Apical localization of the alpha subunit of GTP-binding protein Go in choroidal and ciliated ependymocytes

The presence of GTP-binding proteins (G proteins) has been studied in murine adult choroid plexuses and cultured fetal choroidal or hypothalamic ependymal cells by ADP-ribosylation catalyzed by Bordetella pertussis toxin (PTX) and by immunodetection using affinity-purified polyclonal antibodies against the alpha subunit of the Go protein (Go alpha), the major brain G protein. ADP-ribosylation with 32P-NAD and PTX of choroid plexus revealed an intense labeling at the 40 kDa level in addition to the known PTX-substrates at 41 kDa (Gi alpha) and 39 kDa (Go alpha). This 40 kDa substrate was also predominant in cultured ependymal cells. However, a positive immunoreactivity with the anti-Go alpha antibodies was detected at the level of the 39 kDa faster component, indicating the presence of Go alpha in both choroid plexuses and cultured ependymal cells. In thin frozen sections as well as in cultured cells, Go alpha was mainly immunolocalized at the apical pole of choroidal ependymocytes and in the kinocilia of ciliated ependymal cells. At the ultrastructural level, using gold immunoprobes, the immunoreactivity of a Go alpha-like protein was detected on the cytoplasmic face of the apical plasma membrane, coated pits and vesicles, and in the apical cytoplasmic matrix. In ciliated ependymal cells, the positive immunostaining displayed a dotted pattern at the surface of demembranated axonema of apical kinocilia. These findings strongly suggest that G proteins, especially Go, are involved in transducing chemical signals that modulate traffic and exchanges between cerebrospinal fluid and ependyma through the apical membrane of ependymocytes.