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Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands.

Sergeev E, Hansen AH, Pandey SK, MacKenzie AE, Hudson BD, Ulven T, Milligan G - J. Biol. Chem. (2015)

Bottom Line: Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two.Moreover, different chemical series of antagonist interact preferentially with different arginine residues.A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom and.

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GLPG0974 inhibits the actions of C3 and Cmp 1 at hFFA2. hFFA2-eYFP and β-arrestin-2 Renilla luciferase were co-transfected transiently into HEK293T cells. The capacity of GLPG0974 to inhibit interactions between hFFA2-eYFP and β-arrestin-2 Renilla luciferase induced by an approximately EC80 concentration of C3 (3 mm) (filled symbols) or Cmp 1 (10 μm) (open symbols) was then assessed (A). Flp-InTM T-RExTM 293 cells stably harboring hFFA2-eYFP at the Flp-InTM T-RExTM locus were induced to express the receptor. The capacity of GLPG0974 to inhibit elevation of [Ca2+]i produced by EC80 concentrations of C3 or Cmp 1 is shown (B). Membranes from such induced cells were used to assess the ability of GLPG0974 to inhibit the binding of [35S]GTPγS stimulated by EC80 concentrations of C3 (300 μm) and or Cmp 1 (1 μm) (C). Although C3 inhibited forskolin-induced cAMP production in membranes of Flp-InTM T-RExTM 293 cells induced to express hFFA3-eYFP, GLPG0974 did not inhibit this (D). Moreover, unlike at hFFA2, GLPG0974 was unable to inhibit either C3 (filled symbols) or Cmp 1 (open symbols)-mediated inhibition of forskolin-induced cAMP production in membranes from Flp-InTM T-RExTM 293 cells induced to express mFFA2-eYFP (E).
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Figure 2: GLPG0974 inhibits the actions of C3 and Cmp 1 at hFFA2. hFFA2-eYFP and β-arrestin-2 Renilla luciferase were co-transfected transiently into HEK293T cells. The capacity of GLPG0974 to inhibit interactions between hFFA2-eYFP and β-arrestin-2 Renilla luciferase induced by an approximately EC80 concentration of C3 (3 mm) (filled symbols) or Cmp 1 (10 μm) (open symbols) was then assessed (A). Flp-InTM T-RExTM 293 cells stably harboring hFFA2-eYFP at the Flp-InTM T-RExTM locus were induced to express the receptor. The capacity of GLPG0974 to inhibit elevation of [Ca2+]i produced by EC80 concentrations of C3 or Cmp 1 is shown (B). Membranes from such induced cells were used to assess the ability of GLPG0974 to inhibit the binding of [35S]GTPγS stimulated by EC80 concentrations of C3 (300 μm) and or Cmp 1 (1 μm) (C). Although C3 inhibited forskolin-induced cAMP production in membranes of Flp-InTM T-RExTM 293 cells induced to express hFFA3-eYFP, GLPG0974 did not inhibit this (D). Moreover, unlike at hFFA2, GLPG0974 was unable to inhibit either C3 (filled symbols) or Cmp 1 (open symbols)-mediated inhibition of forskolin-induced cAMP production in membranes from Flp-InTM T-RExTM 293 cells induced to express mFFA2-eYFP (E).

Mentions: GLPG0974 (Fig. 1) has recently been described as a selective FFA2 receptor antagonist that is able to block C2-mediated chemotaxis of human neutrophils (19). We synthesized this compound and demonstrated that it was able to antagonize, in a concentration-dependent manner, both C3 and Cmp 1-mediated activation of a form of hFFA2 that had been C-terminally tagged with enhanced yellow fluorescent protein (hFFA2-eYFP). This was the case whether employing a BRET-based β-arrestin-2 recruitment assay (Fig. 2A), a Gq/11-dependent [Ca2+]i mobilization (Fig. 2B), or a Gi/o-dependent [35S]GTPγS incorporation (Fig. 2C) assay. In each case, when employing 80% maximally effective concentrations of either C3 or Cmp 1, GLPG0974 displayed high and similar potency inhibition against each of the agonists (Table 1). GLPG0974 was highly selective for hFFA2, showing no inhibitory effect on C3-mediated activation of the closely related SCFA receptor hFFA3 (Fig. 2D). However, as found previously for CATPB, another FFA2 antagonist (10), GLPG0974 displayed marked species selectivity and was unable to block the effects of either C3 or Cmp 1 at mFFA2 (Fig. 2E).


Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands.

Sergeev E, Hansen AH, Pandey SK, MacKenzie AE, Hudson BD, Ulven T, Milligan G - J. Biol. Chem. (2015)

GLPG0974 inhibits the actions of C3 and Cmp 1 at hFFA2. hFFA2-eYFP and β-arrestin-2 Renilla luciferase were co-transfected transiently into HEK293T cells. The capacity of GLPG0974 to inhibit interactions between hFFA2-eYFP and β-arrestin-2 Renilla luciferase induced by an approximately EC80 concentration of C3 (3 mm) (filled symbols) or Cmp 1 (10 μm) (open symbols) was then assessed (A). Flp-InTM T-RExTM 293 cells stably harboring hFFA2-eYFP at the Flp-InTM T-RExTM locus were induced to express the receptor. The capacity of GLPG0974 to inhibit elevation of [Ca2+]i produced by EC80 concentrations of C3 or Cmp 1 is shown (B). Membranes from such induced cells were used to assess the ability of GLPG0974 to inhibit the binding of [35S]GTPγS stimulated by EC80 concentrations of C3 (300 μm) and or Cmp 1 (1 μm) (C). Although C3 inhibited forskolin-induced cAMP production in membranes of Flp-InTM T-RExTM 293 cells induced to express hFFA3-eYFP, GLPG0974 did not inhibit this (D). Moreover, unlike at hFFA2, GLPG0974 was unable to inhibit either C3 (filled symbols) or Cmp 1 (open symbols)-mediated inhibition of forskolin-induced cAMP production in membranes from Flp-InTM T-RExTM 293 cells induced to express mFFA2-eYFP (E).
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Figure 2: GLPG0974 inhibits the actions of C3 and Cmp 1 at hFFA2. hFFA2-eYFP and β-arrestin-2 Renilla luciferase were co-transfected transiently into HEK293T cells. The capacity of GLPG0974 to inhibit interactions between hFFA2-eYFP and β-arrestin-2 Renilla luciferase induced by an approximately EC80 concentration of C3 (3 mm) (filled symbols) or Cmp 1 (10 μm) (open symbols) was then assessed (A). Flp-InTM T-RExTM 293 cells stably harboring hFFA2-eYFP at the Flp-InTM T-RExTM locus were induced to express the receptor. The capacity of GLPG0974 to inhibit elevation of [Ca2+]i produced by EC80 concentrations of C3 or Cmp 1 is shown (B). Membranes from such induced cells were used to assess the ability of GLPG0974 to inhibit the binding of [35S]GTPγS stimulated by EC80 concentrations of C3 (300 μm) and or Cmp 1 (1 μm) (C). Although C3 inhibited forskolin-induced cAMP production in membranes of Flp-InTM T-RExTM 293 cells induced to express hFFA3-eYFP, GLPG0974 did not inhibit this (D). Moreover, unlike at hFFA2, GLPG0974 was unable to inhibit either C3 (filled symbols) or Cmp 1 (open symbols)-mediated inhibition of forskolin-induced cAMP production in membranes from Flp-InTM T-RExTM 293 cells induced to express mFFA2-eYFP (E).
Mentions: GLPG0974 (Fig. 1) has recently been described as a selective FFA2 receptor antagonist that is able to block C2-mediated chemotaxis of human neutrophils (19). We synthesized this compound and demonstrated that it was able to antagonize, in a concentration-dependent manner, both C3 and Cmp 1-mediated activation of a form of hFFA2 that had been C-terminally tagged with enhanced yellow fluorescent protein (hFFA2-eYFP). This was the case whether employing a BRET-based β-arrestin-2 recruitment assay (Fig. 2A), a Gq/11-dependent [Ca2+]i mobilization (Fig. 2B), or a Gi/o-dependent [35S]GTPγS incorporation (Fig. 2C) assay. In each case, when employing 80% maximally effective concentrations of either C3 or Cmp 1, GLPG0974 displayed high and similar potency inhibition against each of the agonists (Table 1). GLPG0974 was highly selective for hFFA2, showing no inhibitory effect on C3-mediated activation of the closely related SCFA receptor hFFA3 (Fig. 2D). However, as found previously for CATPB, another FFA2 antagonist (10), GLPG0974 displayed marked species selectivity and was unable to block the effects of either C3 or Cmp 1 at mFFA2 (Fig. 2E).

Bottom Line: Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two.Moreover, different chemical series of antagonist interact preferentially with different arginine residues.A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom and.

Show MeSH
Related in: MedlinePlus