Cross-regulation between G-protein-mediated pathways: Acute activation of the inhibitory pathway of adenylylcyclase reduces β₂-adrenergic receptor phosphorylation and increases β-adrenergic responsiveness

Cross-regulation from the stimulatory to the inhibitory adenylylcyclase pathways has been described (Hadcock, J. R., Ros, M., Watkins, D. C., and Malbon, C. C. (1990) J. Biol. Chem. 265, 14784-14790). More recently, persistent activation (48 h) of the inhibitory adenylylcyclase pathway has been shown to cross-regulate the stimulatory pathway (i) enhancing the maximal response of β-adrenergic agonists, (ii) increasing the expression of β-adrenergic receptor, and (iii) reducing the ED₅₀ for the isoproterenol-stimulated response by 50-fold (Hadcock, J. R., Port, J. D., and Malbon, C. C. (1991) J. Biol. Chem. 266, 11915-11922). Here, we report that short term activation (60 min) of the inhibitory adenylylcyclase pathway of hamster smooth muscle DDT₁MF-2 cells with the A1-adenosine receptor agonist N⁶-phenylisopropyladenosine (PIA) likewise enhances the stimulatory adenylylcyclase response to the β-adrenergic agonist isoproterenol. The PIA effect was exerted at the level of the receptor, i.e. the β-adrenergic receptor-mediated response was enhanced, whereas the guanosine 5′-O-(thiotriphosphate)- and forskolin-stimulated adenylyl-cyclase activities were largely unaffected. In contrast to longer term persistent activation of the inhibitory pathway, receptor number and affinity for ¹²⁵I-labeled cyanopindolol were unaffected. Metabolic labeling of cells with [³²P]orthophosphate and immuneprecipitation of β-adrenergic receptors detected phosphorylation of the receptor in unstimulated cells and marked phosphorylation in cells challenged with epinephrine. When cells were challenged short term with PIA, the basal state of β-adrenergic receptor phosphorylation was reduced by 75%. Treating cells with PIA in combination with the cAMP analog 8-(4-chlorophenyl-thio)adenosine cyclic AMP attenuated the enhanced receptor-mediated adenylylcyclase response observed in cells treated with PIA alone. These data suggest that short term cross-regulation from the inhibitory to stimulatory adenylylcyclase pathways results in the following: (i) decreased intracellular cAMP levels and protein kinase A activity, (ii) reduced phosphorylation of the β₂-adrenergic receptor in the "basal" (i.e. unstimulated) state, and (iii) enhanced receptor-mediated activation of G_s.