Short term desensitization of the A(1) adenosine receptors in DDT1MF-2 cells

Previous studies have indicated that desensitization of the A(1) adenosine receptor (A(1)AR), unlike other adenosine receptor subtypes and G protein-coupled receptors, required prolonged exposure to agonists. We more closely studied this observation by focusing on changes in the A(1)AR signal transduction pathway after short term agonist exposure (0.5-4 hr) in the hamster vas deferens smooth muscle cell line (DDT1MF-2 cells). Incubation of these cells with 1 mu M (R)-phenylisopropyladenosine [(R)-PIA] produced a time-dependent loss in binding of the agonist radioligand [I-125]N-6-2-(4-amino-3-iodophenyl)ethyladenosine but not of the antagonist radioligand [H-3]8-cyclopentyl-1,3-dipropylxanthine. This was accompanied by a reduction in the high affinity (G protein-coupled) state of this receptor from 63 +/- 8% to 37 +/- 12% after treatment for 4 hr. Moreover, cells treated with (R)-PIA demonstrated reduced agonist-stimulated GTPase activity and diminished inhibition of adenylyl cyclase activity but no change in expression of alpha(i) and beta subunits. The decreases in agonist binding in the desensitized cells were reversible after treatment of DDT1MF-2 cell membranes with alkaline phosphatase or protein phosphatases 1 and 2A, suggesting a role of phosphorylation in the uncoupling and desensitization of the A(1)AR. Incubation of cells with (R)-PIA led to rapid translocation of G protein-coupled receptor kinase (GRK) from the cytosol to the plasma membrane within 1 hr of exposure. In addition, purified preparations of the A(1)AR that were phosphorylated with purified recombinant GRK-2 demonstrated enhanced affinity for arrestin over G(i)/G(o). These results indicate rapid and functional desensitization of the A(1)AR by brief exposure to agonist. The mechanism underlying this event seems to involve phosphorylation of the A(1)AR, presumably by the GRK or GRKs.