Differential interaction with and regulation of multiple G-proteins by the rat A3 adenosine receptor

Interaction of the rat A3 adenosine receptor (A3AR) with G-proteins has been assessed using a stably transfected Chinese hamster ovary cell system. The non-selective AR agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased the labeling of a 41-kDa membrane protein by 4-azidoanilido-[alpha-32P]guanosine 5'-triphosphate (AA-[32P]GTP), a photolabile GTP analogue. Subsequent immunoprecipitation of Gi alpha-subunits indicated that NECA stimulated incorporation of label into both Gi alpha-2 and Gi alpha-3. Additional experiments revealed an A3AR stimulation of label into Gq and/or G11 alpha-subunits, albeit to a lesser degree than that elicited by endogenous P2U purinergic receptors. No interaction with Gs could be detected. Sustained cellular exposure to NECA induced A3AR desensitization and specific down-regulation of Gi alpha-3 and G-protein beta-subunits without changing levels of Gi alpha-2, Gs alpha, or Gq+11 alpha-subunits. Therefore the A3AR can interact with Gi alpha-2, Gi alpha-3, and, to some extent, Gq-like proteins, but sustained agonist exposure down-regulates only one of the G-proteins with which it interacts. This is the first description of the differing specificities of A3AR/G-protein coupling versus down-regulation in situ and provides a potential mechanism by which the A3AR could elicit the heterologous desensitization of signaling events mediated by Gi3.