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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.

Han X, Feng Y, Chen X, Gerard C, Boisvert WA - FEBS Open Bio (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, United States.

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.

No MeSH data available.


Related in: MedlinePlus

CXCR1 and mutants coupled to Gαi2. (A) COS-7 cells were co-transfected with equal amounts of cDNA (0.1 μg per well per component) encoding Gαi2, Gβ1, Gγ2, PLCβ2, as well as WT CXCR1 or its mutants. (A and B) The influence of PTX on the release of inositol phosphates. The cells were treated with or without PTX (100 ng/ml) for 18 h as indicated. The release of inositol phosphates was measured 1 h after the treatment in the absence (A) or presence (B) of 40 nM IL-8. “M” denotes cells co-transfected with cDNA components encoding Gαi2, Gβ1, Gγ2, and PLCβ2. (C) COS-7 cells were co-transfected with 0.3 μg of cDNA encoding Gαqi5 and the WT CXCR1 or its mutants. The release of inositol phosphates, induced by 40 nM IL-8, was measured 1 h after the treatment as described in detail under “Section 4”. Data are mean ± SEM of replicate wells from a representative experiment.
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f0040: CXCR1 and mutants coupled to Gαi2. (A) COS-7 cells were co-transfected with equal amounts of cDNA (0.1 μg per well per component) encoding Gαi2, Gβ1, Gγ2, PLCβ2, as well as WT CXCR1 or its mutants. (A and B) The influence of PTX on the release of inositol phosphates. The cells were treated with or without PTX (100 ng/ml) for 18 h as indicated. The release of inositol phosphates was measured 1 h after the treatment in the absence (A) or presence (B) of 40 nM IL-8. “M” denotes cells co-transfected with cDNA components encoding Gαi2, Gβ1, Gγ2, and PLCβ2. (C) COS-7 cells were co-transfected with 0.3 μg of cDNA encoding Gαqi5 and the WT CXCR1 or its mutants. The release of inositol phosphates, induced by 40 nM IL-8, was measured 1 h after the treatment as described in detail under “Section 4”. Data are mean ± SEM of replicate wells from a representative experiment.

Mentions: To examine their coupling to Gαi protein, WT CXCR1 and mutants were co-transfected into COS-7 cells with Gαi2, Gβ1, Gγ2 and PLCβ2. This five-component co-transfection system (Gαi2-Gβ1-Gγ2-PLCβ2) has been successfully applied to investigate IL-8 signaling pathways in previous studies by our group and others [36,37]. This unique co-transfection system allows Gαi2 activation in response to IL-8, leading to the release of Gβγ subunits from Gαi2 and resulting in PLCβ2 activation and subsequent IP accumulation. Our results showed that mutants S132A, M241V, F251H and F251Y stimulated IP accumulation in response to IL-8, similar to CXCR1 WT (Fig. 8C). IP production in response to IL-8 was totally abolished by PTX, a specific inhibitor of Gαi, suggesting that CXCR1 and its mutants investigated here are coupled to Gαi2 (Fig. 8A and B). In contrast, IP production was mostly retained in response to IL-8 when Gα15 was expressed (data not shown). Thus, IP accumulation could be achieved via the PTX-insensitive Gα15 pathway or the PTX-sensitive Gαi2 pathway.


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.

Han X, Feng Y, Chen X, Gerard C, Boisvert WA - FEBS Open Bio (2015)

CXCR1 and mutants coupled to Gαi2. (A) COS-7 cells were co-transfected with equal amounts of cDNA (0.1 μg per well per component) encoding Gαi2, Gβ1, Gγ2, PLCβ2, as well as WT CXCR1 or its mutants. (A and B) The influence of PTX on the release of inositol phosphates. The cells were treated with or without PTX (100 ng/ml) for 18 h as indicated. The release of inositol phosphates was measured 1 h after the treatment in the absence (A) or presence (B) of 40 nM IL-8. “M” denotes cells co-transfected with cDNA components encoding Gαi2, Gβ1, Gγ2, and PLCβ2. (C) COS-7 cells were co-transfected with 0.3 μg of cDNA encoding Gαqi5 and the WT CXCR1 or its mutants. The release of inositol phosphates, induced by 40 nM IL-8, was measured 1 h after the treatment as described in detail under “Section 4”. Data are mean ± SEM of replicate wells from a representative experiment.
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f0040: CXCR1 and mutants coupled to Gαi2. (A) COS-7 cells were co-transfected with equal amounts of cDNA (0.1 μg per well per component) encoding Gαi2, Gβ1, Gγ2, PLCβ2, as well as WT CXCR1 or its mutants. (A and B) The influence of PTX on the release of inositol phosphates. The cells were treated with or without PTX (100 ng/ml) for 18 h as indicated. The release of inositol phosphates was measured 1 h after the treatment in the absence (A) or presence (B) of 40 nM IL-8. “M” denotes cells co-transfected with cDNA components encoding Gαi2, Gβ1, Gγ2, and PLCβ2. (C) COS-7 cells were co-transfected with 0.3 μg of cDNA encoding Gαqi5 and the WT CXCR1 or its mutants. The release of inositol phosphates, induced by 40 nM IL-8, was measured 1 h after the treatment as described in detail under “Section 4”. Data are mean ± SEM of replicate wells from a representative experiment.
Mentions: To examine their coupling to Gαi protein, WT CXCR1 and mutants were co-transfected into COS-7 cells with Gαi2, Gβ1, Gγ2 and PLCβ2. This five-component co-transfection system (Gαi2-Gβ1-Gγ2-PLCβ2) has been successfully applied to investigate IL-8 signaling pathways in previous studies by our group and others [36,37]. This unique co-transfection system allows Gαi2 activation in response to IL-8, leading to the release of Gβγ subunits from Gαi2 and resulting in PLCβ2 activation and subsequent IP accumulation. Our results showed that mutants S132A, M241V, F251H and F251Y stimulated IP accumulation in response to IL-8, similar to CXCR1 WT (Fig. 8C). IP production in response to IL-8 was totally abolished by PTX, a specific inhibitor of Gαi, suggesting that CXCR1 and its mutants investigated here are coupled to Gαi2 (Fig. 8A and B). In contrast, IP production was mostly retained in response to IL-8 when Gα15 was expressed (data not shown). Thus, IP accumulation could be achieved via the PTX-insensitive Gα15 pathway or the PTX-sensitive Gαi2 pathway.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, United States.

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.

No MeSH data available.


Related in: MedlinePlus