Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)-dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide-binding proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-β3 bound to activated Gα q reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein-dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine triphosphate hydrolysis by Gα q , causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs.Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] to the second messengers inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and diacylglycerol in an essential step for the physiological action of many hormones, neurotransmitters, growth factors, and other extracellular stimuli (1-3). These cascades use

G protein-coupled receptor kinase 2 (GRK2) plays a key role in the desensitization of G protein-coupled receptor signaling by phosphorylating activated heptahelical receptors and by sequestering heterotrimeric G proteins. We report the atomic structure of GRK2 in complex with Galphaq and Gbetagamma, in which the activated Galpha subunit of Gq is fully dissociated from Gbetagamma and dramatically reoriented from its position in the inactive Galphabetagamma heterotrimer. Galphaq forms an effector-like interaction with the GRK2 regulator of G protein signaling (RGS) homology domain that is distinct from and does not overlap with that used to bind RGS proteins such as RGS4.

The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the G␣ 12/13 and G␣ q/11 families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing G␣ 12/13 -mediated RhoA activation, proteins that functionally couple G␣ q/11 to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of G␣ q/11 signaling to RhoA based on its ability to synergize with G␣ q/11 resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated G␣ q directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated G␣ q relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rhospecific DH-PH cassette of Trio is similarly activated by G␣ q .Rho GTPases are integral regulators of gene transcription and actin cytoskeletal remodeling during many dynamic cellular processes (1, 2). Signal transduction cascades mediated by Rho GTPases originate via the extracellular stimulation of transmembrane receptors such as G protein-coupled receptors (GPCRs), 4 receptor tyrosine kinases, cytokine receptors, and integrins. Of the 22 human Rho family members, RhoA, Rac1, and Cdc42 are the most characterized, stemming from their ability to induce striking changes in cellular morphology upon activation (3). Numerous studies have established that RhoA activation downstream of GPCRs is vital for a multitude of diverse physiological responses including cell migration (4), lipid metabolism (5), vascular smooth muscle cell contraction (6 -8), and cell survival/apoptosis (9 -12). GPCR-mediated activation of RhoA effectively couples signaling pathways mediated by two distinct groups of guanine nucleotide-binding proteins: the heterotrimeric G␣-subunits and the monomeric small GTPases. These two groups of G proteins share a universal mechanism for guanine nucleotide binding, GTP hydrolysis, and conformational switching between two discrete states: a GDPbound inactive state and a GTP-bound active state (13). Guanine nucleotide exchange factors (GEFs) activate G proteins by promoting the release of bound GDP, allowing the subsequent binding of GTP. Active, GTP-bound G proteins can then interact with numerous downstream effector molecules, further propagating the signal initiated at the plasma membrane.GPCRs function as GEFs for heterotrimeric G␣-subunits, whereas Dbl-family GEFs are the major class of exchange factors for Rho GTPases. Dbl-family GEFs are defined by the presence of a Dbl homology domain (DH doma...

Heterotrimeric G proteins stimulate the activities of two stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase in mammalian cells. In this study, we examined whether ␣ subunits of G i family activate JNK using transient expression system in human embryonal kidney 293 cells. Constitutively activated mutants of G␣ i1 , G␣ i2 , and G␣ i3 increased JNK activity. In contrast, constitutively activated G␣ o and G␣ z mutants did not stimulate JNK activity. To examine the mechanism of JNK activation by G␣ i , kinase-deficient mutants of mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7), which are known to be JNK activators, were transfected into the cells. However, G␣ i -induced JNK activation was not blocked effectively by kinase-deficient MKK4 and MKK7. In addition, activated G␣ i mutant failed to stimulate MKK4 and MKK7 activities. Furthermore, JNK activation by G␣ i was inhibited by dominant-negative Rho and Cdc42 and tyrosine kinase inhibitors, but not dominant-negative Rac and phosphatidylinositol 3-kinase inhibitors. These results indicate that G␣ i regulates JNK activity dependent on small GTPases Rho and Cdc42 and on tyrosine kinase but not on MKK4 and MKK7.

Hydrogen-oxidizing bacterium, Alcaligenes eutrophus autotrophically produces biodegradable plastic material, poly(D-3-hydroxybutyrate), P(3HB), from carbon dioxide, hydrogen, and oxygen. In autotrophic cultivation of the microorganism, it is essential to eliminate possible occurrence of gas explosions from the fermentation process. We developed a bench-plant scale, recycled-gas, closed-circuit culture system equipped with several safety features to perform autotrophic cultivation of A. eutrophus by maintaining the oxygen concentration in the substrate gas phase below the lower limit for a gas explosion (6.9%). The culture vessel utilized a baskettype agitator, resulting in a K(L) a value of 2970 h(-1). Oxygen gas was also directly fed to the fermentor separately from the other gases. As a result, 91.3 g . dm(-3) of the cells and 61.9 g . dm(-3) of P(3HB) were obtained after 40 h of cultivation under this oxygen-limited condition. The results compared favorably with those reported for mass production of P(3HB) by heterotrophic fermentation. (c) 1995 John Wiley & Sons, Inc.