The active conformation of beta-arrestin1: direct evidence for the phosphate sensor in the N-domain and conformational differences in the active states of beta-arrestins1 and -2

J Biol Chem. 2007 Jul 20;282(29):21370-81. doi: 10.1074/jbc.M611483200. Epub 2007 May 18.

Abstract

beta-Arrestins are multifunctional adaptor proteins that regulate seven transmembrane-spanning receptor (7TMR) desensitization and internalization and also initiate alternative signaling pathways. Studies have shown that beta-arrestins undergo a conformational change upon interaction with agonist-occupied, phosphorylated 7TMRs. Although conformational changes have been reported for visual arrestin and beta-arrestin2, these studies are not representative of conformational changes in beta-arrestin1. Accordingly, in this study, we determine conformational changes in beta-arrestin1 using limited tryptic proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis in the presence of a phosphopeptide derived from the C terminus of the V(2) vasopressin receptor (V(2)Rpp) or the corresponding unphosphorylated peptide (V(2)Rnp). V(2)Rpp binds specifically to beta-arrestin1 causing significant conformational changes, whereas V(2)Rnp does not alter the conformation of beta-arrestin1. Upon V(2)Rpp binding, we show that the previously shielded Arg(393) becomes accessible, which indicates release of the C terminus. Moreover, we show that Arg(285) becomes more accessible, and this residue is located in a region of beta-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1, and we also show a functional consequence of the release of the C terminus of beta-arrestin1 by enhanced clathrin binding. In addition, we show marked protection of the N-domain of beta-arrestin1 in the presence of V(2)Rpp, which is consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. A striking difference in conformational changes is observed in beta-arrestin1 when compared with beta-arrestin2, namely the flexibility of the interdomain hinge region. This study represents the first direct evidence that the "receptor-bound" conformations of beta-arrestins1 and 2 are different.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Arrestins / chemistry*
  • Arrestins / metabolism*
  • Cattle
  • Clathrin / chemistry
  • Lysine / chemistry
  • Molecular Conformation
  • Molecular Sequence Data
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Rats
  • Recombinant Proteins / chemistry
  • beta-Arrestins

Substances

  • Arrestins
  • Clathrin
  • Recombinant Proteins
  • beta-Arrestins
  • Lysine