Mammotroph Autoregulation: The Differential Roles of the 24K Isoforms of Prolactin

In this study we have attempted to determine which of the secreted 24K isoforms was responsible for autocrine regulation of PRL secretion by comparing the isoforms synthesized and secreted by normal cells, which do autoregulate, with those synthesized and secreted by GH3 cells, which do not normally autoregulate. Comparable numbers of cells were washed free of serum and then extracted into Tris-buffered saline by sonication and detergent treatment. Proteins present in these cell extracts and in samples of culture medium were then precipitated with cold acetone (_20 C; 48 h) and subsequently dissolved in urea-lysis buffer for 2-dimensional (2-D) electrophoresis. The 2-D patterns for normal cells showed four 24K PRL isoforms inside the cells and three 24K PRL isoforms (designated 2, 3, and 3') secreted into the medium. The 2-D patterns for GH3 cells showed very little intracellular storage of PRL, but what was present was identified as 24K PRL isoform 2. The GH3 cells secreted large amounts of only 24K PRL ...