Distinct patterns of cytoplasmic protein phosphorylation related to regulation of synthesis and release of prolactin by GH cells.

The actions of thyrotropin-releasing hormone (TRH) on protein phosphorylation in the GH4C1 clonal strain of rat pituitary cells were examined by two-dimensional polyacrylamide gel electrophoresis and related to the ability of this tripeptide to stimulate prolactin (PRL) synthesis and release, respectively. Between 2 and 10 min after addition of the hormone, TRH increased the rate of phosphorylation of a small number of specific cytoplasmic proteins. The dose-response characteristics for TRH-induced protein phosphorylation were closely similar to those for its biological actions (ED50 congruent to 2 nM). TRH did not stimulate protein phosphorylation in the related GH12C1 cell strain which lacks TRH receptors. The proteins modified by TRH treatment were present in low amounts in the cell; they were not detectable by silver staining of the gels, except for protein 1 (Mr = 80,000) whose TRH-induced conversion from its unphosphorylated to its more acidic phosphorylated form was observed directly on silver-stained gels. Two distinct sets of phosphoproteins were identified among the seven affected by TRH: set I, five proteins whose phosphorylation was increased only by agents which stimulated PRL synthesis; and set II, two proteins whose phosphorylation was also increased by vasoactive intestinal peptide, which stimulates PRL release but not its synthesis in GH4C1 cells. This distinction between phosphoproteins related either to the release or synthesis of PRL was substantiated further by the use of an analog of TRH, A42658, which stimulated PRL synthesis and phosphorylation of proteins of set I at a lower concentration (0.2-0.5 microM) than was required to stimulate PRL release and phosphorylation of proteins of set II (greater than 0.5 microM). Finally, pretreatment of the cells with 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, which prevents TRH-induced PRL release but not synthesis, also suppressed TRH-stimulated phosphorylation of proteins of set II, but not of set I. These results relate stimulation by TRH of the phosphorylation of two distinct sets of proteins to the two major biological action of TRH in GH4C1 cells; proteins of set I appear to be related to the regulation of PRL synthesis, whereas those of set II appear related to modulation of PRL release.