Rapid agonist-induced desensitization and internalization of the A(2B) adenosine receptor is mediated by a serine residue close to the COOH terminus

The G.-coupled rat A(2B) adenosine receptor (A(2B)-AR) was epitope-tagged a t the NH2 terminus with hemagglutinin (HA) and subjected to progressive del etions or point mutations of the COOH terminus in order to determine region s of the receptor that contribute to agonist-induced desensitization and in ternalization. When expressed stably in Chinese hamster ovary cells, a muta nt receptor in which the final 2 amino acids were deleted, the Leu(330)-sto p mutant, underwent rapid agonist-induced desensitization and internalizati on as did the wild type (WT) receptor. However, the Phe(328) and the Gln(32 5)-stop mutants were resistant to rapid agonist-induced desensitization and internalization. Co-expression of arrestin-2-green fluorescent protein (ar restin-2-GFP) with WT receptor or Leu(330)-stop mutant resulted in rapid tr anslocation of arrestin-2-GFP from cytosol to membrane upon agonist additio n. On the other hand, agonist activation of the Phe(328)-stop or Gln(325)-s top mutant did not result in translocation of arrestin-2-GFP from cytosol. A COOH terminus point mutant, S329G, was also unable to undergo rapid agoni st-induced desensitization and internalization, indicating that Ser(329) is a critical residue for these processes. A further deletion mutant (Ser(326 )- stop) unexpectedly underwent rapid agonist-induced desensitization and i nternalization. However, activation of this mutant did not promote transloc ation of arrestin-2-GFP from cytosol to membrane. In addition, whereas WT r eceptor internalization was markedly inhibited by co-expression of dominant negative mutants of arrestin-2 (arrestin-2-(319-418)), dynamin (dynamin K4 4A), or Eps-15 (E Delta 95-295), Ser(326)-stop receptor internalization was only inhibited by dominant negative mutant dynamin. Taken together these r esults indicate that Ser(329), close to the COOH terminus of the rat A2B-AR , is critical for the rapid agonist-induced desensitization and internaliza tion of the receptor. However, deletion of the COOH terminus also uncovers a motif that is able to redirect internalization of the receptor to an arre stin- and clathrin-independent pathway.