Regulated interactions of the α2A adrenergic receptor with spinophilin, 14-3-3ζ, and arrestin 3

Qin Wang, Lee E. Limbird

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

Abstract

The present studies demonstrate that no single stretch of sequence in the third intracellular (3i) loop of the α2A adrenergic receptor (α2A-AR) can fully account for its previously described interactions with spinophilin (Richman, J. G., Brady, A. E., Wang, Q., Hensel, J. L., Colbran, R. J., and Limbird, L. E. (2001) J. Biol. Chem. 276, 15003-15008), 14-3-3ζ (Prezeau, L., Richman, J. G., Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 13462-13469), and arrestin 3 (Wu, G., Krupnick, J. G., Benovic, J. L., and Lanier, S. M. (1997) J. Biol. Chem. 272, 17836-17842), suggesting that a three-dimensional surface, rather than a linear sequence, provides the basis for these interactions as proposed for 3i loop tethering of the α2A-AR to the basolateral surface of Madin-Darby canine kidney cells (Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 16331-16336). Sequences at the extreme N-terminal and C-terminal ends of the 3i loop are critical for interaction with spinophilin but not for interaction with 14-3-3ζ or arrestin 3, for which the C-terminal half of the loop is more important. Competition binding for 35S-labeled α2A-AR 3i loop binding to glutathione S-transferase (GST)-spinophilin amino acids 151-444 revealed a relative affinity of spinophilin ≅ arrestin > 14-3-3ζ for the unphosphorylated α2A-AR 3i loop. Agonist occupancy of the α2A-AR increases receptor association with spinophilin, and arrestin 3 appears to compete for this enrichment. However, when the G protein-coupled receptor kinase 2 substrate sequence was deleted from the 3i loop, arrestin 3 could not compete for the agonist-enriched binding of spinophilin to the mutant α2A-AR. These data are consistent with a model where sequential or competitive interactions among spinophilin, arrestin, and/or 14-3-3ζ play a role in α2A-AR functions.

Original languageEnglish (US)
Pages (from-to)50589-50596
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number52
DOIs
StatePublished - Dec 27 2002
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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