Processing of the insulin-like growth factor-II–mannose 6-phosphate receptor in isolated liver subcellular fractions

Abstract

A truncated, soluble form of the insulin-like growth factor-II–mannose 6-phosphate (IGF-II–M6P) receptor has been identified in serum and shown to be released from cultured tissues and cells, liver being the main contributor to serum receptor in adult rats. In the present study, the processing of the IGF-II–M6P receptor has been characterized in isolated liver subcellular fractions using ligand binding, affinity crosslinking, and Western immunoblotting techniques. The receptor in plasma membrane fractions differed from that in Golgi-endosomal fractions by: (i) a lower molecular size upon reducing polyacrylamide gel electrophoresis (245 vs. 255 kDa); (ii) a less tight membrane association as judged upon extractibility by NaCl; and (iii) the inability to recognize antibody anti-22C, directed against the cytoplasmic domain of the receptor. Incubation of cell fractions at 30°C led to a pH- and time-dependent release of the receptor into the medium. The pH optimum for release was 5.5 in the Golgi-endosomal fraction and 7.5 in plasma membrane fractions; at this pH, approximately 2% and 20%–30% of total receptors were released per hour, respectively. Receptor release was inhibited in a dose-dependent manner by aprotinin, benzamidine, and leupeptin in the Golgi-endosomal fraction, and by 1,10 phenanthroline in plasma membrane fractions, although high concentrations were required for inhibition. The receptor released from Golgi-endosomes showed a 5–10 kDa reduction in size and a loss of ability to recognize antibody anti-22C, but that released from plasma membranes showed little or no changes in size. We conclude that soluble, carboxy-terminally truncated forms of the IGF-II–M6P receptor are generated from the intact receptor in isolated Golgi-endosomal and plasma membrane fractions. However, receptor processing in these fractions exhibits different properties, suggesting the involvement of different proteases.Key words: insulin-like growth factor-II–mannose 6-phosphate (IGF-II–M6P) receptor, liver, plasma membrane, Golgi apparatus, endosomes.