Selective Interaction of ARF1 with the Carboxy-Terminal Tail Domain of the 5-HT_2A Receptor

Abstract

The 5-hydroxytryptamine 2A receptor (5-HT 2A R) is a member of the class I family of rhodopsin-related G protein-coupled receptors. The receptor is known to activate phospholipase C via the heterotrimeric G proteins G q/11 , but we showed previously that it can also signal through the phospholipase D (PLD) pathway in an ADP-ribosylation factor (ARF)-dependent manner that seems to be independent of G q/11 (Mitchell et al., 1998). Both coimmunoprecipitation experiments and the effects of negative mutant ARF constructs on 5-HT 2A R-induced PLD activation here suggested that ARF1 may play a greater role than ARF6 in the function of this receptor. Furthermore, we demonstrated using glutathione S-transferase (GST)-fusion proteins of receptor domains that ARF1 and ARF6 bind to the third intracellular loop (i3) and the carboxy terminal tail (ct) of the 5-HT 2A R. The association of ARF1 with the ct domain of the receptor was stronger than its interaction with i3, or the interactions of ARF6 with either construct. Experiments using ARF mutants that are deficient in GTP loading, and the in vitro addition of GTP␥S suggested that GTP loading enhances ARF1 binding to the receptor. The N376PxxY motif in the transmembrane 7 domain of the receptor (rather than a N376DPxxY mutant form) was shown to be essential for ARF-dependent PLD signaling and ARF1 coimmunoprecipitation. In GST-fusion proteins of the 5-HT 2A R ct domain, mutation of Asn376 to Asp also markedly reduced ARF1-HA binding, although additional motifs in the Asn376 -Asn384 sequence and to a lesser extent elsewhere, seem also to contribute to the interaction.

Role of Gq protein in behavioral effects of the hallucinogenic drug 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane

“…5-HT 2A receptors are known to couple to multiple G-proteins including G q , G 11 , and G 13 to mediate a wide array of second messenger signaling pathways (Berg et al, 1998;KurraschOrbaugh et al, 2003;Robertson et al, 2003); however, the classical pathway associated with 5-HT 2A receptor signaling is stimulation of PLCβ;via Gα q protein. In the present study, we evaluated the role of the Gα q protein in several behavioral and biochemical assays, including the elevated plus maze, head-twitch response, 5-HT 2A receptor binding, and c-fos immunohistochemistry.…”

Cytohesin-1 Regulates the Arf6-Phospholipase D Signaling Axis in Human Neutrophils: Impact on Superoxide Anion Production and Secretion

“…There are several reports of G protein-coupled receptor activation of Arf6 because of the ability of GTP-bound Arf1 or Arf6 to directly bind to the activated receptors (64,65) or to interact with activated Gq and ARNO (66). Arf6 has also been shown to stimulate PLD through activation of fMLF receptors in differentiated PLB-985 cells (4), whereas the addition of myristoylated Arf1 or Arf6 and GTPgS in permeabilized HL60 cells stimulates PLD activity (67).…”

Overexpression of ARF1 is associated with cell proliferation and migration through PI3K signal pathway in ovarian cancer

“…Several studies have suggested that ARF1 can also localize to the plasma membrane transmitting signals from transmembrane proteins (12,13). In addition to their conventional roles of vesicle formation, ARF1 plays important roles in a wide range of physiological and pathophysiological processes in tumor formation, including inter-and intra cellular signaling, transcription, DNA repair pathways, cell cycle regulation, and mitosis, as well as necrosis and apoptosis (14).…”