{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/362679"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/362679","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Investigating the immunosuppressive functions of the GPCR adapter protein Norbin","abstract":"This project investigated the roles of Norbin, an adaptor protein which regulates trafficking and signalling of G protein-coupled receptors (GPCRs). Norbin was originally expected to be exclusively neuronal. However, previous PhD students in our laboratory found that Norbin is also expressed in myeloid cells and made mice with myeloid Norbin deficiency (NcdnΔmye) to investigate its role in these cells. They showed that NcdnΔmye mice have increased immunity during pulmonary *S. pneumoniae* infection, and that neutrophils from these mice kill bacteria better, in a reactive oxygen species (ROS)-dependent manner, and produce more ROS in response to a wide range of stimuli.
I followed up on their work using immune cell depletion to demonstrate that the elevated immunity of NcdnΔmye mice is neutrophil-derived rather than depending on macrophages. I showed that neutrophils arrive more rapidly in the airways of NcdnΔmye mice during infection with *S. pneumoniae*, and they degranulate in the lungs to a greater extent, although the systemic levels of inflammatory cytokines are normal. I also found ten-fold elevated immunity of NcdnΔmye mice during septic peritonitis. I showed that NcdnΔmye neutrophils degranulate more and produce more neutrophils extracellular traps (NETs) than control neutrophils, and their elevated killing of *S. aureus* is dependent on NETs, whereas their production of inflammatory cytokines is normal. I showed that Norbin controls the Rac-GEFs Vav, which is constitutively active in NcdnΔmye neutrophils, and Tiam1 which is upregulated to cell surface upon C5a-stimulation. I also showed that the elevated ROS production in these cells can be reversed by titrating inhibitors of Rac and Erk.
Arguably my most important contribution was to investigate the role of Norbin in neutrophil GPCR trafficking. Norbin is known to interact directly with numerous GPCRs, influencing their trafficking and signalling. However, apart from Norbin needing to interact directly with a GPCR, little is known about the mechanisms. I identified novel direct interactions between Norbin and the C-terminal tails of the GPCRs CXCR4 and C5aR1. I showed that the cell surface levels of CXCR4 and C5aR1 are elevated in NcdnΔmye neutrophils whereas the total cellular levels of these receptors are normal. I used various pulse-chase experiments to show that constitutive trafficking of these GPCRs is unaffected, but their agonist-induced internalisation is altered in NcdnΔmye neutrophils, and their recycling back to the plasma membrane faster. Altered binding kinetics of β-arrestin with stimulated C5aR1 may underlie the mechanisms behind these trafficking differences, and the resulting elevated GPCR cell surface levels can explain some of the increased responsiveness of NcdnΔmye neutrophils.","abstract_html":"This project investigated the roles of Norbin, an adaptor protein which regulates trafficking and signalling of G protein-coupled receptors (GPCRs). Norbin was originally expected to be exclusively neuronal. However, previous PhD students in our laboratory found that Norbin is also expressed in myeloid cells and made mice with myeloid Norbin deficiency (Ncdn<sup>Δmye</sup>) to investigate its role in these cells. They showed that Ncdn<sup>Δmye</sup> mice have increased immunity during pulmonary *S. pneumoniae* infection, and that neutrophils from these mice kill bacteria better, in a reactive oxygen species (ROS)-dependent manner, and produce more ROS in response to a wide range of stimuli.<br> I followed up on their work using immune cell depletion to demonstrate that the elevated immunity of Ncdn<sup>Δmye</sup> mice is neutrophil-derived rather than depending on macrophages. I showed that neutrophils arrive more rapidly in the airways of Ncdn<sup>Δmye</sup> mice during infection with *S. pneumoniae*, and they degranulate in the lungs to a greater extent, although the systemic levels of inflammatory cytokines are normal. I also found ten-fold elevated immunity of Ncdn<sup>Δmye</sup> mice during septic peritonitis. I showed that Ncdn<sup>Δmye</sup> neutrophils degranulate more and produce more neutrophils extracellular traps (NETs) than control neutrophils, and their elevated killing of *S. aureus* is dependent on NETs, whereas their production of inflammatory cytokines is normal. I showed that Norbin controls the Rac-GEFs Vav, which is constitutively active in Ncdn<sup>Δmye</sup> neutrophils, and Tiam1 which is upregulated to cell surface upon C5a-stimulation. I also showed that the elevated ROS production in these cells can be reversed by titrating inhibitors of Rac and Erk.<br> Arguably my most important contribution was to investigate the role of Norbin in neutrophil GPCR trafficking. Norbin is known to interact directly with numerous GPCRs, influencing their trafficking and signalling. However, apart from Norbin needing to interact directly with a GPCR, little is known about the mechanisms. I identified novel direct interactions between Norbin and the C-terminal tails of the GPCRs CXCR4 and C5aR1. I showed that the cell surface levels of CXCR4 and C5aR1 are elevated in Ncdn<sup>Δmye</sup> neutrophils whereas the total cellular levels of these receptors are normal. I used various pulse-chase experiments to show that constitutive trafficking of these GPCRs is unaffected, but their agonist-induced internalisation is altered in NcdnΔmye neutrophils, and their recycling back to the plasma membrane faster. Altered binding kinetics of β-arrestin with stimulated C5aR1 may underlie the mechanisms behind these trafficking differences, and the resulting elevated GPCR cell surface levels can explain some of the increased responsiveness of Ncdn<sup>Δmye</sup> neutrophils.","abstract_has_math":false,"creators":["Chetwynd, Stephen"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Welch, Heidi"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-12-20","date_published":"2023-12-20","updated_at":"2026-07-22T22:23:54Z","subjects":["GPCR","NCDN","Neurochondrin","Norbin","trafficking"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/c4fa0a73-3e81-4ba4-ad31-b18daa5492ce/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.104740","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Welch, Heidi"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["MRC"]},{"key":"dc:creator","label":"Author","values":["Chetwynd, Stephen"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-12-20"]},{"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/1810/362679"]},{"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":["GPCR","NCDN","Neurochondrin","Norbin","trafficking"]}]},{"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/c4fa0a73-3e81-4ba4-ad31-b18daa5492ce/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.104740"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/97c98035-d8b0-4a4d-8c3d-2fcdc155746d/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["This project investigated the roles of Norbin, an adaptor protein which regulates trafficking and signalling of G protein-coupled receptors (GPCRs). Norbin was originally expected to be exclusively neuronal. However, previous PhD students in our laboratory found that Norbin is also expressed in myeloid cells and made mice with myeloid Norbin deficiency (NcdnΔmye) to investigate its role in these cells. They showed that NcdnΔmye mice have increased immunity during pulmonary *S. pneumoniae* infection, and that neutrophils from these mice kill bacteria better, in a reactive oxygen species (ROS)-dependent manner, and produce more ROS in response to a wide range of stimuli.
I followed up on their work using immune cell depletion to demonstrate that the elevated immunity of NcdnΔmye mice is neutrophil-derived rather than depending on macrophages. I showed that neutrophils arrive more rapidly in the airways of NcdnΔmye mice during infection with *S. pneumoniae*, and they degranulate in the lungs to a greater extent, although the systemic levels of inflammatory cytokines are normal. I also found ten-fold elevated immunity of NcdnΔmye mice during septic peritonitis. I showed that NcdnΔmye neutrophils degranulate more and produce more neutrophils extracellular traps (NETs) than control neutrophils, and their elevated killing of *S. aureus* is dependent on NETs, whereas their production of inflammatory cytokines is normal. I showed that Norbin controls the Rac-GEFs Vav, which is constitutively active in NcdnΔmye neutrophils, and Tiam1 which is upregulated to cell surface upon C5a-stimulation. I also showed that the elevated ROS production in these cells can be reversed by titrating inhibitors of Rac and Erk.
Arguably my most important contribution was to investigate the role of Norbin in neutrophil GPCR trafficking. Norbin is known to interact directly with numerous GPCRs, influencing their trafficking and signalling. However, apart from Norbin needing to interact directly with a GPCR, little is known about the mechanisms. I identified novel direct interactions between Norbin and the C-terminal tails of the GPCRs CXCR4 and C5aR1. I showed that the cell surface levels of CXCR4 and C5aR1 are elevated in NcdnΔmye neutrophils whereas the total cellular levels of these receptors are normal. I used various pulse-chase experiments to show that constitutive trafficking of these GPCRs is unaffected, but their agonist-induced internalisation is altered in NcdnΔmye neutrophils, and their recycling back to the plasma membrane faster. Altered binding kinetics of β-arrestin with stimulated C5aR1 may underlie the mechanisms behind these trafficking differences, and the resulting elevated GPCR cell surface levels can explain some of the increased responsiveness of NcdnΔmye neutrophils."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["87eda9de84448d1f82354d60eee3eb5f","9a2a9af0f96921b1bf1b672bdb18fab5"]},{"key":"dc:title","label":"Title","values":["Investigating the immunosuppressive functions of the GPCR adapter protein Norbin"]}]}],"canonical_facts":{"dc:contributor.advisor":["Welch, Heidi"],"dc:contributor.sponsor":["MRC"],"dc:creator":["Chetwynd, Stephen"],"dc:date.issued":["2023-12-20"],"dc:description.abstract":["This project investigated the roles of Norbin, an adaptor protein which regulates trafficking and signalling of G protein-coupled receptors (GPCRs). Norbin was originally expected to be exclusively neuronal. However, previous PhD students in our laboratory found that Norbin is also expressed in myeloid cells and made mice with myeloid Norbin deficiency (NcdnΔmye) to investigate its role in these cells. They showed that NcdnΔmye mice have increased immunity during pulmonary *S. pneumoniae* infection, and that neutrophils from these mice kill bacteria better, in a reactive oxygen species (ROS)-dependent manner, and produce more ROS in response to a wide range of stimuli.
I followed up on their work using immune cell depletion to demonstrate that the elevated immunity of NcdnΔmye mice is neutrophil-derived rather than depending on macrophages. I showed that neutrophils arrive more rapidly in the airways of NcdnΔmye mice during infection with *S. pneumoniae*, and they degranulate in the lungs to a greater extent, although the systemic levels of inflammatory cytokines are normal. I also found ten-fold elevated immunity of NcdnΔmye mice during septic peritonitis. I showed that NcdnΔmye neutrophils degranulate more and produce more neutrophils extracellular traps (NETs) than control neutrophils, and their elevated killing of *S. aureus* is dependent on NETs, whereas their production of inflammatory cytokines is normal. I showed that Norbin controls the Rac-GEFs Vav, which is constitutively active in NcdnΔmye neutrophils, and Tiam1 which is upregulated to cell surface upon C5a-stimulation. I also showed that the elevated ROS production in these cells can be reversed by titrating inhibitors of Rac and Erk.
Arguably my most important contribution was to investigate the role of Norbin in neutrophil GPCR trafficking. Norbin is known to interact directly with numerous GPCRs, influencing their trafficking and signalling. However, apart from Norbin needing to interact directly with a GPCR, little is known about the mechanisms. I identified novel direct interactions between Norbin and the C-terminal tails of the GPCRs CXCR4 and C5aR1. I showed that the cell surface levels of CXCR4 and C5aR1 are elevated in NcdnΔmye neutrophils whereas the total cellular levels of these receptors are normal. I used various pulse-chase experiments to show that constitutive trafficking of these GPCRs is unaffected, but their agonist-induced internalisation is altered in NcdnΔmye neutrophils, and their recycling back to the plasma membrane faster. Altered binding kinetics of β-arrestin with stimulated C5aR1 may underlie the mechanisms behind these trafficking differences, and the resulting elevated GPCR cell surface levels can explain some of the increased responsiveness of NcdnΔmye neutrophils."],"dc:format.checksum.md5":["87eda9de84448d1f82354d60eee3eb5f","9a2a9af0f96921b1bf1b672bdb18fab5"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.104740"],"dc:identifier.uri":["https://www.repository.cam.ac.uk/bitstreams/97c98035-d8b0-4a4d-8c3d-2fcdc155746d/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/362679"],"dc:rights":["https://www.repository.cam.ac.uk/bitstreams/c4fa0a73-3e81-4ba4-ad31-b18daa5492ce/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["GPCR","NCDN","Neurochondrin","Norbin","trafficking"],"dc:title":["Investigating the immunosuppressive functions of the GPCR adapter protein Norbin"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:23:54Z"}