Multiple mechanisms of self-association of chemokine receptors CXCR4 and CCR5 demonstrated by deep mutagenesis

• Published in: The Journal of biological chemistry Vol. 299; no. 10; p. 105229
• Main Authors: Gill, Kevin S., Mehta, Kritika, Heredia, Jeremiah D., Krishnamurthy, Vishnu V., Zhang, Kai, Procko, Erik
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-10-2023; American Society for Biochemistry and Molecular Biology
• Subjects: bimolecular fluorescence complementation; CCR5; Cell Line; CXCR4; deep mutational scan; Dimerization; G protein–coupled receptor; Humans; Models, Molecular; Mutagenesis; Mutation; Protein Structure, Tertiary; receptor dimer; Receptors, CCR5 - chemistry; Receptors, CCR5 - genetics; Receptors, CCR5 - metabolism; Receptors, CXCR4 - chemistry; Receptors, CXCR4 - genetics; Receptors, CXCR4 - metabolism; Signal Transduction; YFP; deep mutational scan; CXCR4; FACS; receptor dimer; SSM; BSA; MA; GPCR; TM; G protein–coupled receptor; BiFC; CCR5; bimolecular fluorescence complementation; TIR
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1016/j.jbc.2023.105229

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.