Modulation of the insulin growth factor II/mannose 6-phosphate receptor in microvascular endothelial cells by phorbol ester via protein kinase C.
Phosphorylation of hormone receptors by protein kinase C (PKC) may be involved in the regulation of receptor recycling. We have studied the recycling and the phosphorylation state of the insulin growth factor (IGF) II/mannose 6-phosphate (Man-6-P) receptor in microvascular endothelial cells from rat adipose tissue. Scatchard analysis showed these cells have over 2 x 10(6) receptors/cell with an affinity constant of 1 x 10(9) M-1. In the presence of phorbol myristate acetate (PMA), an activator of PKC and analog of diacylglycerol, IGF-II receptor number increased in the plasma membrane by 60% without changes in the binding affinity. This increase in cell surface receptor number was confirmed by affinity cross-linking and 125I-surface labeling studies, occurred with a half-time of 20 min, and was reversible upon withdrawal of PMA. The redistribution of IGF-II/Man-6-P receptors was not due to an inhibition of internalization which was in fact stimulated by PMA. The effect of PMA on IGF-II receptor recycling correlated with its stimulation of PKC activity. Furthermore, after down-regulation of cellular PKC levels by preincubation with PMA, PMA was unable to activate residual PKC activity in the membranous pool or increase IGF-II receptor number at the cell surface. The phosphorylation state of the IGF-II/Man-6-P receptor was determined by 32P labeling of intact cells and immunoprecipitation with anti-receptor antibodies. In the basal state, the receptor was phosphorylated only on serine residues which was increased by 75% after treatment with PMA. In contrast, IGF-II decreased receptor phosphorylation and plasma membrane binding in a parallel and dose-dependent manner. Thus, PKC-stimulated serine phosphorylation of IGF-II/Man-6-P receptor may promote the translocation of the receptor to the cell surface, whereas IGF-II-stimulated dephosphorylation of the receptor may lead to a decrease in the number of cell surface receptors. These data suggest a role for PKC-mediated serine phosphorylation in the regulation of intracellular trafficking of receptors in endothelial cells.
Hu
K Q
KQ
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215.
Backer
J M
JM
Sahagian
G
G
Feener
E P
EP
King
G L
GL
D000273:Adipose Tissue / Q000098:blood supply; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004730:Endothelium, Vascular / Q000378:metabolism*; D006600:Hexosephosphates / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008360:Mannosephosphates / Q000378:metabolism*; D008833:Microcirculation; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D017451:Receptors, Somatomedin; D013002:Somatomedins / Q000378:metabolism*; D013696:Temperature; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000596:Amino Acids; D006600:Hexosephosphates; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D013002:Somatomedins; D007335:Insulin-Like Growth Factor II; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate