Agonist-stimulated desensitization of the ␤ 2 -adrenergic receptor (␤ 2 AR) is caused by both a potent cAMP-dependent protein kinase (PKA)-mediated phosphorylation and a less potent, occupancy-dependent, G protein-coupled receptor kinase (GRK)-mediated phosphorylation that leads to ␤-arrestin binding and internalization. In this study the kinetics of phosphorylation of the third intracellular loop PKA site Ser262 and the putative C-tail GRK sites Ser355, Ser356 of the human ␤ 2 AR overexpressed in human embryonic kidney (HEK) 293 cells were characterized using phosphoserine-specific antibodies. Specificity of the antibodies was shown by their lack of reactivity with mutant ␤ 2 ARs lacking the respective sites. In addition, overexpression of GRK2 and GRK5 increased basal levels of phosphorylation of the GRK sites Ser355, Ser356 in both COS-7 and HEK 293 cells. Epinephrine, prostaglandin E 1 , and forskolin at maximum concentrations stimulated phosphorylation of the ␤ 2 AR PKA site (Ser262) by 4-fold, whereas PMA stimulated it by 2-fold. Epinephrine stimulated PKA site phosphorylation with an EC 50 of 20 to 40 pM. In contrast, epinephrine stimulated GRK site phosphorylation (Ser355,Ser356) with an EC 50 of 200 nM (1-min treatments), which is more than 4000-fold higher relative to PKA site phosphorylation, consistent with an occupancy-driven process. After 10 to 30 min, the EC 50 for epinephrine stimulation of GRK site phosphorylation was reduced to 10 to 20 nM but was still Ϸ200-fold greater than for the PKA site. The EC 50 for internalization correlated with GRK site phosphorylation and showed a similar shift with time of epinephrine stimulation. The kinetics of epinephrine-stimulated GRK site phosphorylation were not altered in a mutant of the ␤ 2 AR lacking the PKA consensus sites. The initial levels (2 min) of a range of agoniststimulated GRK site phosphorylations were correlated with their efficacy for activation of adenylyl cyclase, namely epinephrine Ն formoterol ϭ fenoterol Ͼ terbutaline ϭ zinterol ϭ albuterol Ͼ salmeterol Ͼ Ͼ dobutamine Ն ephedrine. However, after 20 to 30 min of treatment, agonists with intermediate strengths, such as albuterol and salmeterol, stimulate GRK site phosphorylations that are approximately equal to that produced by epinephrine, and the correlation breaks down. The GRK and PKA site antibodies were also effective in detecting phosphorylation of the endogenous ␤ 2 AR expressed in A431 human epidermoid carcinoma cells. To summarize, our results show a remarkable amplification of PKA site phosphorylation relative to the putative GRK site phosphorylation, heterologous stimulation of the PKA site phosphorylation, no dependence of GRK site phosphorylation on PKA sites, and a reasonable correlation of initial levels of GRK site phosphorylation with the strength of a range of agonists.The ␤ 2 -adrenergic receptor (␤ 2 AR) plays significant roles in relaying signals from the autonomic sympathetic nervous system to the cardiovascular and pulmonary systems in particular. There are...

Spatiotemporal specificity of cAMP action is best explained by targeting protein kinase A (PKA) to its substrates by A-kinase-anchoring proteins (AKAPs). At synapses in the brain, AKAP79/150 incorporates PKA and other regulatory enzymes into signal transduction networks that include ␤-adrenergic receptors, ␣-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), and N-methyl-D-aspartic acid receptors. We previously showed that AKAP79/150 clusters PKA with type 5 adenylyl cyclase (AC5) to assemble a negative feedback loop in which the anchored kinase phosphorylates AC5 to dynamically suppress cAMP synthesis. We now show that AKAP79 can associate with multiple AC isoforms. The N-terminal regions of AC5, -6, and -9 mediate this protein-protein interaction. Localized activation of PKA 2 triggers a plethora of intracellular signaling processes (1). Precise control of these phosphorylation events is often achieved by restricted activation of PKA in discrete microenvironments. AKAPs participate in this process by tethering the kinase close to preferred substrates. AKAPs now represent a family of 43 diverse but functionally related proteins that bind the regulatory subunit dimer of the PKA holoenzyme (2).AKAPs have been identified in a range of species, tissues, and cellular compartments. The AKAP79/150 group of anchoring proteins is perhaps the best understood member of this class of signal-organizing proteins. AKAP79/150 consists of three orthologs: bovine AKAP75, human AKAP79, and murine AKAP150. Although originally identified in the postsynaptic densities of neurons, this group of anchoring proteins is also expressed in a variety of other tissues. In addition to binding PKA, AKAP79 has the ability to bind protein phosphatase 2B (3) and protein kinase C (PKC) (4, 5). By organizing these signal transduction and signal termination enzymes in the same location, AKAP79 provides a platform to facilitate the bidirectional control of cAMP-and calcium-mediated signaling events.Although anchoring of PKA with its substrates provides an efficient mechanism for the spatial regulation necessary for selectivity of cAMP signaling, it was not clear how local pools of cAMP are managed. We have shown that AC isoforms can specifically interact with three different AKAP complexes, AKAP79, Yotiao, and mAKAP, to regulate events downstream of cAMP production (6 -8). We have also demonstrated that anchoring of AC5 to an AKAP79/150 complex provides negative feedback on AC5 via PKA phosphorylation of AC5 within the complex (6).Although characterization of the AKAP79-AC5 interaction has shed some light on the advantages gained by localizing different components of cAMP signaling pathways, several key issues remain unresolved. First of all, do other AC isoforms interact with AKAP79 or other anchoring proteins? Secondly, are AC isoforms recruited into larger signaling networks via their protein-protein interactions with AKAP79? AKAP79/150 has been shown to form a multiprotein signaling complex with AMPA and NMDA receptors (9 -11), adhesion mol...

Background: WT non-visual arrestins are promiscuous, binding numerous GPCRs. Results:Mutations of very few receptor discriminator residues greatly increase receptor specificity of arrestin-3. Conclusion: Targeted manipulation of key residues that determine receptor preference is a viable approach to the construction of arrestins with high specificity for particular GPCR subtypes. Significance: Non-visual arrestins with high receptor specificity make therapeutic use of signaling-biased arrestin mutants feasible.

cAMP signaling plays a key role in regulating pain sensitivity. Here, we uncover a previously unidentified molecular mechanism in which direct phosphorylation of the exchange protein directly activated by cAMP 1 (EPAC1) by G protein kinase 2 (GRK2) suppresses Epac1-to-Rap1 signaling, thereby inhibiting persistent inflammatory pain. Epac1 −/− mice are protected against inflammatory hyperalgesia in the complete Freund's adjuvant (CFA) model. Moreover, the Epac-specific inhibitor ESI-09 inhibits established CFA-induced mechanical hyperalgesia without affecting normal mechanical sensitivity. At the mechanistic level, CFA increased activity of the Epac target Rap1 in dorsal root ganglia of WT, but not of Epac1 −/− , mice. Using sensory neuronspecific overexpression of GRK2 or its kinase-dead mutant in vivo, we demonstrate that GRK2 inhibits CFA-induced hyperalgesia in a kinase activity-dependent manner. In vitro, GRK2 inhibits Epac1-to-Rap1 signaling by phosphorylation of Epac1 at Ser-108 in the Disheveled/Egl-10/pleckstrin domain. This phosphorylation event inhibits agonist-induced translocation of Epac1 to the plasma membrane, thereby reducing Rap1 activation. Finally, we show that GRK2 inhibits Epac1-mediated sensitization of the mechanosensor Piezo2 and that Piezo2 contributes to inflammatory mechanical hyperalgesia. Collectively, these findings identify a key role of Epac1 in chronic inflammatory pain and a molecular mechanism for controlling Epac1 activity and chronic pain through phosphorylation of Epac1 at Ser-108. Importantly, using the Epac inhibitor ESI-09, we validate Epac1 as a potential therapeutic target for chronic pain.

Background: Arrestin-1 selectively binds light-activated phosphorhodopsin. Results: Deletions in the 139-loop or disruptions of its interactions with the body of arrestin-1 greatly reduce arrestin-1 stability and selectivity. Conclusion: The central 139-loop supports basal arrestin-1 conformation and reduces its binding to non-preferred forms of rhodopsin. Significance: The central 139-loop is an earlier unappreciated element contributing to the thermal stability and binding selectivity of arrestins.