently of its Rac-GEF activity. Furthermore, P-Rex1 facilitates the Fc receptor-mediated activation of Rac and Syk. To investigate the underlying mechanism by which P-Rex1 mediates GPCR trafficking, we found that P-Rex1 does not associate with the GPCR S1PR1 in HEK293-S1PR1 cells to exert this effect. Instead, P-Rex1 binds GRK2, both in vitro and in cells, but does not appear to regulate GRK2 activity. Moreover, P-Rex1 binds GRK2 directly in vitro through its DEP domains, independently of its GEF activity, and the catalytic domain of GRK2 is not essential for this interaction. P-Rex2, a close homologue of P-Rex1, similarly binds GRK2 in a Rac-GEF-independent manner. These data suggest that P-Rex1 may regulate GPCR internalisation by sequestering GRK2, thereby maintaining receptor presence at the plasma membrane and enhancing GPCR signalling. In summary, this work identifies P-Rex1 as a key modulator of neutrophil effector functions such as NET formation and phagocytosis, with some distinct roles beyond its catalytic activity. 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