Serum Form of the Rat Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor Is Truncated in the Carboxyl-terminal Domain

Abstract

The insulin-like growth factor (IGF)-II/mannose 6-phosphate (Man-6-P) receptor present in mammalian tissues as an apparent molecular mass = 250 kDa glycoprotein has recently been detected in fetal rat serum in a lower molecular mass form (240 kDa). In the present studies the serum receptor was affinity labeled with 125I-IGF-II after its adsorption onto pentamannosyl 6-phosphate-Sepharose, demonstrating that it can also bind both ligands simultaneously. The receptors in both serum and fresh plasma exhibited the lower molecular mass compared to tissue receptors, indicating this form circulates in vivo. In order to probe the structural basis of the serum receptor's lower mass, we raised antipeptide antibodies against cytoplasmic and extracellular domains of the tissue form of the rat receptor deduced from complementary DNA clones (MacDonald, R. G., Pfeffer, S. R., Coussens, L., Tepper, M. A., Brocklebank, C. M., Mole, J. E., Anderson, J. K., Chen, E., Czech, M. P., and Ullrich, A. (1988) Science 239, 1134-1137). Peptide 22C, Glu-Glu-Glu-Thr-Asp-Glu-Asn-Glu-Thr-Glu-Trp-Leu-Met-Glu-Glu-Ile-Gln-Val- Pro-Ala - Pro-Arg, located in the cytoplasmic domain 32 residues carboxyl-terminal to the transmembrane region, and peptide 13D, Tyr-Tyr-Leu-Asn-Val-Cys-Arg-Pro-Leu-Asn-Pro-Val-Pro-Gly-Cys-Asp, located 1476 residues amino-terminal to the transmembrane domain were synthesized and used as immunogens in rabbits. IGF-II/Man-6-P receptors were first immunoprecipitated from either rat serum or a Triton X-100 extract of rat placental plasma membranes using a polyclonal antireceptor antibody. The immunoadsorbed receptors were then reduced, alkylated, electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and probed with antipeptide antibodies. Anti-13D revealed the major receptor band in all the membrane and serum samples tested as well as several minor species of lower apparent mass in serum. Fetal and neonatal rat sera contained 3-4 times as much of the receptor as adult serum. In contrast, anti-22C recognized the membrane IGF-II/Man-6-P receptor but failed to recognize any of the serum receptor species. These results indicate that the serum IGF-II/Man-6-P receptor is truncated or altered in its cytoplasmic domain, consistent with the hypothesis that it is derived from cells by proteolytic cleavage.