Phosphorylation of the ␤2 adrenoreceptor (␤2AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (Gs) to inhibitory guanine nucleotide regulatory protein (G i). ␤-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the ␤2AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the ␤2AR. In human embryonic kidney 293 cells overexpressing a recombinant ␤2AR, stimulation with isoprenaline recruits ␤-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the ␤2AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the ␤2AR. Rolipram and PDE4D5-D556A also enhance ␤2AR-mediated activation of extracellular signal-regulated kinases ERK1͞2. This is consistent with a switch in coupling of the receptor from G s to Gi, because the ERK1͞2 activation is sensitive to both inhibitors of PKA (H89) and G i (pertussis toxin). In cardiac myocytes, the ␤2AR also switches from G s to Gi coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK1͞2. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK1͞2 activation. Thus, receptor-stimulated ␤-arrestinmediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the ␤2AR in a physiological system, the cardiac myocyte.

A ntidiuretic hormone (arginine vasopressin [AVP]) induces fusion of vesicles that contain the water channel aquaporin-2 (AQP2) with the plasma membrane of renal collecting duct principal cells. This "AQP2 shuttle" increases the osmotic water permeability (Pf) of the cells, facilitating water reabsorption from the collecting duct (1). The AQP2 shuttle is initiated upon binding of AVP to vasopressin-2 receptors (V2R) and triggered by the consequent cAMP elevation and protein kinase A (PKA) activation. It is the PKA phosphorylation of AQP2 that elicits redistribution of AQP2-bearing vesicles. Pivotal to this redistribution is the compartmentalization of PKA by A kinase anchoring proteins (AKAP) (2). Phosphodiesterases (PDE), which are the sole means of degrading cAMP, are poised to regulate PKA signaling (3-6). The PDE4 family has attracted great interest because of its link to stroke (7), schizophrenia (8), and the therapeutic potential of selective inhibitors for treating inflammatory diseases (9-12). The four subfamilies (PDE4A through D) are encoded by separate genes, generating approximately 20 isoforms (9,11) that can interact with scaffolding proteins, including AKAP and ␤-arrestin (12-16), positioning them for a role in compartmentalized cAMP/PKA signaling.Here we show that compartmentalization of cAMP/PKA signaling by PDE4 is involved in the regulation of the AQP2 shuttle and the Pf. This is of particular pertinence because PDE4 hyperactivity causes nephrogenic diabetes insipidus in a mouse model (17).