Role of the conserved NPxxY motif of the 5-HT2A receptor in determining selective interaction with isoforms of ADP-ribosylation factor (ARF).
In this study we have shown that N376 to D mutation in the conserved NPxxY motif within the carboxy terminal tail domain (CT) of the 5-HT2A receptor alters the binding preference of GST-fusion protein constructs of the CT domain from ARF1 to an alternative isoform, ARF6. These findings were corroborated by experiments investigating co-immunoprecipitation of the wild type (WT) and N376D mutant of the 5-HT2A receptor with ARF1 or 6 or dominant negative ARF1/6 constructs co-expressed in COS7 cells. In functional assays of 5-HT-induced phospholipase D (PLD) activation responses of the WT receptor were inhibited by a dominant negative mutant of ARF1 but not ARF6, whereas responses of the N376D mutant were strongly inhibited by negative mutant ARF6. No equivalent effect of the ARF mutants was seen on phospholipase C activation. In experiments assaying 5-HT-induced increases in [35S]GTPgammaS binding to ARF 1/6 immunoprecipitates as a measure of ARF activation, increased ARF6 activation was seen only with the mutant receptor. When cellular PLD responses of other NPxxY- or a DPxxY-containing GPCRs were measured in the presence of dominant negative ARF1/6 constructs, the majority, but not all, fitted the pattern exemplified by the 5-HT2A receptor and its N376D mutant. These data suggest that the presence of the N or a D in this highly conserved motif is an important, but not exclusive, determinant of which ARF isoform interacts with the GPCR.
Membrane Biology Interdisciplinary Research Group, Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, Scotland, EH8 9XD, UK.
Melanie S
MS
Johnson
Derek N
DN
Robertson
Pamela J
PJ
Holland
Eve M
EM
Lutz
Rory
R
Mitchell
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D006801:Humans; D047468:Immunoprecipitation; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D044402:Receptor, Serotonin, 5-HT2A / Q000737:chemistry* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D000089982:ADP-Ribosylation Factor 6; D050505:Mutant Proteins; D020033:Protein Isoforms; D044402:Receptor, Serotonin, 5-HT2A; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D010739:Phospholipase D; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human