Post-translational prenylation of heterotrimeric G protein ␥ subunits is essential for high affinity ␣-␤␥ and ␣-␤␥-receptor interactions, suggesting that the prenyl group is an important domain in the ␤␥ dimer. To determine the role of the prenyl modification in the interaction of ␤␥ dimers with effectors, the CAAX (where A indicates alipathic amino acid) motifs in the ␥ 1 , ␥ 2 , and ␥ 11 subunits were altered to direct modification with different prenyl groups. Six recombinant ␤␥ dimers were overexpressed in baculovirus-infected Sf9 insect cells, purified, and examined for their ability to stimulate three phospholipase C-␤ isozymes and type II adenylyl cyclase. The native ␤ 1 ␥ 2 dimer (␥ subunit modified with geranylgeranyl) is more potent and effective in activating phospholipase C-␤ than either the ␤ 1 ␥ 1 (farnesyl) or the ␤ 1 ␥ 11 (farnesyl) dimers. However, farnesyl modification of the ␥ subunit in the ␤ 1 ␥ 2 dimer (␤ 1 ␥ 2-L71S) caused a decrement in its ability to activate phospholipase C-␤. In contrast, both the ␤ 1 ␥ 1-S74L (geranylgeranyl) and the ␤ 1 ␥ 11-S73L (geranylgeranyl) dimers were more active than the native forms. The ␤ 1 ␥ 2 dimer activates type II adenylyl cyclase about 12-fold; however, neither the ␤ 1 ␥ 1 nor the ␤ 1 ␥ 11 dimers activate the enzyme. As was the case with phospholipase C-␤, the ␤ 1 ␥ 2-L71S dimer was less able to activate adenylyl cyclase than the native ␤ 1 ␥ 2 dimer. Interestingly, neither the ␤ 1 ␥ 1-S74L nor the ␤ 1 ␥ 11-S73L dimers stimulated adenylyl cyclase. The results suggest that both the amino acid sequence of the ␥ subunit and its prenyl group play a role in determining the activity of the ␤␥-effector complex.Heterotrimeric G proteins 1 are transducers of numerous extracellular signals from seven transmembrane receptors to intracellular effectors (1-4). G proteins are composed of ␣, ␤, and ␥ subunits and associate with the inner side of the plasma membrane. Receptor activation catalyzes the exchange of GDP for GTP on the ␣ subunit, resulting in dissociation of the GTP-liganded ␣ subunit from the ␤␥ dimer (1). Both the GTPbound form of the ␣ subunit and the released ␤␥ dimer regulate a variety of effectors, including PLC-␤ (5, 6) and adenylyl cyclases (7,8). To date, 7 ␤ subunits and 11 ␥ subunits have been identified in mammalian systems (9 -15). Selective assembly of ␤␥ heterodimers from these proteins may produce a large number of unique complexes that differ in their interactions with ␣ subunits, receptors, and effectors. While the first four ␤ subunits identified, ␤ 1 -␤ 4 , are 85-90% identical in primary amino acid sequence (9), the ␥ subunits are much more divergent. For example, the ␥ 1 and ␥ 5 subunits are only 25% identical. Thus, the ␥ subunits may impart some specificity to the ␤␥ signal. The G protein ␥ subunits are subject to post-translational modification by the addition of isoprenoid lipids to an invariant cysteine residue in the CAAX motif at their C terminus (15-17). The last amino acid (X) of the CAAX motif is an important determinant for...

The G protein ␤ 5 subunit differs substantially in amino acid sequence from the other known ␤ subunits suggesting that ␤␥ dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the ␤ 5 subunit, recombinant ␤ 5 ␥ 2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the ␥ 2 subunit (␥ 2HF ). The function of the pure ␤ 5 ␥ 2HF dimers was examined in three assays: activation of pure phospholipase C-␤ in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of ␣ subunits to the endothelin B (ET B ) and M 1 muscarinic receptors. In each case, the efficacy of the ␤ 5 ␥ 2HF dimer was compared with that of the ␤ 1 ␥ 2HF dimer, which has demonstrated activity in these assays. The ␤ 5 ␥ 2HF dimer activated phospholipase C-␤ with a potency and efficacy similar to that of ␤ 1 ␥ 2 or ␤ 1 ␥ 2HF ; however, it was markedly less effective than the ␤ 1 ␥ 2HF or ␤ 1 ␥ 2 dimer in its ability to activate type II adenylyl cyclase (EC 50 of ϳ700 nM versus 25 nM). Both the ␤ 5 ␥ 2HF and the ␤ 1 ␥ 2HF dimers supported coupling of M 1 muscarinic receptors to the G q ␣ subunit. The ET B receptor coupled effectively to both the G i and G q ␣ subunits in the presence of the ␤ 1 ␥ 2HF dimer. In contrast, the ␤ 5 ␥ 2HF dimer only supported coupling of the G q ␣ subunits to the ET B receptor and did not support coupling of the G i ␣ subunit. These results suggest that the ␤ 5 ␥ 2HF dimer binds selectively to G q ␣ subunits and does not activate the same set of effectors as dimers containing the ␤ 1 subunit. Overall, the data support a specialized role for the ␤ 5 subunit in cell signaling.

Eriodictyol [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-2,3-dihydrochromen-4-one] is a flavonoid with anti-inflammatory and antioxidant activities. Because inflammation and oxidative stress play critical roles in the pathogenesis of diabetes mellitus, the present study was designed to explore whether eriodictyol has therapeutic potential for the treatment of type 2 diabetes. The results show that eriodictyol increased insulin-stimulated glucose uptake in both human hepatocellular liver carcinoma cells (HepG2) and differentiated 3T3-L1 adipocytes under high-glucose conditions. Eriodictyol also up-regulated the mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and adipocyte-specific fatty acid-binding protein (aP2) as well as the protein levels of PPARγ2 in differentiated 3T3-L1 adipocytes. Furthermore, it reactivated Akt in HepG2 cells with high-glucose-induced insulin resistance. This response was strongly inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, indicating that eriodictyol increased Akt phosphorylation by activating the PI3K/Akt pathway. These results imply that eriodictyol can increase glucose uptake and improve insulin resistance, suggesting that it may possess antidiabetic properties.

The ability of G protein ␣ and ␤␥ subunits to activate the p110␥ isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110␥ form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTPactivated G s , G i , G q , or G o ␣ subunits were unable to activate PtdIns 3-kinase. Dimers containing G␤ 1-4 complexed with ␥ 2 -stimulated PtdIns 3-kinase activity about 26-fold with EC 50 values ranging from 4 to 7 nM. G␤ 5 ␥ 2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase C␤. A series of dimers with ␤ subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of ␤␥ with phosducin (␤1H311A␥2, ␤1R314A␥2, and ␤1W332A␥2) was tested, and only ␤1W332A␥2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the ␤ 1 subunit complexed with a panel of different G␥ subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The ␤ 1 ␥ 2 , ␤ 1 ␥ 10 , ␤ 1 ␥ 12 , and ␤ 1 ␥ 13 dimers all activated PtdIns 3-kinase about 26-fold with 4 -25 nM EC 50 values. The ␤ 1 ␥ 11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110␥ form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the ␥ subunit in determining the activation of PtdIns 3-kinase was examined using ␥ subunits with altered CAAX boxes directing the addition of farnesyl to the ␥ 2 subunit and geranylgeranyl to the ␥ 1 and ␥ 11 subunits. Replacement of the geranylgeranyl group of the ␥ 2 subunit with farnesyl inhibited the activity of ␤ 1 ␥ 2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the ␥ 1 and ␥ 11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all ␤ subunits, with the exception of ␤ 5 , interact equally well with PtdIns 3-kinase. In contrast, the composition of the ␥ subunit and its prenyl group markedly affects the ability of the ␤␥ dimer to stimulate PtdIns 3-kinase.The generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) 1 in the inner leaflet of the plasma membrane is critical to the regulation of cell function (1-3). The phosphorylated inositol head group provides a docking site for proteins containing pleckstrin homology domains (PH domains) and leads to activation of many enzymes including the phosphoinositol-dependent protein kinase and protein kinase B (Akt). Activation of protein kinase B regulates multiple cellular functions including differentiation, regulation of metabolic events, cell survival, and motility (1-3). In keeping with this central role, the level of PIP3 is tightly regulated, it can be elevated by multiple classes of receptors (1, 2), and there are specific phosphatidylinositol 5-phosphatases (SHIP and PTEN) that degrade the signal (4 -8).A large family of phosphatidylinositol 4,5-bisphosphate 3-kinases (PtdIns 3-kinases) is responsible for generating PIP3 by phosphorylating the D3 ...

Age-related changes in hepatic expression and activity of cytochrome P450 (CYP) were investigated in male rats aged 3 (weanling), 12 (young), 26 (adult), and 104 (old) weeks. Levels of microsomal protein, total CYP, and cytochrome b(5) increased fully after puberty. CYP1A1 was detected only in 3-week-old rats, and CYP1A2, CYP2B1, and CYP2E1 were maximally expressed at 3 weeks but decreased at 12 and 26 weeks. CYP2C11 and CYP3A2 increased markedly after puberty and decreased with aging. Ethoxyresorufin-O-deethylase, methoxyresorufin-O-demethylase, pentoxyresorufin-O-depenthylase, and p-nitrophenol hydroxylase activities were at their highest in 3-week-old rats, and midazolam hydroxylase activity was at a maximum in 12-week-old rats but decreased with aging. The present results show that increasing age caused significant alterations in hepatic expression/activity of CYP isoforms in an isoform-specific manner. These results suggest that age-related changes in hepatic CYP isoforms may be an important factor for deciding the efficacy and safety of xenobiotics.