Elimination of excess climbing fiber (CF)-Purkinje cell synapses during cerebellar development involves a signaling pathway that includes type 1 metabotropic glutamate receptor, G␣q, and the ␥ isoform of protein kinase C. To identify phospholipase C (PLC) isoforms involved in this process, we generated mice deficient in PLC␤4, one of two major isoforms expressed in Purkinje cells. PLC␤4 mutant mice are viable but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple CF innervation clearly is impaired in the rostral portion of the cerebellar vermis, in which PLC␤4 mRNA is predominantly expressed. By contrast, CF synapse elimination is normal in the caudal cerebellum, in which low levels of PLC␤4 mRNA but reciprocally high levels of PLC␤3 mRNA are found. These results indicate that PLC␤4 transduces signals that are required for CF synapse elimination in the rostral cerebellum.The mRNA for phospholipase C (PLC) ␤4 has been reported to be particularly enriched in cerebellar Purkinje cells (PCs) (1, 2, 3). PCs express high levels of type 1 metabotropic glutamate receptor (mGluR1) (4, 5), which stimulates PLC through the activation of heterotrimeric G-proteins of the G␣q͞11 family (6, 7). PCs are also very rich in the ␥ isoform of protein kinase C (PKC␥) (8, 9, 10) and in inositol 1,4,5-trisphosphate receptors (11), both of which are activated after hydrolysis of phosphatidylinositol 4,5-bisphosphate by the PLC␤ enzymes. G␣q has been shown to colocalize with mGluR1 in PC dendritic spines (S. Nakagawa, J. Tanaka, M.W., M.K., M.I.S., and Y.I., unpublished data). It is, therefore, likely that the signal transduction cascade from mGluR1 to PKC␥ and inositol 1,4,5-trisphosphate receptor activation proceeds via G␣q and PLC␤4 and that this cascade specifically plays an important role in PC function.PCs receive distinct types of excitatory inputs from parallel fibers (PFs) and climbing fibers (CFs) (12, 13). Each PF synapse is weak, but one PC receives inputs from many (Ϸ10 5 ) PF synapses. In contrast, CFs originate from the inferior olive and form strong excitatory synapses on proximal dendrites of PCs. In an adult mouse, Ͼ85% of PCs are innervated by single CFs. Massive elimination of supernumerary CFs occurs during the second and third postnatal weeks until a one-to-one relation between CFs and PCs is attained at approximately postnatal day 21 (P21). This relationship then is maintained through adult life. It was reported previously that mutant mice deficient in PKC␥, mGluR1, or G␣q retain persistent multiple CFs into adulthood and display motor discoordination (14,15,16). These results suggest that G␣q mediates signals from mGluR1 that are necessary for regression of multiple CFs during cerebellar development.To identify the isoform(s) of PLC␤ that transduces these signals, we used mutant mice deficient in PLC␤4. These mice were used previously to investigate the physiological significance o...

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...

The C-C chemokines are major mediators of chemotaxis of monocytes and some T cells in inflammatory reactions. The pathways by which the C-C chemokine receptors activate phospholipase C (PLC) were investigated in cotransfected COS-7 cells. The C-C chemokine receptor-1 (CKR-1), the MCP-1 receptor-A (MCP-1Ra), and MCP-1Rb can reconstitute ligand-induced accumulation of inositol phosphates with PLC beta2 in a pertussis toxin-sensitive manner, presumably through G beta gamma released from the Gi proteins. However, these three receptors demonstrated different specificity in coupling to the alpha subunits of the Gq class. While none of the receptors can couple to Galphaq/11, MCP-1Rb can couple to both Galpha14 and Galpha16, but its splicing variant, MCP-1Rb, cannot. Since MCP-1Ra and -b differ only in their C-terminal intracellular domains, the C-terminal ends of MCP-1Rs determine G protein coupling specificity. CKR-1 can couple to Galpha14 but not to Galpha16, suggesting some of the C-C chemokine receptors, unlike the C-X-C chemokine receptors, discriminate against Galpha16, a hematopoietic-specific Galpha subunit. The intriguing specificity in coupling of the Gq class of G proteins implies that the chemokines may be involved in some distinct functions in vivo. The commonality of the chemokine receptors in coupling to the Gi-Gbetagamma-PLC beta2 pathway provides a potential target for developing broad spectrum anti-inflammatory drugs.