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

Long-term (or =12 h) treatment of cultured bovine adrenal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), caused a time (t(1/2)=16.3 h)- and concentration (IC50=37.8 nM)-dependent decrease of cell surface 125I-insulin binding by 35%, but did not change the Kd value. TG caused a sustained increase of cytoplasmic concentration of Ca2+ ([Ca2+]c) in a biphasic manner, and the effect of TG on 125I-insulin binding was abolished by BAPTA-AM. Western blot analysis showed that TG lowered insulin receptor (IR) beta-subunit level in membrane, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network (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.