Characterization of G protein coupling mediated by the conserved D1343.49 of DRY motif, M2416.34, and F2516.44 residues on human CXCR1. Issue 1 (7th March 2015)

• Record Type: Journal Article
• Authors: Han, Xinbing; Feng, Yan; Chen, Xinhua; Gerard, Craig; Boisvert, William A.
• Is Part Of: FEBS open bio. Volume 5:Issue 1(2015)
• Page Start: 182
• Page End: 190
• Publication Date: 2015-03-07
• Subjects: CXCR1 -- CXC receptor 1 -- GPCR -- G protein-coupled receptor -- DRY motif -- Asp-Arg-Tyr motif -- IL-8 -- interleukin 8 -- TMs -- transmembrane domain -- i2 -- intracellular loop 2 -- i3 -- intracellular loop 3 -- PLC -- phospholipase C -- WT -- wild type -- Kd -- affinity constants -- IP -- inositol phosphate -- PTX -- pertussis toxin -- Chemokine receptor -- CXCR1 -- G protein coupled receptor -- Gα15 -- Gαi -- Constitutive activity; Molecular biology -- Periodicals; Cytology -- Periodicals; Life sciences -- Periodicals; Biological Science Disciplines -- Periodicals; Molecular Biology -- Periodicals; Cell Biology -- Periodicals; Cytology; Life sciences; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.fob.2015.03.001
• Languages: English
• ISSNs: 2211-5463

Abstract

Abstract : CXCR1, a receptor for interleukin‐8 (IL‐8), plays an important role in defending against pathogen invasion during neutrophil‐mediated innate immune response. Human CXCR1 is a G protein‐coupled receptor (GPCR) with its characteristic seven transmembrane domains (TMs). Functional and structural analyses of several GPCRs have revealed that conserved residues on TM3 (including the highly conserved Asp‐Arg‐Tyr (DRY) motif) and TM6 near intracellular loops contain domains critical for G protein coupling as well as GPCR activation. The objective of this study was to elucidate the role of critical amino acid residues on TM3 near intracellular loop 2 (i2) and TM6 near intracellular loop 3 (i3), including S132 3.47 (Baldwin location), D134 3.49, M241 6.34, and F251 6.44, in G protein coupling and CXCR1 activation. The results demonstrate that mutations of D134 3.49 at DRY motif of CXCR1 (D134N and D134V) completely abolished the ligand binding and functional response of the receptor. Additionally, point mutations at positions 241 and 251 between TM6 and i3 loop generated mutant receptors with modest constitutive activity via Gα15 signaling activation. Our results show that D134 3.49 on the highly conserved DRY motif has a distinct role for CXCR1 compared to its homologues (CXCR2 and KSHV‐GPCR) in G protein coupling and receptor activation. In addition, M241 6.34 and F251 6.44 along with our previously identified V247 6.40 on TM6 are spatially located in a "hot spot" likely essential for CXCR1 activation. Identification of these amino acid residues may be useful for elucidating mechanism of CXCR1 activation and designing specific antagonists for the treatment of CXCR1‐mediated diseases. Abstract : Mutations in the DRY motif of CXCR1 abolish ligand binding and receptor activation. Point mutations between TM6 and i3 loop result in constitutive activity of CXCR1. Constitutive activity of mutant CXCR1 occurs via Gα15 signaling activation. The highly conserved DRY motifs have distinct roles in CXCR1 and CXCR2.