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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 mutants coupled to Gα15. Basal and IL-8-stimulated inositol phosphate (IP) accumulation is shown in COS-7 cells transiently co-transfected with WT or mutant CXCR1 and pSG5 or Gα15. The release of inositol phosphate, induced by 40 nM IL-8, was measured 1 h after the treatment. Data for mutants are summarized from 3–7 experiments, each performed in triplicate, and are expressed as a percentage of WT CXCR1 baseline determined in parallel. The results are mean ± SEM. ∗∗P < 0.001, vs. WT + Gα15 (in the absence of IL-8).
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f0030: CXCR1 mutants coupled to Gα15. Basal and IL-8-stimulated inositol phosphate (IP) accumulation is shown in COS-7 cells transiently co-transfected with WT or mutant CXCR1 and pSG5 or Gα15. The release of inositol phosphate, induced by 40 nM IL-8, was measured 1 h after the treatment. Data for mutants are summarized from 3–7 experiments, each performed in triplicate, and are expressed as a percentage of WT CXCR1 baseline determined in parallel. The results are mean ± SEM. ∗∗P < 0.001, vs. WT + Gα15 (in the absence of IL-8).

Mentions: CXCR1 is coupled to both PTX-sensitive Gαi2 as well as PTX-resistant Gα15 in transfected COS-7 cells [37]. Mutation in D134 of DRY motif of CXCR1 did not retain G protein coupling or agonist-induced response (Fig. 6). Despite the high sequence identity between CXCR2 and KSHV-GPCR, both capable of binding IL-8 with high affinity, our results suggest that the conservative D on DRY motif plays a distinct role in G protein coupling from these two receptor homologues. This is probably responsible for the unique regulation and activation of effectors triggered by binding to IL-8. In contrast, among mutants detected compared with WT CXCR1, individual replacement of methionine (M241V) and phenylalanine (F251H) increased basal signal activity by 43.4% and 46.1%, respectively, in the absence of IL-8 (p < 0.001 vs. WT without IL-8 treatment), but not agonist-stimulated IP accumulation of the receptor. This suggests that substitution of M241V and F251H constitutively activates Gα15 protein (Fig. 6). In addition, M241V6.34 increased the IP accumulation basally as well as in response to IL-8 in a dose-dependent manner (Fig. 7). Moreover, in the absence of IL-8, IP accumulation was significantly higher in cells expressing M241V (p < 0.001) (Fig. 7). Two mutations on V247 (TM6:40), V247A and V247N, displayed constitutive activity as reported recently [36]. Although F251A mutant showed increased ligand affinity, it was nearly dysfunctional, which is likely attributed to its instability and its poor expression on cell surface (Figs. 3–5).


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 mutants coupled to Gα15. Basal and IL-8-stimulated inositol phosphate (IP) accumulation is shown in COS-7 cells transiently co-transfected with WT or mutant CXCR1 and pSG5 or Gα15. The release of inositol phosphate, induced by 40 nM IL-8, was measured 1 h after the treatment. Data for mutants are summarized from 3–7 experiments, each performed in triplicate, and are expressed as a percentage of WT CXCR1 baseline determined in parallel. The results are mean ± SEM. ∗∗P < 0.001, vs. WT + Gα15 (in the absence of IL-8).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372616&req=5

f0030: CXCR1 mutants coupled to Gα15. Basal and IL-8-stimulated inositol phosphate (IP) accumulation is shown in COS-7 cells transiently co-transfected with WT or mutant CXCR1 and pSG5 or Gα15. The release of inositol phosphate, induced by 40 nM IL-8, was measured 1 h after the treatment. Data for mutants are summarized from 3–7 experiments, each performed in triplicate, and are expressed as a percentage of WT CXCR1 baseline determined in parallel. The results are mean ± SEM. ∗∗P < 0.001, vs. WT + Gα15 (in the absence of IL-8).
Mentions: CXCR1 is coupled to both PTX-sensitive Gαi2 as well as PTX-resistant Gα15 in transfected COS-7 cells [37]. Mutation in D134 of DRY motif of CXCR1 did not retain G protein coupling or agonist-induced response (Fig. 6). Despite the high sequence identity between CXCR2 and KSHV-GPCR, both capable of binding IL-8 with high affinity, our results suggest that the conservative D on DRY motif plays a distinct role in G protein coupling from these two receptor homologues. This is probably responsible for the unique regulation and activation of effectors triggered by binding to IL-8. In contrast, among mutants detected compared with WT CXCR1, individual replacement of methionine (M241V) and phenylalanine (F251H) increased basal signal activity by 43.4% and 46.1%, respectively, in the absence of IL-8 (p < 0.001 vs. WT without IL-8 treatment), but not agonist-stimulated IP accumulation of the receptor. This suggests that substitution of M241V and F251H constitutively activates Gα15 protein (Fig. 6). In addition, M241V6.34 increased the IP accumulation basally as well as in response to IL-8 in a dose-dependent manner (Fig. 7). Moreover, in the absence of IL-8, IP accumulation was significantly higher in cells expressing M241V (p < 0.001) (Fig. 7). Two mutations on V247 (TM6:40), V247A and V247N, displayed constitutive activity as reported recently [36]. Although F251A mutant showed increased ligand affinity, it was nearly dysfunctional, which is likely attributed to its instability and its poor expression on cell surface (Figs. 3–5).

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