Characterization of G protein coupling mediated by the conserved D134(3.49) of DRY motif, M241(6.34), and F251(6.44) residues on human CXCR1

Xinbing Han; Yan Feng; Xinhua Chen; Craig Gerard; William A Boisvert

doi:10.1016/j.fob.2015.03.001

Characterization of G protein coupling mediated by the conserved D134(3.49) of DRY motif, M241(6.34), and F251(6.44) residues on human CXCR1

FEBS Open Bio. 2015 Mar 7:5:182-90. doi: 10.1016/j.fob.2015.03.001. eCollection 2015.

Authors

Xinbing Han¹, Yan Feng², Xinhua Chen³, Craig Gerard¹, William A Boisvert⁴

Affiliations

¹ Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, United States.
² First Affiliated Hospital, Xinjiang Medical University, Urumqi, Xinjiang 830054, China.
³ Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, United States.
⁴ Center for Cardiovascular Research, John A. Burns School of Medicine, University of Hawaii, 651 Ilalo Street, Honolulu, HI 96813, United States ; Kazan Federal University, Kazan, Russia.

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.

Keywords: CXCR1; CXCR1, CXC receptor 1; Chemokine receptor; Constitutive activity; DRY motif, Asp-Arg-Tyr motif; G protein coupled receptor; GPCR, G protein-coupled receptor; Gα15; Gαi; IL-8, interleukin 8; IP, inositol phosphate; Kd, affinity constants; PLC, phospholipase C; PTX, pertussis toxin; TMs, transmembrane domain; WT, wild type; i2, intracellular loop 2; i3, intracellular loop 3.

Abstract

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