Toshiaki Katada

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110 alpha or p110 beta). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to alpha beta gamma-trimeric G proteins also stimulate the lipid kinase activity of a novel p110 gamma isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110 gamma PI 3-kinase appears to be mediated through the beta gamma subunits of the G protein (G beta gamma) In addition, rat liver heterodimeric PI 3-kinases containing the p110 beta catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus G beta gamma. Such enzymatic properties were also observed with a recombinant p110 beta/p85 alpha expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110 alpha and p85 in the same rat liver, together with a recombinant p110 alpha/p85 alpha, was not activated by G beta gamma, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide (N-formyl-Met-Leu-Phe) plus insulin (or Fc gamma II) receptors in differentiated THP-1 and CHO cells and adenosine (Al) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110 beta catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a 'cross-talk' enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.

CD38 is a type II transmembrane glycoprotein possessing an NAD(+) glycohydrolase activity in its extracellular domain, We previously reported that the ligation of CD38 by a monoclonal antibody (mAb), HE-7, induces the tyrosine phosphorylation of cellular proteins including p120(c-cbl) in differentiated human myeloid cell lines and that the phosphorylated p120(c-cbl) is capable of binding to phosphatidylinositol (PI) 3-kinase. In the present study, we found that the agonistic anti-CD38 mAb markedly potentiates superoxide generation stimulated by chemotactic formyl-Met-Leu-Phe receptors in the CD38-producing cells, HE-7 neither generated superoxide by itself nor enhanced the cell response induced by phorbol 12-myristate acetate, indicating that the potentiating action of the anti-CD38 mAb is specific for the stimulation by the GTP-binding protein (G(1))-coupled membrane receptors, The potentiation by HE-7 was abolished by prior treatment of the cells with a tyrosine kinase inhibitor, pertussis toxin, or a potent PI 3-kinase inhibitor, wortmannin. HE-7 also enhanced the product formation of PI 3-kinase in response to the chemotactic receptor stimulation, without significant changes in the receptor-stimulated accumulations of inositol-1,4,5-trisphosphate, arachidonate release, and intracellular Ca2+. These results indicate that the CD38-induced tyrosine phosphorylation has a crosstalk with the chemotactic receptor/G(i)-mediated signal transduction pathway resulting in the enhancement of superoxide generation, probably through the activation of PI 3-kinase.

Differentiated HL-60 cells acquire responsiveness to fMet-Leu-Phe (fMLP), which activates phospholipase C and O-2(-) generation in a pertussis toxin-sensitive manner. Addition of retinoic acid (RA) for the last 24 h during dimethyl sulfoxide (Me(2)SO)-induced differentiation enhanced fMLP dependent signals and interaction between fMLP receptor and G(i). RA modifies both the function and subunit composition of G(i2), the predominant G(i) of HL-60 membranes, as shown by comparing purified G(i2) from membranes of Me(2)SO-treated cells (D-G(i2)) to G(i) from membranes of cells treated with both Me(2)SO and RA (DR-G(i2)). As compared to D-G(i2), DR-G(i2) induced more fMLP binding when added to membranes of pertussis toxin treated HL-60 cells and, in the presence of GTP gamma S, stimulated beta gamma-sensitive phospholipase C in extracts of HL-60 cells to a much greater extent and at lower concentrations. Immunoblots revealed that RA induced expression of the gamma(2) subunit, which was otherwise undetectable in G(i2) purified from HL-60 cells or in HL-60 membranes. Possibly by inducing expression of gamma(2), RA alters two functions of the G(i) beta gamma subunit, modulation of fMLP receptor-G(i2) coupling and activation of the effector, phospholipase C.

To determine whether G proteins activate cardiac ATP-sensitive K+ (K-ATP) channels by regulating intracellular ATP (ATP(i))-dependent gating, currents were measured in inside-out patches. When ATP(i) closed K-ATP channels, activators of endogenous G proteins, GTP (plus adenosine or acetylcholine), GTP gamma S, or AlF4- stimulated channels, an effect prevented by GDP beta S. In the absence of ATP(i), G protein activators were ineffective. Intracellular nucleoside diphosphates restored K-ATP channel openings after the ''rundown'' of spontaneous activity. Only when ATP(i) suppressed nucleoside diphosphate-induced openings, GTP gamma S or AlF4- enhanced K-ATP channel activity. Active forms of exogenous G protein subunits (G(alpha i-1), G(alpha i-2), or G(alpha o)) activated only K-ATP channels closed by ATP(i). G proteins stimulate cardiac K-ATP channels apparently by antagonizing ATP(i)-dependent inhibitory gating. Regulation of ligand-dependent gating represents a distinct type of G protein modulation of ion channels.

We recently identified four isoforms of bovine prostaglandin E receptor EP3 subtype, which are coupled to different signaling pathways; EP3A is coupled to inhibition of adenylate cyclase, while EP3B and EP3C are coupled to its stimulation and EP3D is coupled to phosphatidylinositol turnover, in addition to the adenylate cyclase system (Namba, T., Sugimoto, Y., Negishi, M., Irie, A., Ushikubi, F., Kakizuka, Ito, S., A., Ichikawa, A., and Narumiya, S. (1993) Nature 365, 166-170). We examined here the identity of coupled G proteins and their regulation by one of the isoforms, EP3C, in the membranes of EP3C cDNA-transfected Chinese hamster ovary cells. M&B 28767, an EP3 agonist, stimulated the GTPase activity in the pertussis toxin (PT)-treated cell membrane, but inhibited it in the cholera toxin (CT)-treated cell membrane, while the agonist neither stimulated nor inhibited it in the both PT- and CT-treated cell membrane. In the PT- and CT-treated cell membrane reconstituted with various G proteins, M&B 28767 inhibited the GTPase activity of G(o), but stimulated that of G(s). On the other hand, M&B 28767 did not affect the GTPase activity of G(i1), G(i2), or G(i3). M&B 28767 increased the apparent affinity of G(o) for GDP without any change in that for GTP, as assessed by displacement of [S-35]GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)) binding to G(o). In contrast, M&B 28767 increased the apparent affinity of G(s) for GTP but decreased that for GDP. These results demonstrated that the EP3 receptor isoform is coupled to two different G proteins, and oppositely regulates their activities, inhibition of G(o), and stimulation of G(s).