The chronic stimulation of certain G protein-coupled receptors promotes cardiomyocyte hypertrophy and thus plays a pivotal role in the development of human heart failure. The beta-adrenergic receptors (beta-AR) are unique among these in that they signal via Gs, whereas others, such as the alpha1-adrenergic (alpha1-AR) and endothelin-1 (ET-1) receptors, predominantly act through Gq. In this study, we investigated the potential role of regulator of G protein signalling 2 (RGS2) in modulating the hypertrophic effects of the beta-AR agonist isoproterenol (ISO) in rat neonatal ventricular cardiomyocytes. We found that ISO-induced hypertrophy in rat neonatal ventricular myocytes was accompanied by the selective upregulation of RGS2 mRNA, with little or no change in RGS1, RGS3, RGS4 or RGS5. The adenylyl cyclase activator forskolin had a similar effect suggesting that it was mediated through cAMP production. To study the role of RGS2 upregulation in beta-AR-dependent hypertrophy, cardiomyocytes were infected with adenovirus encoding RGS2 and assayed for cell growth, markers of hypertrophy, and beta-AR signalling. ISO-induced increases in cell surface area were virtually eliminated by the overexpression of RGS2, as were increases in alpha-skeletal actin and atrial natriuretic peptide. RGS2 overexpression also significantly attenuated ISO-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt activation, which may account for, or contribute to, its observed antihypertrophic effects. In contrast, RGS2 overexpression significantly activated JNK MAP kinase, while decreasing the potency but not the maximal effect of ISO on cAMP accumulation. In conclusion, the present results suggest that RGS2 negatively regulates hypertrophy induced by beta-AR activation and thus may play a protective role in cardiac hypertrophy.

To investigate whether G protein-coupled receptor kinases (GRKs) are involved in the regulation of the PTH/PTHrPR, we have established mutant SaOS-2 cells which stably overexpress (> 10-20-fold) a dominant negative form of the beta-adrenergic receptor kinase-1 (beta ARK-1). Acute (< or = 2 h) incubation with hPTH (1-34) induced significantly less (by up to 50%) downregulation of the PTH/PTHrPR in beta ARK-1 mutant SaOS-2 cells than observed in wild-type cells. Pretreatment of wild-type cells with PTH for 2 h induced homologous cAMP desensitisation to a second challenge with PTH, while the effect was blunted by up to 60% in beta ARK-1 mutant cells. We conclude that activation of beta ARK-1 (or a closely related GRK) is a critical component of the acute phase (< or = 2 h) of PTH-induced receptor downregulation and homologous cAMP desensitisation of the PTH/PTHrPR.

Activation of receptor tyrosine kinases (RTKs) is associated with carcinogenesis, but its contribution to smoking-associated lung carcinogenesis is poorly understood. Here we show that a tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced insulin-like growth factor 1 receptor (IGF-1R) activation via β-adrenergic receptor (β-AR) is crucial for smoking-associated lung carcinogenesis. Treatment with NNK stimulated the IGF-1R signaling pathway in a time- and dose-dependent manner, which was suppressed by pharmacological or genomic blockade of β-AR and the downstream signaling including a Gβγ subunit of β-AR and phospholipase C (PLC). Consistently, β-AR agonists led to increased IGF-1R phosphorylation. The increase in IGF2 transcription via β-AR, signal transducer and activator of transcription 3 (STAT3), and nuclear factor-kappa B (NF-κB) was associated with NNK-induced IGF-1R activation. Finally, treatment with β-AR antagonists suppressed the acquisition of transformed phenotypes in lung epithelial cells and lung tumor formation in mice. These results suggest that blocking β-AR-mediated IGF-1R activation can be an effective strategy for lung cancer prevention in smokers.

G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades.

Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.

The G-protein coupled receptor kinase 2 (GRK2) regulates the desensitization of beta-adrenergic receptors (β-AR), and its overexpression has been implicated in heart failure. Hence, the inhibition of GRK2 is considered to be an important drug target for the treatment of heart failure. Due to the high sequence similarity of GRK2 with the A, G, and C family (AGC family) of kinases, the inhibition of GRK2 also leads to the inhibition of AGC kinases such as Rho-associated coiled-coil kinase 1 (ROCK1). Therefore, unraveling the mechanisms to selectively inhibit GRK2 poses an important challenge. We have performed molecular docking, three dimensional quantitative structure activity relationship (3D-QSAR), molecular dynamics (MD) simulation, and free energy calculations techniques on a series of 53 paroxetine-like compounds to understand the structural properties desirable for enhancing the inhibitory activity for GRK2 with selectivity over ROCK1. The formation of stable hydrogen bond interactions with the residues Phe202 and Lys220 of GRK2 seems to be important for selective inhibition of GRK2. Electropositive substituents at the piperidine ring and electronegative substituents near the amide linker between the benzene ring and pyrazole ring showed a higher inhibitory preference for GRK2 over ROCK1. This study may be used in designing more potent and selective GRK2 inhibitors for therapeutic intervention of heart failure.

Activation of myosin light chain kinase (MLCK) and other kinases was studied in the arteries of transgenic mice that express an optical fluorescence resonance energy transfer (FRET) MLCK activity biosensor. Binding of Ca(2+)/calmodulin (Ca(2+)/CaM) induces an increase in MLCK activity and a change in FRET. After exposure to high external [K(+)], intracellular [Ca(2+)] (fura-2 ratio or fluo-4 fluorescence) and MLCK activity both increased rapidly to an initial peak and then declined, rapidly at first and then very slowly. After an initial peak ('phasic') force was constant or increased slowly (termed 'tonic' force). Inhibition of rho-kinase (Y-27632) decreased tonic force more than phasic, but had little effect on [Ca(2+)] and MLCK activation. Inhibition of PKCalpha and PKCbeta with Gö6976 had no effect. KN-93, an inhibitor of CaMK II, markedly reduced force, MLCK FRET and [Ca(2+)]. Applied during tonic force, forskolin caused a rapid decrease in MLCK FRET ratio and force, but no change in Ca(2+), suggesting a cAMP mediated decrease in affinity of MLCK for Ca(2+)/CaM. However, receptor (beta-adrenergic) activated increases in cAMP during KCl were ineffective in causing relaxation, changes in [Ca(2+)], or MLCK FRET. At the same tonic force, MLCK FRET ratio activated by alpha(1)-adrenoceptors was approximately 60% of that activated by KCl. In conclusion, MLCK activity of arterial smooth muscle during KCl-induced contraction is determined primarily by Ca(2+)/CaM. Rho-kinase is activated, by unknown mechanisms, and increases 'Ca(2+) sensitivity' significantly. Forskolin mediated increases in cAMP, but not receptor mediated increases in cAMP cause a rapid decrease in the affinity of MLCK for Ca(2+)/CaM.

During development of disease, complex intracellular signaling pathways regulate an intricate series of events, including resistance to external toxins, the secretion of cytokines and the production of pathological phenomena. Adenosine 3',5'-cyclic monophosphate (cAMP) is a nucleotide that acts as a key second messenger in numerous signal transduction pathways. cAMP regulates various cellular functions, including cell growth and differentiation, gene transcription and protein expression. This review aimed to provide an understanding of the effects of the cAMP signaling pathway and the associated factors on disease occurrence and development by examining the information from a new perspective. These novel insights aimed to promote the development of novel therapeutic approaches and aid in the development of new drugs.