A library of 7TM receptor C-terminal tails: Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

Arne Heydorn, Birgitte P. Søndergaard, Bjarne Ersbøll, Birgitte Holst, Finn Cilius Nielsen, Carol Renfrew Haft, Jennifer Whistler, Thue W. Schwartz

Research output: Contribution to journalArticle

131 Scopus citations

Abstract

Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and down-stream effectors and the adaptors determine post-signaling events such as receptor sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase in fusion proteins for interactions with four different adaptor proteins previously proposed to be involved in post-endocytotic sorting of receptors. Of the two proteins suggested to target receptors for recycling to the cell membrane, which is the route believed to be taken by a majority of receptors, ERM (ezrin-radixin-moesin)-binding phosphoprotein 50 (EBP50) bound only a single receptor tail, i.e. the β2-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein-coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the δ-opioid receptor, confirmed the expected nanomolar affinities for interaction with SNX1. Truncations of the NK 1 receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrum-recycling adaptor.

Original languageEnglish (US)
Pages (from-to)54291-54303
Number of pages13
JournalJournal of Biological Chemistry
Volume279
Issue number52
DOIs
StatePublished - Dec 24 2004
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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