G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor–dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein–coupled receptor signaling that may represent an important therapeutic strategy.

Accumulating evidence suggests that heterotrimeric G protein activation may not require G protein subunit dissociation. Results presented here provide evidence for a subunit dissociation-independent mechanism for G protein activation by a receptor-independent activator of G protein signaling, AGS8. AGS8 is a member of the AGS group III family of AGS proteins thought to activate G protein signaling primarily through interactions with G␤␥ subunits. Results are presented demonstrating that AGS8 binds to the effector and ␣ subunit binding "hot spot" on G␤␥ yet does not interfere with G␣ subunit binding to G␤␥ or phospholipase C ␤2 activation. AGS8 stimulates activation of phospholipase C ␤2 by heterotrimeric G␣␤␥ and forms a quaternary complex with G␣ i1 , G␤ 1 ␥ 2 , and phospholipase C ␤2. AGS8 rescued phospholipase C ␤ binding and regulation by an inactive ␤ subunit with a mutation in the hot spot (␤ 1 (W99A)␥ 2 ) that normally prevents binding and activation of phospholipase C ␤2. This demonstrates that, in the presence of AGS8, the hot spot is not used for G␤␥ interactions with phospholipase C ␤2. Mutation of an alternate binding site for phospholipase C ␤2 in the amino-terminal coiled-coil region of G␤␥ prevented AGS8-dependent phospholipase C binding and activation. These data implicate a mechanism for AGS8, and potentially other G␤␥ binding proteins, for directing G␤␥ signaling through alternative effector activation sites on G␤␥ in the absence of subunit dissociation.G protein-coupled receptor signaling systems play key roles in a number of biological processes (1). When bound to specific ligands, G protein-coupled receptors facilitate exchange of GDP for GTP on the G␣ subunit, leading to conformational changes in the G␣"switch" regions (2). These changes decrease the affinity of G␣ for G␤␥ and are thought to result in subunit dissociation such that G␣ and G␤␥ subunits are free to interact with downstream effectors (3). In this model, structural elements in G␣ and G␤␥ that bind downstream effectors are masked at the interface between the G␤␥ and G␣ subunits and revealed upon subunit dissociation.Alternatively it has been argued that subunit dissociation is not a required step in the G protein activation cycle, instead, a distinct GTP-bound conformation of the ␣␤␥ complex may represent the activated form of the G protein (4). Considerable experimental evidence has accumulated in favor of this idea. Most recently, cell-based fluorescence resonance energy transfer and bioluminescence energy transfer analyses suggest that G protein heterotrimers may not dissociate upon activation but rather undergo specific conformational rearrangements (5-7). Other evidence includes: A covalently linked G␣-␤␥ fusion protein is signaling competent in yeast (8); the kinetic coupling model for G protein activation suggests that, in the presence of regulators of G protein signaling proteins, GTP hydrolysis is too rapid to allow subunit dissociation prior to effector activation (9) and the ␤2-adrenergic receptor can activate adenyly...

Summary Adenylyl cyclase type 5 knockout mice (AC5 KO) live longer and are stress resistant, similar to calorie restriction (CR). AC5 KO mice eat more, but actually weigh less and accumulate less fat compared to WT mice. CR applied to AC5 KO result in rapid decrease in body weight, metabolic deterioration and death. These data suggest that despite restricted food intake in CR, but augmented food intake in AC5 KO, the two models affect longevity and metabolism similarly. To determine shared molecular mechanisms, mRNA expression was examined genome-wide for brain, heart, skeletal muscle and liver. Significantly more genes were regulated commonly rather than oppositely in all the tissues in both models, indicating commonality between AC5 KO and CR. Gene Ontology analysis identified many significantly regulated, tissue-specific pathways shared by the two models, including sensory perception in heart and brain, muscle function in skeletal muscle, and lipid metabolism in liver. Moreover, when comparing gene expression changes in the heart under stress, the glutathione regulatory pathway was consistently upregulated in the longevity models but downregulated with stress. In addition, AC5 and CR shared changes in genes and proteins involved in the regulation of longevity and stress resistance, including Sirt1, ApoD and olfactory receptors in both young and intermediate age mice. Thus, the similarly regulated genes and pathways in AC5 KO and CR, particularly related to the metabolic phenotype, suggest a unified theory for longevity and stress resistance.

We tested whether a high fat diet (HFD) containing the inflammatory dietary fatty acid palmitate or insulin deficient diabetes altered the remodeling of atherosclerotic plaques in LDL receptor knockout (Ldlr-/-) mice. Cholesterol reduction was achieved by using a helper-dependent adenovirus (HDAd) carrying the gene for the low-density lipoprotein receptor (Ldlr; HDAd-LDLR). After injection of the HDAd-LDLR, mice consuming either HFD, which led to insulin resistance but not hyperglycemia, or low fat diet (LFD), showed regression compared to baseline. However there was no difference between the two groups in terms of atherosclerotic lesion size, or CD68+ cell and lipid content. Because of the lack of effects of these two diets, we then tested whether viral-mediated cholesterol reduction would lead to defective regression in mice with greater hyperglycemia. In both normoglycemic and streptozotocin (STZ)-treated hyperglycemic mice, HDAd-LDLR significantly reduced plasma cholesterol levels, decreased atherosclerotic lesion size, reduced macrophage area and lipid content, and increased collagen content of plaque in the aortic sinus. However, reductions in anti-inflammatory and ER stress-related genes were less pronounced in STZ-diabetic mice compared to non-diabetic mice. In conclusion, HDAd-mediated Ldlr gene therapy is an effective and simple method to induce atherosclerosis regression in Ldlr-/- mice in different metabolic states.

Adenylyl cyclase type 5 knockout (AC5KO) mice have increased longevity and share a similar phenotype with calorie-restricted wild-type (WT) mice. To determine the in vivo metabolic properties of AC5 deficiency, we compared the effects of standard diet (SD) and high-fat diet (HFD) on obesity, energy balance, glucose regulation, and insulin sensitivity. AC5KO mice on SD had reduced body weight and adiposity compared with WT mice. Blood cholesterol and triglyceride levels were also significantly reduced in AC5KO mice. Indirect calorimetry demonstrated increased oxygen consumption, respiratory exchange ratio, and energy expenditure in AC5KO compared with WT mice on both SD and HFD. AC5KO mice also displayed improved glucose tolerance and increased whole-body insulin sensitivity, accompanied by decreased liver glycogen stores. Euglycemic-hyperinsulinemic clamp studies confirmed the marked improvement of glucose homeostasis and insulin sensitivity in AC5KO mice primarily through increased insulin sensitivity in skeletal muscle. Moreover, the genes involved in mitochondrial biogenesis and function were significantly increased in AC5KO skeletal muscle. These data demonstrate that deficiency of AC5 protects against obesity, glucose intolerance, and insulin resistance, supporting AC5 as a potential novel therapeutic target for treatment of obesity and diabetes.