Kevin S Gill, Kritika Mehta, Jeremiah D Heredia, Vishnu V Krishnamurthy, Kai Zhang, Erik Procko
{"title":"通过深度诱变证明了趋化因子受体CXCR4和CCR5的多种自缔合机制。","authors":"Kevin S Gill, Kritika Mehta, Jeremiah D Heredia, Vishnu V Krishnamurthy, Kai Zhang, Erik Procko","doi":"10.1016/j.jbc.2023.105229","DOIUrl":null,"url":null,"abstract":"<p><p>Chemokine receptors are members of the rhodopsin-like class A GPCRs whose signaling through G proteins drives the directional movement of cells in response to a chemokine gradient. Chemokine receptors CXCR4 and CCR5 have been extensively studied due to their roles in leukocyte development and inflammation and their status as coreceptors for HIV-1 infection, among other roles. Both receptors form dimers or oligomers of unclear function. While CXCR4 has been crystallized in a dimeric arrangement, available atomic resolution structures of CCR5 are monomeric. To investigate their dimerization interfaces, we used a bimolecular fluorescence complementation (BiFC)-based screen and deep mutational scanning to find mutations that change how the receptors self-associate, either via specific oligomer assembly or alternative mechanisms of clustering in close proximity. Many disruptive mutations promoted self-associations nonspecifically, suggesting they aggregated in the membrane. A mutationally intolerant region was found on CXCR4 that matched the crystallographic dimer interface, supporting this dimeric arrangement in living cells. A mutationally intolerant region was also observed on the surface of CCR5 by transmembrane helices 3 and 4. Mutations predicted from the scan to reduce BiFC were validated and were localized in the transmembrane domains as well as the C-terminal cytoplasmic tails where they reduced lipid microdomain localization. A mutation in the dimer interface of CXCR4 had increased binding to the ligand CXCL12 and yet diminished calcium signaling. There was no change in syncytia formation with cells expressing HIV-1 Env. The data highlight that multiple mechanisms are involved in self-association of chemokine receptor chains.</p>","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105229"},"PeriodicalIF":0.0000,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10551899/pdf/","citationCount":"0","resultStr":"{\"title\":\"Multiple mechanisms of self-association of chemokine receptors CXCR4 and CCR5 demonstrated by deep mutagenesis.\",\"authors\":\"Kevin S Gill, Kritika Mehta, Jeremiah D Heredia, Vishnu V Krishnamurthy, Kai Zhang, Erik Procko\",\"doi\":\"10.1016/j.jbc.2023.105229\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Chemokine receptors are members of the rhodopsin-like class A GPCRs whose signaling through G proteins drives the directional movement of cells in response to a chemokine gradient. Chemokine receptors CXCR4 and CCR5 have been extensively studied due to their roles in leukocyte development and inflammation and their status as coreceptors for HIV-1 infection, among other roles. Both receptors form dimers or oligomers of unclear function. While CXCR4 has been crystallized in a dimeric arrangement, available atomic resolution structures of CCR5 are monomeric. To investigate their dimerization interfaces, we used a bimolecular fluorescence complementation (BiFC)-based screen and deep mutational scanning to find mutations that change how the receptors self-associate, either via specific oligomer assembly or alternative mechanisms of clustering in close proximity. Many disruptive mutations promoted self-associations nonspecifically, suggesting they aggregated in the membrane. A mutationally intolerant region was found on CXCR4 that matched the crystallographic dimer interface, supporting this dimeric arrangement in living cells. A mutationally intolerant region was also observed on the surface of CCR5 by transmembrane helices 3 and 4. Mutations predicted from the scan to reduce BiFC were validated and were localized in the transmembrane domains as well as the C-terminal cytoplasmic tails where they reduced lipid microdomain localization. A mutation in the dimer interface of CXCR4 had increased binding to the ligand CXCL12 and yet diminished calcium signaling. There was no change in syncytia formation with cells expressing HIV-1 Env. The data highlight that multiple mechanisms are involved in self-association of chemokine receptor chains.</p>\",\"PeriodicalId\":22621,\"journal\":{\"name\":\"The Journal of Biological Chemistry\",\"volume\":\" \",\"pages\":\"105229\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2023-10-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10551899/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"The Journal of Biological Chemistry\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1016/j.jbc.2023.105229\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2023/9/9 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"The Journal of Biological Chemistry","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.jbc.2023.105229","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/9/9 0:00:00","PubModel":"Epub","JCR":"","JCRName":"","Score":null,"Total":0}
Multiple mechanisms of self-association of chemokine receptors CXCR4 and CCR5 demonstrated by deep mutagenesis.
Chemokine receptors are members of the rhodopsin-like class A GPCRs whose signaling through G proteins drives the directional movement of cells in response to a chemokine gradient. Chemokine receptors CXCR4 and CCR5 have been extensively studied due to their roles in leukocyte development and inflammation and their status as coreceptors for HIV-1 infection, among other roles. Both receptors form dimers or oligomers of unclear function. While CXCR4 has been crystallized in a dimeric arrangement, available atomic resolution structures of CCR5 are monomeric. To investigate their dimerization interfaces, we used a bimolecular fluorescence complementation (BiFC)-based screen and deep mutational scanning to find mutations that change how the receptors self-associate, either via specific oligomer assembly or alternative mechanisms of clustering in close proximity. Many disruptive mutations promoted self-associations nonspecifically, suggesting they aggregated in the membrane. A mutationally intolerant region was found on CXCR4 that matched the crystallographic dimer interface, supporting this dimeric arrangement in living cells. A mutationally intolerant region was also observed on the surface of CCR5 by transmembrane helices 3 and 4. Mutations predicted from the scan to reduce BiFC were validated and were localized in the transmembrane domains as well as the C-terminal cytoplasmic tails where they reduced lipid microdomain localization. A mutation in the dimer interface of CXCR4 had increased binding to the ligand CXCL12 and yet diminished calcium signaling. There was no change in syncytia formation with cells expressing HIV-1 Env. The data highlight that multiple mechanisms are involved in self-association of chemokine receptor chains.