Role of the conserved NPxxY motif of the 5-HT2A receptor in determining selective interaction with isoforms of ADP-ribosylation factor (ARF)

Melanie S Johnson, Derek N Robertson, Pamela J Holland, Eve M Lutz, Rory Mitchell

Research output: Contribution to journalArticlepeer-review

Abstract

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.
Original languageEnglish
Pages (from-to)1793-800
Number of pages8
JournalCellular Signalling
Volume18
Issue number10
DOIs
Publication statusPublished - Oct 2006

Keywords / Materials (for Non-textual outputs)

  • ADP-Ribosylation Factor 1
  • ADP-Ribosylation Factors
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Cercopithecus aethiops
  • Conserved Sequence
  • Humans
  • Immunoprecipitation
  • Mutant Proteins
  • Phospholipase D
  • Protein Binding
  • Protein Isoforms
  • Receptor, Serotonin, 5-HT2A
  • Receptors, G-Protein-Coupled
  • Recombinant Fusion Proteins
  • Substrate Specificity
  • Time Factors

Fingerprint

Dive into the research topics of 'Role of the conserved NPxxY motif of the 5-HT2A receptor in determining selective interaction with isoforms of ADP-ribosylation factor (ARF)'. Together they form a unique fingerprint.

Cite this