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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

Two-dimensional diagram of human CXCR1. Most rhodopsin-like GPCRs have a conserved Asp-Arg (DR) pair at the cytoplasmic end of TM3. CXCR1 consists of characteristic seven transmembrane helices (TM1–TM7) connected by three extracellular loops (e1∼e3) and three intracellular loops (i1∼i3). The positions of the residues (S1323.47, D1343.49, M2416.34 and F2516.44) that were targeted for mutagenesis in TM3 and TM6 of the CXCR1 are indicated by filled circles. The putative two disulphide bonds (gold), formed by Cys110/Cys187 are indicated by “S–S”.
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f0005: Two-dimensional diagram of human CXCR1. Most rhodopsin-like GPCRs have a conserved Asp-Arg (DR) pair at the cytoplasmic end of TM3. CXCR1 consists of characteristic seven transmembrane helices (TM1–TM7) connected by three extracellular loops (e1∼e3) and three intracellular loops (i1∼i3). The positions of the residues (S1323.47, D1343.49, M2416.34 and F2516.44) that were targeted for mutagenesis in TM3 and TM6 of the CXCR1 are indicated by filled circles. The putative two disulphide bonds (gold), formed by Cys110/Cys187 are indicated by “S–S”.

Mentions: CXCR1 is a member of rhodopsin-like GPCRs (Fig. 1). To elucidate the role of the highly conserved DRY motif in activation and function of CXCR1, we introduced a mutation in D1343.49 of DRY motif of CXCR1. Other amino acid residues on TM3 and TM6 potentially involved in G protein coupling and constitutive activity of GPCRs that were studied include S132 (TM3:47), M241 (TM6:34) and F251 (TM6:44). Among four amino acid residues chosen for our study, D1343.49 and F2516.44 are highly conserved whereas S1323.47 and M2416.34 are modestly conserved (Fig. 2). Selective mutations were made because of their critical roles in G protein coupling in several other GPCRs. These CXCR1 mutants were expressed in COS-7 cells or HEK239 cells and their expression, binding of IL-8, and Gα15- and Gαi coupling-induced IP production were determined.


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)

Two-dimensional diagram of human CXCR1. Most rhodopsin-like GPCRs have a conserved Asp-Arg (DR) pair at the cytoplasmic end of TM3. CXCR1 consists of characteristic seven transmembrane helices (TM1–TM7) connected by three extracellular loops (e1∼e3) and three intracellular loops (i1∼i3). The positions of the residues (S1323.47, D1343.49, M2416.34 and F2516.44) that were targeted for mutagenesis in TM3 and TM6 of the CXCR1 are indicated by filled circles. The putative two disulphide bonds (gold), formed by Cys110/Cys187 are indicated by “S–S”.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0005: Two-dimensional diagram of human CXCR1. Most rhodopsin-like GPCRs have a conserved Asp-Arg (DR) pair at the cytoplasmic end of TM3. CXCR1 consists of characteristic seven transmembrane helices (TM1–TM7) connected by three extracellular loops (e1∼e3) and three intracellular loops (i1∼i3). The positions of the residues (S1323.47, D1343.49, M2416.34 and F2516.44) that were targeted for mutagenesis in TM3 and TM6 of the CXCR1 are indicated by filled circles. The putative two disulphide bonds (gold), formed by Cys110/Cys187 are indicated by “S–S”.
Mentions: CXCR1 is a member of rhodopsin-like GPCRs (Fig. 1). To elucidate the role of the highly conserved DRY motif in activation and function of CXCR1, we introduced a mutation in D1343.49 of DRY motif of CXCR1. Other amino acid residues on TM3 and TM6 potentially involved in G protein coupling and constitutive activity of GPCRs that were studied include S132 (TM3:47), M241 (TM6:34) and F251 (TM6:44). Among four amino acid residues chosen for our study, D1343.49 and F2516.44 are highly conserved whereas S1323.47 and M2416.34 are modestly conserved (Fig. 2). Selective mutations were made because of their critical roles in G protein coupling in several other GPCRs. These CXCR1 mutants were expressed in COS-7 cells or HEK239 cells and their expression, binding of IL-8, and Gα15- and Gαi coupling-induced IP production were determined.

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