Using subtype-specific antisera, we were able to identify the recently described alpha subunits of G12 and G13 in platelet membranes as 43-kDa proteins. Activation of the thromboxane A2 and the thrombin receptors in platelet membranes led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha 12 and alpha 13, indicating that both receptors couple to G12 and G13. In addition, both activated receptors were demonstrated to couple to one or more members of the Gq family. In the absence of receptor agonists, incorporation of [alpha-32P]GTP azidoanilide into alpha 12 and alpha 13 was low over a long time period (up to 45 min) due to an obviously low basal nucleotide exchange rate, whereas an agonist-stimulated photolabeling of alpha 12 and alpha 13 could be observed after 4-8 min and reached a maximum after 30-45 min. Effective activation of G12 and G13 via the thromboxane A2 and the thrombin receptors was not dependent on the presence of GDP. Our results provide evidence that G12 and G13 play a functional role in transmembrane signal transduction and suggest that both proteins are involved in pathways leading to platelet activation.

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3–binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110γ/p101 PI3Kγ is activated by Gβγ on stimulation of G protein–coupled receptors. It is currently unknown whether in living cells Gβγ acts as a membrane anchor or an allosteric activator of PI3Kγ, and which role its noncatalytic p101 subunit plays in its activation by Gβγ. Using GFP-tagged PI3Kγ subunits expressed in HEK cells, we show that Gβγ recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein–mediated activation of PI3Kγ in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3–binding PH domains. Furthermore, membrane-targeted p110γ displayed basal enzymatic activity, but was further stimulated by Gβγ, even in the absence of p101. Therefore, we conclude that in vivo, Gβγ activates PI3Kγ by a mechanism assigning specific roles for both PI3Kγ subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of Gβγ with p110γ contributes to activation of PI3Kγ.

The G protein G o is highly expressed in neurons and mediates effects of a group of rhodopsin-like receptors that includes the opioid, ␣ 2 -adrenergic, M2 muscarinic, and somatostatin receptors. In vitro, G o is also activated by growth cone-associated protein of M r 43,000 (GAP43) and the Alzheimer amyloid precursor protein, but it is not known whether this occurs in intact cells. G o is an ␣␤␥ heterotrimeric G protein discovered in 1984 in brain by Neer and collaborators (1, 2) and by Sternweis and Robishaw (3), who all characterized it as a substrate for the ADP-ribosyltransferase activity of pertussis toxin. G o has received special attention for the following reasons: (i) It is the most abundant G protein in neurons, where it can constitute up to 2% of membrane protein (3). (ii) In addition to neurons, it appears to be expressed only on endocrine cells and heart, albeit at much lower levels comparable to those of the other heterotrimeric G proteins. (iii) G o is activated not only by the same class of seven-transmembrane receptors that activate the inhibitory G proteins G i1 -G i3 (4-9) but also by at least two proteins that do not belong to the rhodopsin-like family of G protein-coupled receptors, GAP43, an intracellular growth cone-associated protein active in neurite outgrowth (10), and the Alzheimer amyloid protein precursor protein responsible for familial forms of this disease (11). (iv) Except for inhibition of neuronal Ca 2ϩ channels, for which the mechanism of action of G o has been elucidated at the molecular level and shown to be due to the interaction of its ␤␥ moiety with the ␣ 1 subunit of the channel (6, 12-16), the mode of action of G o is essentially unknown. Tests for an ␣ i -like function for ␣ o have failed (17,18). Activated ␣ o has transformed NIH-3T3 cells (19) and activated mitogen-activated protein kinase activity in Chinese hamster ovary cells (20), phospholipase C in Xenopus oocytes, and K ϩ channels in neurons (21, 22), but how these effects come about has not been established. In fact, there is scant knowledge of the gamut of effector systems that may be the target(s) of activated G o .Gene ablation in mice is a powerful yet technically complex approach to identify as yet unknown functions of proteins that become manifest in mutated animals and͞or in cell lines derived from them. It has been applied to several G proteins with the following interesting results. G i2 -deficient mice were found to develop ulcerative colitis and adenocarcinomas, revealing an unexpected and as yet unexplained role of G i2 in the development of a chronic inflammatory response and very likely in lymphocyte homing to enteric epithelia (23, 24). Ablation of G q revealed an essential role for this G protein in platelet activation, because G q -deficient mice bleed and their platelets fail to be activated by physiologic activators such as collagen, thrombin, thromboxane, and ADP (25). Also the ablation of G o has been reported (26). Live mice, homozygous for loss of ␣ o , were obtained showing that...

Heterotrimeric G proteins of the Gi class have been implicated in signaling pathways regulating growth and metabolism under physiological and pathophysiological conditions. Knockout mice carrying inactivating mutations in both of the widely expressed G␣ i class genes, G␣i2 and G␣i3, demonstrate shared as well as genespecific functions. The presence of a single active allele of G␣i3 is sufficient for embryonic development, whereas at least one allele of G␣ i2 is required for extrauterine life. Mice lacking both G␣i2 and G␣ i3 are massively growth-retarded and die in utero. We have used biochemical and cell biological methods together with in situ liver perfusion experiments to study G␣ i isoform-specific functions in G␣i2-and G␣i3-deficient mice. The subcellular localization of G␣i3 in isolated mouse hepatocytes depends on the cellular metabolic status. G␣ i3 localizes to autophagosomes upon starvation-induced autophagy and distributes to the plasma membrane upon insulin stimulation. Analysis of autophagic proteolysis in perfused mouse livers showed that mice lacking G␣ i3 are deficient in the inhibitory action of insulin. These data indicate that G␣ i3 is crucial for the antiautophagic action of insulin and suggest an as-yet-unrecognized function for G␣ i3 on autophagosomal membranes.anticatabolic actions ͉ autophagy ͉ mouse knockout ͉ pertussis toxin-sensitive G proteins