Abstract

We have previously shown that alkyl-substituted amino acid derivatives directly activate G_i/o proteins.N-Dodecyl-N ^α,N ^ε-(bis-l-lysinyl)-l-lysine amide (FUB132) is a new representative of this class of compounds with increased efficacy. Here, we characterized the molecular mechanism of action of this class of compounds. FUB132 and its predecessor FUB86 were selective receptomimetics for G_i/obecause they stimulated the guanine nucleotide exchange reaction of purified G_i/o as documented by an increased rate of GDP release, GTPγS binding, and GTP hydrolysis. In contrast to the receptomimetic peptide mastoparan, stimulation of G proteins by lipoamines required the presence of neither Gβγ-dimers nor lipids. On the contrary, Gβγ-dimers suppressed the stimulatory effect of FUB132. The stimulation of G_i/o by lipoamines and by mastoparan was not additive. A peptide derived from the C terminus of Gα_o3, but not a corresponding Gα_q-derived peptide, quenched the FUB132-induced activation of Gα_o. In membranes prepared from human embryonic kidney 293 cells that stably expressed the G_i/o-coupled human A₁-adenosine receptor, lipoamines impeded high-affinity agonist binding. In contrast, antagonist binding was not affected. We conclude that alkyl-substituted amines target a site, most likely at the C terminus of Gα_i/o-subunits, that is also contacted by receptors. However, because Gβγ-dimers blunt rather than enhance their efficacy, their mechanism of action differs fundamentally from that of a receptor. Thus, despite their receptomimetic effect in vitro, alkyl-substituted amines and related polyamines are poor direct G protein activators in vivo. In the presence of Gβγ, they rather antagonize G protein-coupled receptor signaling.