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

MRC Membrane and Adapter Proteins Cooperative Group, Membrane Biology Interdisciplinary Research Group, University of Edinburgh, School of Biomedical and Clinical Laboratory Sciences, Hugh Robson Building, George Square, Edinburgh, Scotland, United Kingdom (D.N.R., M.S.J., L.O.M., P.J.H., R.M.); Department of Bioscience & Biotechnology, University of Strathclyde, Glasgow, Scotland, United Kingdom (E.M.L.)

The 5-hydroxytryptamine 2A receptor (5-HT_2AR) 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_2AR-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_2AR. 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 GTPS 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_2AR 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.

Address correspondence to: Rory Mitchell, MRC Membrane and Adapter Proteins Co-operative Group, Membrane Biology Interdisciplinary Research Group, University of Edinburgh, School of Biomedical and Clinical Laboratory Sciences, Hugh Robson Building, George Square, Edinburgh, Scotland, EH8 9XD, UK. E-mail: rory.mitchell{at}ed.ac.uk

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