Regulation of the avidity of ternary complexes containing the human 5-HT(1A) receptor by mutation of a receptor contact site on the interacting G protein alpha subunit

Abstract

1 Fusion proteins were constructed between the human 5-HT(1A) receptor and pertussis toxin-resistant forms of both G(i1)alpha and G(o1)alpha mutated at residue(351) from cysteine to either glycine or isoleucine. Each of these was expressed stably in HEK293 cells. 2 Increasing concentrations of GDP inhibited binding of the agonist [(3)H]-8-OH-DPAT but not the antagonist [(3)H]-MPPF to each construct. 3 The IC(50) for GDP was greater for constructs containing isoleucine at residue(351) of the G proteins compared to those with glycine at this position. 4 The G protein antagonist suramin had similar effects to GDP on the binding of [(3)H]-8-OH-DPAT. 5 The proportion of 5-HT(1A) receptor binding sites detected by [(3)H]-MPPF that displayed high affinity for 8-OH-DPAT was significantly greater when the interacting G protein contained isoleucine rather than glycine at residue(351). 6 The 5-HT(1A) receptor displayed similar avidity of interaction with G(i1)alpha and G(o1)alpha. 7 These results indicate that a higher avidity ternary complex is formed between 8-OH-DPAT, the 5-HT(1A) receptor and G proteins when isoleucine rather than glycine is located at residue(351) of the interacting G protein.

Figure 1

GDP and pertussis toxin pre-treatment reduces the binding of [³H]-8-OH-DPAT to the 5-HT_1A receptor. Membranes were prepared from untreated and pertussis toxin-pre-treated (25 ng ml⁻¹), 16 h) HEK293 cells stably expressing the human 5-HT_1A receptor. The specific binding of [³H]-8-OH-DPAT (1 nM) was then assessed in the presence of increasing concentrations of GDP.

Figure 2

GDP but not pertussis toxin pre-treatment reduces the binding of [³H]-8-OH-DPAT to pertussis toxin-resistant 5-HT_1A receptor-G_ilα fusion proteins. Membranes were prepared from untreated and pertussis toxin-pretreated (25 ng ml⁻¹, 16 h) HEK293 cells stably expressing 5-HT_1A receptor-G_i1α fusion proteins in which the pertussis toxin-sensitive cysteine was replaced by either glycine or isoleucine. The specific binding of [³H]-8-OH-DPAT was assessed in the presence of varying concentrations of GDP as in Figure 1.

Figure 3

The nature of the pertussis toxin-insensitive mutation alters the potency of GDP to reduce the binding of [³H]-8-OH-DPAT to pertussis toxin-resistant 5-HT_1A receptor-G_o1α fusion proteins. Membranes were prepared from untreated and pertussis toxin-pretreated (25 ng ml⁻¹, 16 h) HEK293 cells stably expressing 5-HT_1A receptor-G_o1α fusion proteins in which the pertussis toxin-sensitive cysteine was replaced by either glycine or isoleucine. The specific binding of [³H]-8-OH-DPAT was assessed in the presence of varying concentrations of GDP as in Figure 2.

Figure 4

Suramin reduces the binding of [³H]-8-OH-DPAT but not [³H]-MPPF to the 5HT_1A receptor. Membranes were prepared from untreated and pertussis toxin pre-treated (25 mg ml⁻¹, 16 h) HEK293 cells stably expressing the human 5-HT_1A receptor. The specific binding of [³H]-8-OH-DPAT (1 nM) or [³H]-MPPF (1 nM) was then assessed in the presence of increasing concentrations of suramin.

Figure 5

The nature of the pertussis toxin-insensitive mutation alters the potency of suramin to reduce the binding of [³H]-8-OH-DPAT to pertussis toxin-resistant 5-HT_1A receptor-G_i1α and −G_o1α fusion proteins. Membranes were prepared from untreated and pertussis toxin-pre-treated (25 ng ml⁻¹, 16 h) HEK293 cells stably expressing 5-HT_1A receptor-G_i1α (a) or 5-HT_1A receptor-G_o1α (b) fusion proteins in which the pertussis toxin sensitive cysteine was replaced by either glycine or isoleucine. The specific binding of [³H]-8-OH-DPAT was assessed in the presence of varying concentrations of suramin as in Figure 4.

Figure 6

A 5HT_1A-(Cys³⁵¹Ile)G_i1α fusion protein displays a greater proportion of high affinity binding sites for 8-OH-DPAT than a 5HT_1A-(Cys³⁵¹Gly)G_i1α fusion protein. Membranes were prepared from pertussis toxin pre-treated HEK293 cells expressing either the 5HT_1A-(Cys³⁵¹Ile)G_i1α fusion protein or the 5HT_1A-(Cys³⁵¹Gly)G_i1α fusion protein. The ability of varying concentrations of 8-OH-DPAT to compete with [³H]-MPPF (1 nM) for the binding site was then assessed.