ABSTRACT
Guanylate cyclase C is the receptor for the bacterial heat-stable enterotoxins and guanylin family of peptides, and mediates its action by elevating intracellular cGMP levels. Potentiation of ligand-stimulated activity of guanylate cyclase C in human colonic T84 cells is observed following activation of protein kinase C as a result of direct phosphorylation of guanylate cyclase C. Here, we show that prolonged exposure of cells to phorbol esters results in a decrease in guanylate cyclase C content in 4beta-phorbol 12-myristate 13-acetate-treated cells, as a consequence of a decrease in guanylate cyclase C mRNA levels. The reduction in guanylate cyclase C mRNA was inhibited when cells were treated with 4beta-phorbol 12-myristate 13-acetate (PMA) in the presence of staurosporine, indicating that a primary phosphorylation event by protein kinase C triggered the reduction in RNA levels. The reduction in guanylate cyclase C mRNA levels was not due to alterations in the half-life of guanylate cyclase C mRNA, but regulation occurred at the level of transcription of guanylate cyclase C mRNA. Expression in T84 cells of a guanylate cyclase C promoter-luciferase reporter plasmid, containing 1973 bp of promoter sequence of the guanylate cyclase C gene, indicated that luciferase activity was reduced markedly on PMA treatment of cells, and the protein kinase C-responsive element was present in a 129-bp region of the promoter, containing a HNF4 binding element. Electrophoretic mobility shift assays using an oligonucleotide corresponding to the HNF4 binding site, indicated a decrease in binding of the factor to its cognate sequence in nuclear extracts prepared from PMA-treated cells. We therefore show for the first time that regulation of guanylate cyclase C activity can be controlled at the transcriptional level by cross-talk with signaling pathways that modulate protein kinase C activity. We also suggest a novel regulation of the HNF4 transcription factor by protein kinase C.
Subject(s)
DNA-Binding Proteins , Gene Expression Regulation, Enzymologic , Guanylate Cyclase/genetics , Protein Kinase C/metabolism , Receptors, Peptide/genetics , Transcription, Genetic , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Blotting, Western , Catalytic Domain , Cell Line , Enzyme Activation , Guanylate Cyclase/metabolism , Hepatocyte Nuclear Factor 4 , Humans , Phosphoproteins/metabolism , Phosphorylation , Promoter Regions, Genetic , Protein Binding , RNA, Messenger/metabolism , Receptors, Enterotoxin , Receptors, Guanylate Cyclase-Coupled , Receptors, Peptide/metabolism , Staurosporine/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factors/metabolismABSTRACT
The mannose-6-phosphate/IGF-II receptor MPR300 mediates sorting of lysosomal enzymes from the trans-Golgi network to endosomes and endocytosis of hormones, for example, of IGF-II. We analyzed transport of MPR300 in mu1A-adaptin-deficient fibroblasts, which lack a functional AP-1 clathrin adaptor complex. In mu1A-adaptin-deficient fibroblasts, the homologous MPR46 accumulates in endosomes due to a block in retrograde transport to the trans-Golgi network. The MPR300-mediated endocytosis is markedly enhanced. We demonstrate that the seven-fold increase in endocytosis is not associated with an increased steady-state concentration of receptors at the plasma membrane, but with an increased internalization rate of MPR300. Internalization of other receptors that are also endocytosed by AP-2 is not affected. More MPR300 receptors are found in clathrin-coated pits of the plasma membrane, whereas outside coated-areas, more MPR300 are concentrated in clusters and all intracellular receptors reside in endosomes, which are in equilibrium with the plasma membrane. Thus AP-1-mediated transport of MPR300 from endosomes to the TGN controls indirectly the recycling rate of the receptor between the plasma membrane and endosomes.