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Development of cerebellar cells in neuron-enriched cultures: cell surface proteins
Abstract:
Lactoperoxidase catalyzed 125I-iodination of 8-day-old rat cerebellar cultures enriched in interneurons, mainly granule cells, was studied during a period 1-8 days in vitro, when the mature appearance of the cultures develop. Autoradiography of the surface iodinated constituents after separation by SDS-polyacrylamide gel electrophoresis showed a limited number of heavily labeled bands, including polypeptides of apparent molecular weight (X 10(3] of 140, 88, 68, 58 and 53 daltons. Their relative proportion in terms of 125I-content changed during the development of the cultures. Initially, the labeled 140 kdaltons band (P140) was dominant. Using crossed immunoelectrophoresis with an antiserum raised against immature rat cerebellar plasma membrane preparations (anti-BPM serum) that primarily recognizes one neuronal surface antigen (D2)33, it was established that the P140 comprises the D2 protein. In contrast to the amount of D2, which increases during the 8-day culture period, the labeling of P140 decreased sharply after 2 DIV. This decline coincided with a developmental change in the molecular forms of D2 involving desialylation. Treatment of 2 DIV cultures with neuraminidase, which reproduces the D2 developmental change, prior to 125I-iodination resulted in a marked reduction in the labeling of P140, whereas the other major labeled group of polypeptides in the 50 kdalton range were little affected. Further experiments showed that the D2 protein is phosphorylated in the plasma membrane. It was found that some of the surface labeled proteins, including P140, are released into the culture medium, but apparently in a non-phosphorylated form. Thus it would appear that a significant part of the polypeptide chain of D2, which is an integral membrane constituent, is exposed on the cell surface, and that either D2 has an anchorage within the membrane that is phosphorylated but is not released or D2 is rapidly dephosphorylated when it is shed from the membrane.