SUMMARY Formation of the neuromuscular junction (NMJ) requires agrin, a factor released from motoneurons, and MuSK, a transmembrane tyrosine kinase that is activated by agrin. However, how signal is transduced from agrin to MuSK remains unclear. Here we report that low-density lipoprotein receptor (LDLR)-related protein (LRP) 4 (LRP4) functions as a co-receptor of agrin. LRP4 is specifically expressed in myotubes and is concentrated at the NMJ. The extracellular domain of LRP4 interacts with neuronal, but not muscle, agrin. Expression of LRP4 enables agrin binding activity and MuSK signaling in cells that otherwise does not respond to agrin. Suppression of LRP4 expression attenuates agrin binding activity, agrin-induced MuSK tyrosine phosphorylation and AChR clustering in muscle cells. LRP4 also interacts with MuSK in a manner that is stimulated by agrin. Finally, we showed that LRP4 becomes tyrosine-phosphorylated in agrin-stimulated muscle cells. These observations identify LRP4 as a functional co-receptor of agrin that is necessary for agrin-induced MuSK signaling and AChR clustering.

The ubiquitous multifunctional protein kinase, Ca2+/calmodulin-dependent kinase II (CaMKII), is a cytosolic enzyme activated by the signal-transduction pathways that elevate intracellular free Ca2+. CaMKII is important in diverse cellular functions from modulating ionic channels in muscles (1-3) to encoding "memory" in neurons (4, 5). In heart muscle, ryanodine receptors (3) and phospholamban (6)

and several other cell types, including endothelial cells and The c-met proto-oncogene encodes the tyrosine kinase melanocytes. 4,5 receptor for hepatocyte growth factor (HGF), a potent It has been reported that the expression of c-met is enmitogen and motogen for epithelial cells. Because of its hanced in thyroid, 6 gastric, 7 colorectal, 8 and prostatic canprofound effects on cell growth and motility, HGF may cers, 9 which suggests that an altered expression of c-met may be important in the development of cancer metastases be involved in cancer development. With respect to HCC, in hepatocellular carcinoma (HCC). In this study, we exseveral reports have been written on c-met expression. Prat amined HGF concentration and expression of the c-metet al. 10 reported the expression of c-met protein in 11 of 14 proto-oncogene product (c-met) in 62 patients with HCC HCCs using immunofluorescence, and Boix et al. 11 reported to determine the relationship between the level of exthat overexpression of c-met messenger RNA (mRNA) was pression and clinicopathological features, and patient detected in 8 of 18 HCCs. Suzuki et al. showed that c-met outcome following hepatectomy. Western blotting was protein was correlated with differentiation of HCC cells. 12used to examine the c-met expression, and HGF concenHowever, the relationship between c-met expression and tutration in tumors was measured using an enzyme-linked mor development, metastases, and patient outcome has not immunosorbent assay. c-met was found to be overexbeen clarified. pressed in HCC compared with nontumorous liver tissueIn this study, we examined c-met expression and HGF con-(P õ .01), and correlated with an increased incidence of centration in HCC and in nontumorous hepatic tissue, and intrahepatic metastases (P Å Hepatocyte growth factor (HGF) is a pleiotropic polypep-resection. Chronic liver disease was noted as follows: cirrhosis, 51 tide growth factor with a number of biological activities, in-(82.3%); chronic hepatitis, fibrosis, or both, 6 (9.6%). Twelve patients (19%) indicated the presence of hepatitis B surface antigen, and 23 cluding mitogenic, motogenic, and/or morphogenic proper-(37%) showed anti-hepatitis B surface antibody and/or anti-hepatitis ties, in a variety of epithelial tissues. Because HGF is a potent core antibody by enzyme-linked immunosorbent assay. Fifty-five of mitogen in hepatocytes, its involvement in the growth and 62 patients were tested with a second-generation hepatitis C virus development of metastases in hepatocellular carcinoma antibody (Ortho Diagnostics, Raritan, NJ), and 70.9% revealed the (HCC) is suspected. Recently, purified HGF has been shown presence of hepatitis C virus. A tumor sample and nontumorous to stimulate invasive characteristics in various cancer cells tissue were obtained immediately after the liver resection and were in vitro, 1,2 therefore, it is possible that HGF plays an im-snap-frozen in liquid nitrogen and stored at 080ЊC. Histological secportant role in the establishment of intrahepatic m...

mal and malignant epithelial cells and several other cell The c-met proto-oncogene encodes the tyrosine kinase types, including endothelial cells and melanocytes. 4,5 receptor for hepatocyte growth factor (HGF), a potent It has been reported that the expression of c-Met is enmitogen and motogen for epithelial cells. Because of its hanced in thyroid, 6 gastric, 7 colorectal, 8 and prostatic canprofound effects on cell growth and motility, HGF may cers, 9 which suggests that an altered expression of c-Met may be important in the development of cancer metastases be involved in cancer development. With respect to HCC, few in hepatocellular carcinoma (HCC). In this study, we exreports have been written on c-met expression. Prat et al. 10 amined HGF concentration and expression of the c-met have reported the expression of c-met protein in 11 of 14 proto-oncogene product (c-Met) in 62 patients with HCC HCCs using immunofluorescence, and Boix et al. 11 have reto determine the relationship between the level of exported that overexpression of c-met messenger RNA (mRNA) pression and clinicopathological features, and patient was detected in 8 of 18 HCCs. Suzuki et al. have shown outcome following hepatectomy. Western blotting was that c-met protein was correlated with differentiation of HCC used to examine the c-Met expression, and HGF concencells. 12 However, the relationship between c-Met expression tration in tumors was measured using an enzyme-linked and tumor development, metastases, and patient outcome immunosorbent assay. c-Met was found to be overexhas not been clarified. pressed in HCC compared with nontumorous liver tissueIn this study, we examined c-Met expression and HGF con-(P õ .01), and correlated with an increased incidence of centration in HCC and in nontumorous hepatic tissue, and intrahepatic metastases (P Å Hepatocyte growth factor (HGF) is a pleiotropic polypep-resection. Chronic liver disease was noted as follows: cirrhosis, 51 tide growth factor with a number of biological activities, in-(82.3%); chronic hepatitis, fibrosis, or both, 6 (9.6%). Twelve patients (19%) indicated the presence of hepatitis B surface antigen, and 23 cluding mitogenic, motogenic, and/or morphogenic proper-(37%) showed anti-hepatitis B surface antibody and/or anti-hepatitis ties, in a variety of epithelial tissues. Because HGF is a potent core antibody by enzyme-linked immunosorbent assay. Fifty-five of mitogen in hepatocytes, its involvement in the growth and 62 patients were tested with a second-generation hepatitis C virus development of metastases in hepatocellular carcinoma antibody (Ortho Diagnostics, Raritan, NJ), and 70.9% revealed the (HCC) is suspected. Recently, purified HGF has been shown presence of hepatitis C virus. A tumor sample and nontumorous to stimulate invasive characteristics in various cancer cells tissue were obtained immediately after the liver resection and were in vitro, 1,2 therefore, it is possible that HGF plays an im-snap-frozen in liquid nitrogen and stored at 080ЊC. Histological secportant ro...