Abstract
Adenosine activates four different receptors, the A1, A2A, A2B, and the A3 receptors, all of which are G protein-coupled. We have previously shown that stimulation of the human adenosine A3receptor can induce phosphorylation of extracellular signal-regulated kinase (ERK1/2). Here we show that the adenosine receptor agonist 5′N-ethylcarboxamidoadenosine (NECA) induces phosphorylation and activation of ERK1/2 in Chinese hamster ovary (CHO) cells expressing the human adenosine A3 receptor (CHO A3 cells) with the same potency. Pretreatment with pertussis toxin abolished the effect, which also could be blunted by overexpressing the βγ-sequestering peptide β-adrenergic receptor kinase-ct, implicating the involvement of βγ subunits released from Gi/o proteins. Activation of phosphatidylinositol-3-kinase (PI3K) by adenosine A3 receptors is inferred from a dose-dependent Ser-phosphorylation of the protein kinase B (Akt). Furthermore the ERK1/2 phosphorylation was sensitive to the PI3K inhibitors wortmannin and LY294002 (2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride) and the MEK inhibitor PD98059 (2′-amino-3′-methoxyflavone), whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) and long-term treatment with phorboldibutyrate did not decrease the adenosine A3receptor-mediated ERK1/2 phosphorylation. Thus, Ca2+mobilization and conventional and novel protein kinase C (PKC) isoforms are not involved in this pathway. The atypical PKCζ was not activated by NECA and thus not involved in the A3receptor-mediated ERK1/2 phosphorylation. NECA stimulation of CHO A3 cells activated the small G protein Ras and the dominant negative mutant RasS17N prevented the phosphorylation of ERK1/2. In conclusion, the adenosine A3 receptor recruits a pathway that involves βγ release from Gi/o, PI3K, Ras, and MEK to induce ERK1/2 phosphorylation and activation, whereas signaling is independent of Ca2+, PKC, and c-Src.
- The American Society for Pharmacology and Experimental Therapeutics
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