Arrestin isoforms dictate differential kinetics of A2B adenosine receptor trafficking

Biochemistry. 2000 Oct 24;39(42):12828-36. doi: 10.1021/bi0010928.

Abstract

Adenosine mediates the activation of adenylyl cyclase via its interaction with specific A(2A) and A(2B) adenosine receptors. Previously, we demonstrated that arrestins are involved in rapid agonist-promoted desensitization of the A(2B) adenosine receptor (A(2B)AR) in HEK293 cells. In the present study, we investigate the role of arrestins in A(2B)AR trafficking. Initial studies demonstrated that HEK293 cells stably expressing arrestin antisense constructs, which reduce endogenous arrestin levels, effectively reduced A(2B)AR internalization. A(2B)AR recycling after agonist-induced endocytosis was also significantly impaired in cells with reduced arrestin levels. Interestingly, while overexpression of arrestin-2 or arrestin-3 rescued A(2B)AR internalization and recycling, arrestin-3 promoted a significantly faster rate of recycling as compared to arrestin-2. The specificity of arrestin interaction with A(2B)ARs was further investigated using arrestins fused to the green fluorescent protein (arr-2-GFP and arr-3-GFP). Both arrestins underwent rapid translocation (<1 min) from the cytosol to the plasma membrane following A(2B)AR activation. However, longer incubations with agonist (>10 min) revealed that arr-2-GFP but not arr-3-GFP colocalized with the A(2B)AR in rab-5 and transferrin receptor containing early endosomes. At later times, the A(2B)AR but not arr-2-GFP was observed in an apparent endocytic recycling compartment. Thus, while arrestin-2 and arrestin-3 mediate agonist-induced A(2B)AR internalization with relative equal potency, arrestin isoform binding dictates the differential kinetics of A(2B)AR recycling and resensitization.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine-5'-(N-ethylcarboxamide) / agonists
  • Animals
  • Arrestin / genetics
  • Arrestin / physiology*
  • Cell Line
  • Genetic Vectors / metabolism
  • Green Fluorescent Proteins
  • Humans
  • Kinetics
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Protein Isoforms / genetics
  • Protein Isoforms / physiology
  • Purinergic P1 Receptor Agonists
  • Rats
  • Receptor, Adenosine A2B
  • Receptors, Purinergic P1 / metabolism*
  • Transfection
  • rab GTP-Binding Proteins / genetics
  • rab GTP-Binding Proteins / metabolism

Substances

  • Arrestin
  • Luminescent Proteins
  • Protein Isoforms
  • Purinergic P1 Receptor Agonists
  • Receptor, Adenosine A2B
  • Receptors, Purinergic P1
  • Green Fluorescent Proteins
  • Adenosine-5'-(N-ethylcarboxamide)
  • rab GTP-Binding Proteins