The roles of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) in chemoattractant-elicited responses were studied in mice lacking these key enzymes. PI3Kγ was required for chemoattractant-induced production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5)P 3 ] and has an important role in chemoattractant-induced superoxide production and chemotaxis in mouse neutrophils and in production of T cell–independent antigen-specific antibodies composed of the immunoglobulin λ light chain (TI-Igλ L ). The study of the mice lacking PLC-β2 and -β3 revealed that the PLC pathways have an important role in chemoattractant-mediated production of superoxide and regulation of protein kinases, but not chemotaxis. The PLC pathways also appear to inhibit the chemotactic activity induced by certain chemoattractants and to suppress TI-Igλ L production.

Regulation of serum response factor (SRF)-mediated gene transcription by G protein subunits and G proteincoupled receptors was investigated in transfected NIH3T3 cells and in a cell line that was derived from mice lacking G␣ q and G␣ 11 . We found that the constitutively active forms of the ␣ subunits of the G q and G 12 class of G proteins, including G␣ q , G␣ 11 , G␣ 14 , G␣ 16 , G␣ 12 , and G␣ 13 , can activate SRF in NIH3T3 cells. We also found that the type 1 muscarinic receptor (m1R) and ␣ 1 -adrenergic receptor (AR)-mediated SRF activation is exclusively dependent on G␣ q/11 , while the receptors for thrombin, lysophosphatidic acid (LPA), thromboxane A2, and endothelin can activate SRF in the absence of G␣ q/11 . Moreover, RGS12 but not RGS2, RGS4, or Axin was able to inhibit G␣ 12 and G␣ 13 -mediated SRF activation. And RGS12, but not other RGS proteins, blocked thrombin-and LPA-mediated SRF activation in the G␣ q/11 -deficient cells. Therefore, the thrombin, LPA, thromboxane A2, and endothelin receptors may be able to couple to G␣ 12/13 . On the contrary, receptors including ␤ 2 -and ␣ 2 -ARs, m2R, the dopamine receptors type 1 and 2, angiotensin receptors types 1 and 2, and interleukin-8 receptor could not activate SRF in the presence or absence of G␣ q/11 , suggesting that these receptors cannot couple to endogenous G proteins of the G 12 or G q classes.Hormones, neurotransmitters, and many other biologically active molecules, such as lysophosphatidic acid (LPA), 1 thrombin, catecholamines, endothelin, etc., transduce their signals through heterotrimeric G proteins (1, 2). Molecular cloning has revealed at least four classes of G protein ␣ subunits: G␣ s , G␣ i , G␣ q , and G␣ 12 (3). The G␣ s subunits and G␣ i subunits regulate adenylyl cyclase activities, while the G␣ q subunits regulate phospholipase C activities. However, the function of the G␣ 12 class of G proteins, which includes G␣ 12 and G␣ 13 , remains to be elucidated. Activated forms of G␣ 12 and G␣ 13 , when transfected into fibroblast cells, were shown to induce transformation phenotypes (4 -6), suggesting that this class of G proteins may be involved in cell growth regulation. Moreover, G␣ 12 and G␣ 13 were shown to induce formation of stress fibers in fibroblast cells through small G protein RhoA (7). This observation was supported by the report that G␣ 12 activated serum response factor (SRF) through RhoA (8). The in vivo function of G␣ 13 was also investigated using the gene-targeting technique in mice. Mice lacking G␣ 13 are embryonic lethal apparently due to the failure to develop vasculature structures, indicating that G␣ 13 may be involved in the function of endothelial cells (9). In the same study, thrombin-mediated chemotaxis of fibroblasts lacking G␣ 13 was blocked, indicating that the thrombin receptor couples to G␣ 13 . This is consistent with the observation that thrombin as well as a thromboxane A2 receptor agonist could stimulate the binding of a photo-affinity GTP analog to G␣ 13 (10). However, there were contradictory r...

The physiological roles of phospholipase C (PLC) ␤2 in hematopoiesis, leukocyte function, and host defense against infection were investigated using a mouse line that lacks PLC ␤2. PLC ␤2 deficiency did not affect hematopoiesis, but it blocked chemoattractant-induced Ca 2؉ release, superoxide production, and MAC-1 up-regulation in neutrophils. In view of these effects, it was surprising that the absence of PLC ␤2 enhanced chemotaxis of different leukocyte populations and sensitized the in vivo response of the PLC ␤2-deficient mice to bacteria, viruses, and immune complexes. These data raise questions about the roles that PLC ␤2 may play in signal transduction induced by chemoattractants in leukocytes.Phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate to produce two important second messengers, inositol trisphosphates and diacylglycerol (1). There are four different PLC ␤ isoforms that have been cloned. They are all regulated by heterotrimeric G proteins, and there is evidence suggesting that different isoforms may be involved in a variety of signaling circuits. The ␤2 isoform is found primarily in hematopoietic cells (2, 3), and it can be activated by both the G␣ subunits of the Gq class and by the ␤␥ subunits generated by a number of different heterotrimeric G proteins (3-9). Cotransfection experiments in COS-7 and HEK cells suggest that PLC ␤2 may function downstream of chemoattractant receptors. Transfection of receptors for complement component C5a and fMet-Leu-Phe (fMLP) (10), interleukin (IL)-8 receptors a and b (11), and CKR-1 and -2 (12) demonstrated that each of the receptors activates PLC ␤2 through the pertussis toxin (PTx)-sensitive release of ␤␥ from the G i class of heterotrimeric G proteins. In addition, this may be a primary signaling pathway in neutrophils, because much of the PLC activity elicited through chemoattractant receptors also appears to function through the G i -mediated release of ␤␥ (13-17).To confirm the existence of the G␤␥-PLC ␤2 pathway in vivo and to investigate the function of the pathway in hematopoiesis and leukocyte function, we generated a mouse line that lacks PLC ␤2. We found that PLC ␤2 is the major isoform that mediates PTx-sensitive PLC activation induced by chemoattractants and that PLC ␤2 is critical to many chemoattractant-elicited responses, including Ca 2ϩ efflux, superoxide production, and up-regulation of MAC-1. However, PLC ␤2 deficiency does not attenuate chemoattractant-induced chemotaxis; surprisingly, it was found to enhance the process. MATERIALS AND METHODS Generation of PLC ␤2-NullMice. An 8-kb genomic DNA from a 129SV agouti mouse strain library contains two exons of the PLC ␤2 gene, and it was used to make the gene-targeting construct. The exons encoded residues 378-464, which are located in the C terminus of the X box. Parts of the exons were replaced with a neomycin-resistance gene. The gene-targeting construct was transfected into embryonic stem (ES) cells (CJ7 clone) by electroporation. After selection with Geneticin, e...

Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the lambda phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redalpha, a 5' to 3' exonuclease, Redbeta, an annealing protein, and Redgamma, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance the stability of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total number of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double- or single-stranded DNA, published to date.