Down-regulation of cell surface insulin receptors by sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor in adrenal chromaffin cells

Long-term (greater than or equal to 12 h) treatment of cultured bovine adre nal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)pla smic reticulum Ca2+-ATPase (SERCA), caused a time (t(1/2)=16.3 h)- and conc entration (IC50=37.8 nM)-dependent decrease of cell surface I-125-insulin b inding by 35%, but did not change the K-d value. TG caused a sustained incr ease of cytoplasmic concentration of Ca2+ ([Ca2+](c)) in a biphasic manner, and the effect of TG on I-125-insulin binding was abolished by BAPTA-AM. W estern blot analysis showed that TG lowered insulin receptor (IR) beta -sub unit level in membrane, but did not alter total cellular levels of IR precu rsor and IR beta -subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi n etwork (TGN), was not changed by TG. These results suggest that inhibition of SERCA by TG and the subsequent increase of [Ca2+](c) down-regulates cell surface IR by retarding externalization of IR from the TGN. (C) 2001 Elsev ier Science B.V. All rights reserved.