Limits...
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

Confocal analysis of expression of CXCR1 and its mutants. Surface expression of CXCR1 and its mutants on COS-7 cells were measured as described. The green color represents the surface expression of CXCR1. The cells were counterstained with Dapi as shown in blue.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4372616&req=5

f0020: Confocal analysis of expression of CXCR1 and its mutants. Surface expression of CXCR1 and its mutants on COS-7 cells were measured as described. The green color represents the surface expression of CXCR1. The cells were counterstained with Dapi as shown in blue.

Mentions: The flow cytometry data demonstrated the expression of WT CXCR1 and mutants on transfected HEK293 cells (Fig. 3A and B) except F251A which was barely expressed. The surface expression of CXCR1 and its mutants were further supported by confocal imaging results (Fig. 4). Consistently, F251A mutant was poorly expressed, whereas other mutants, including S132A, D134N, D134V, M241V, F251H, and F251Y, were detectable on the cell surface (Figs. 3A, B, and 4).


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)

Confocal analysis of expression of CXCR1 and its mutants. Surface expression of CXCR1 and its mutants on COS-7 cells were measured as described. The green color represents the surface expression of CXCR1. The cells were counterstained with Dapi as shown in blue.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0020: Confocal analysis of expression of CXCR1 and its mutants. Surface expression of CXCR1 and its mutants on COS-7 cells were measured as described. The green color represents the surface expression of CXCR1. The cells were counterstained with Dapi as shown in blue.
Mentions: The flow cytometry data demonstrated the expression of WT CXCR1 and mutants on transfected HEK293 cells (Fig. 3A and B) except F251A which was barely expressed. The surface expression of CXCR1 and its mutants were further supported by confocal imaging results (Fig. 4). Consistently, F251A mutant was poorly expressed, whereas other mutants, including S132A, D134N, D134V, M241V, F251H, and F251Y, were detectable on the cell surface (Figs. 3A, B, and 4).

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