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 ...

Ggamma11 is an unusual guanine nucleotide-binding regulatory protein (G protein) subunit. To study the effect of different Gbeta-binding partners on gamma11 function, four recombinant betagamma dimers, beta1gamma2, beta4gamma2, beta1gamma11, and beta4gamma11, were characterized in a receptor reconstitution assay with the G(q)-linked M1 muscarinic and the G(i1)-linked A1 adenosine receptors. The beta4gamma11 dimer was up to 30-fold less efficient than beta4gamma2 at promoting agonist-dependent binding of [35S]GTPgammaS to either alpha(q) or alpha(i1). Using a competition assay to measure relative affinities of purified betagamma dimers for alpha, the beta4gamma11 dimer had a 15-fold lower affinity for G(i1) alpha than beta4gamma2. Chromatographic characterization of the beta4gamma11 dimer revealed that the betagamma is stable in a heterotrimeric complex with G(i1) alpha; however, upon activation of alpha with MgCl2 and GTPgammaS under nondenaturing conditions, the beta4 and gamma11 subunits dissociate. Activation of purified G(i1) alpha:beta4gamma11 with Mg+2/GTPgammaS following reconstitution into lipid vesicles and incubation with phospholipase C (PLC)-beta resulted in stimulation of PLC-beta activity; however, when this activation preceded reconstitution into vesicles, PLC-beta activity was markedly diminished. In a membrane coupling assay designed to measure the ability of G protein to promote a high-affinity agonist-binding conformation of the A1 adenosine receptor, beta4gamma11 was as effective as beta4gamma2 when coexpressed with G(i1) alpha and receptor. However, G(i1) alpha:beta4gamma11-induced high-affinity binding was up to 20-fold more sensitive to GTPgammaS than G(i1) alpha:beta4gamma2-induced high-affinity binding. These results suggest that the stability of the beta4gamma11 dimer can modulate G protein activity at the receptor and effector.

Tangential flow microfiltration (MF) is a cost-effective and robust bioprocess separation technique, but successful full scale implementation is hindered by the empirical, trial-and-error nature of scale-up. We present an integrated approach leveraging at-line process analytical technology (PAT) and mass balance based modeling to de-risk MF scale-up. Chromatography-based PAT was employed to improve the consistency of an MF step that had been a bottleneck in the process used to manufacture a therapeutic protein. A 10-min reverse phase ultra high performance liquid chromatography (RP-UPLC) assay was developed to provide at-line monitoring of protein concentration. The method was successfully validated and method performance was comparable to previously validated methods. The PAT tool revealed areas of divergence from a mass balance-based model, highlighting specific opportunities for process improvement. Adjustment of appropriate process controls led to improved operability and significantly increased yield, providing a successful example of PAT deployment in the downstream purification of a therapeutic protein. The general approach presented here should be broadly applicable to reduce risk during scale-up of filtration processes and should be suitable for feed-forward and feed-back process control.