Meltrin a/ADAM12 is a member of the ADAM/MDC family proteins characterized by the presence of metalloprotease and disintegrin domains. This protein also contains a single transmembrane domain and a relatively long cytoplasmic domain containing several proline-rich sequences. These sequences are compatible with the consensus sequences for binding the Src homology 3 (SH3) domains. To determine whether the proline-rich sequences interact with SH3 domains in several proteins, binding of recombinant SH3 domains to the meltrin a cytoplasmic domain was analysed by pulldown assays. The SH3 domains of Src and Yes bound strongly, but that of Abl or phosphatidylinositol 3-kinase p85 subunit did not. Full-length Grb2/Ash bound strongly, whereas its N-terminal SH3 domain alone did less strongly. Src and Grb2 in bovine brain extracts also bound to meltrin a cytoplasmic domain on anity resin. Furthermore, immunoprecipitation with a monoclonal antibody to meltrin a resulted in coprecipitation of Src and Grb2 with meltrin a in cell extracts, suggesting that Src and Grb2 are associated in vivo with meltrin a cytoplasmic domain. This notion was also supported by the ®ndings that exogenously expressed meltrin a cytoplasmic domain coexisted with Src and Grb2 on the membrane rues. The C-terminal Tyr901 of meltrin a was phosphorylated both in vitro and in cultured cells by v-Src. These results may imply that meltrin a cytoplasmic domain is involved in a signal transduction for some biological function through the interaction with SH3-containing proteins. Oncogene (2000) 19, 5842 ± 5850.

The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.

Meltrin alpha (ADAM12) is a member of the ADAM (MDC) protein family characterized by the presence of metalloprotease and disintegrin domains. ADAM proteins contain single transmembrane domains, and the processed mature proteins are postulated to span the plasma membrane. It has been reported that transfection of a truncated meltrin alpha cDNA lacking the prodomain and metalloprotease domain promotes skeletal muscle cell fusion. We show here that meltrin alpha was constitutively expressed in both undifferentiated and differentiated C2 skeletal muscle cells and also in fibroblasts. Both its precursor and processed mature forms were present in these cells. Thus, meltrin alpha may play general roles in addition to its roles in myogenesis. Since endogenous meltrin alpha cannot be detected by immunofluorescence microscopy, we examined the location of the exogenously expressed protein by transfection. Unexpectedly, the exogenously expressed meltrin alpha was located to a network structure of the endoplasmic reticulum (ER) but not to the plasma membrane. Cell fractionation revealed that the intrinsic mature protein was associated with the plasma membrane. However, the exogenously expressed protein remained unprocessed. These results seem to imply that the exogenously expressed meltrin alpha is not translocated from the ER to the trans-Golgi network, where a processing enzyme resides, and that it is consequently not converted to the mature form. Thus, the transfected meltrin alpha is unlikely to exert its physiological functions. Conversely, the ER may serve as a reservoir of the latent form of intrinsic meltrin alpha.

Monkey embryonic stem (ES) cells are a useful tool for studying early human development and evaluating the efficacy of stem cell therapy. Monkey ES cells show closer similarity to human ES cells than their mouse counterparts regarding morphology, cell surface markers, and the maintenance of pluripotency, including the leukemia inhibitory factor requirement. The generation of genetically modified monkey ES cells with a biomarker such as green fluorescent protein, which allows noninvasive monitoring of progeny ES cells, is invaluable for the development of cell transplantation therapy and the study of differentiation mechanisms in primates. Here, we describe the generation of green fluorescent protein-expressing monkey ES cells using a conventional electroporation method.