Understanding the precise structure and function of the intracellular domains of G protein-coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5-hydroxytryptamine,, (5-HT2A) receptor, a model G,,-coupl ed receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5-HT, , receptor, a region previously implicated in G-protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with a-helical and p-loop, -turn, and -sheet structure. Using random peptide phage libraries, we identified several arrestin-like sequences as i3-interacting peptides. We subsequently found that all three known arrestins (p-arrestin, arrestin-3, and visual arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5-HT2, receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3-arrestin interactions. Dual-label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed arrestins (p-arrestin or arrestin-3) and 5-HTz, receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5-HT, , receptor represents a structurally ordered domain composed of a-helical and p-loop, -turn, and -sheet regions, (b) that this loop interacts with arrestins in vitro, and is hence active, and (c) that arrestins are colocalized with 5-HT, , receptors in vivo.
