Characterization of hepatic cellular uptake of α1-acid glycoprotein (AGP), part 2: involvement of hemoglobin β-chain on plasma membranes in the uptake of human AGP by liver parenchymal cells.

Human α(1) -acid glycoprotein (AGP), a lipocalin family member, serves as a carrier for basic drugs and endogenous hormones. It is mainly distributed in the liver and also has anti-inflammatory effects. We previously discovered a protein in liver parenchymal cells that interacts with AGP and it was identified as hemoglobin β-chain (HBB). The purpose of this study was to clarify the role of HBB in the hepatic cellular uptake of AGP. Ligand blotting experiments showed that the interaction of (125) I-AGP with hemoglobin was saturable and was significantly suppressed in the presence of excess unlabeled AGP. In addition, the cellular uptake of fluorescein isothiocianate-AGP by HepG2 cells was saturable and temperature dependent. This uptake was inhibited by fillipin and methyl-β-cyclodextrin, but not chlorpromazine, suggesting that AGP is taken up via caveolae/lipid rafts endocytic pathway. Immunostaining showed that HBB and caveolin-1, exclusively expressed in caveolae, were partially colocalized on the plasma membranes of HepG2 cells. HBB knockdown with siRNA decreased the uptake of AGP by HepG2 cells by 40%, and exogenous hemoglobin inhibited the uptake by 40%-50%. These findings indicate that HBB is located on the liver plasma membrane and that it contributes to the intracellular uptake of AGP.