Phosphorylation of eukaryotic initiation factor (eIF) 2 alpha and inhibition of eIF-2B in GH3 pituitary cells by perturbants of early protein processing that induce GRP78.

Agents that mobilize sequestered intracellular Ca2+, including ionophore A23187, EGTA, thapsigargin, and Cbz-Gly-Phe-NH2 (where Cbz is benzyloxycarbonyl), or mild reducing agents, such as dithiothreitol, disrupt early protein processing in the endoplasmic reticulum (ER), inhibit translational initiation, and trigger the induction of GRP78, an ER resident protein. Inhibition of translational initiation in response to acute treatment (15-30 min) of intact GH3 pituitary cells with each of these agents was accompanied by an average 5-fold increase in the amount of phosphorylated eukaryotic initiation factor (eIF) 2 alpha and a 50% reduction in eIF-2B activity. With continued exposure to A23187 (3 h) rates of amino acid incorporation partially recovered, eIF-2 alpha became dephosphorylated, and the inhibition of eIF-2B activity was abolished. These chronic effects were blocked by actinomycin D. Accumulating evidence that the ER may regulate rates of translational initiation through a signaling system altering the activity of eIF-2 is discussed.

Stimulation of GRP78 gene transcription by phorbol ester and cAMP in GH3 pituitary cells. The accommodation of protein synthesis to chronic deprivation of intracellular sequestered calcium.

GRP78/BiP resides in the lumen of the endoplasmic reticulum (ER), a major site of Ca2+ sequestration and early protein processing. Agents, such as ionophore A23187, that mobilize sequestered ER Ca2+ suppress translational initiation within minutes and induce GRP78 within 1-3 h accompanied by development of translational tolerance to the inhibitor. Accommodation is prevented by actinomycin D and reduced by antisense oligonucleotides directed against GRP78 mRNA. In GH3 cells, optimal induction of GRP78 and translational accommodation depended on cAMP elevation and phorbol ester. GRP78 mRNA was induced 3-6-fold with A23187 alone as compared with 12-20-fold with ionophore plus cAMP-elevating agent and phorbol ester, but was not markedly induced without A23187. GRP78 gene transcription in nuclei isolated from A23187-treated cells was increased 2-4-fold by cAMP and phorbol ester. A nucleotide sequence homologous to the cAMP-responsive element consensus potentially exists in the promoter region of the GRP78 gene. GRP78 mRNA in ionophore-treated cells was largely associated with mono- and polysomal fractions rather than ribonuclear protein particles, a distribution different from actin and tubulin mRNAs. While polysomal content increased in cells undergoing translational recovery, cAMP and phorbol esters did not affect GRP78 mRNA stability. Translational accommodation in ionophore-treated GH3 cells is proposed to involve enhanced transcription of GRP78 mRNA promoted by cAMP/phorbol ester in conjunction with preferential polysomal loading of the message.