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The first indication that the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6PR) is developmentally regulated came from studies of the serum form of the receptor in the rat. By immunoblotting, the circulating form of the receptor, which was 10 kDa smaller than the tissue receptor, was high in 19 day fetal and 3, 10, and 20 day postnatal sera and then declined sharply. We next used quantitative immunoblotting to measure the total tissue IGF-II/M6PR in the rat. The receptor levels were high in fetal tissues and in most tissues declined dramatically in late gestation and/or in the early postnatal period. The rank order of receptor expression was heart > placenta > lung = intestine > muscle = kidney > liver > brain. In heart, the receptor was 1.7% of total protein in the extract. More recently, we have examined the expression of IGF-II/M6PR mRNA using Northern blotting and a solution hybridization/RNase protection assay. The rank order of receptor mRNA concentration among fetal tissues agreed with the rank order of receptor protein. The concentration of receptor mRNA was significantly lower in postnatal tissue than in fetal tissue. Thus IGF-II/M6PR mRNA concentration is an important determinant of receptor protein in most tissues. What is the function of the IGF-II/M6PR in embryonic and fetal tissues? The M6PR in birds and frogs does not bind IGF-II. It is intriguing that the rat IGF-II/M6PR is prominent during the embryonic and fetal periods, times at which the differences between mammals, on the one hand, and frogs and birds, on the other, are most striking.(ABSTRACT TRUNCATED AT 250 WORDS)

The insulin-like growth-factor-II/mannose-6-phosphate receptor binds two classes of ligands, insulin-like growth factors and lysosomal enzymes. We have examined the ability of the lysosomal enzyme, Pgalactosidase, to modulate the binding of 251-IGF-II to the receptor. P-Galactosidase purified from bovine testis was fractionated on a DEAE-Sephacel ion-exchange column. Column fractions were assayed for enzymatic activity and for ability to inhibit the binding of 2'I-IGF-II to the IGF-IIIMan6P receptor. Enzyme fractions eluting at higher NaCl concentrations which had previously been shown to exhibit greater uptake by cells in culture, exhibited greater potency in inhibiting the binding of lZ5I-IGF-II to the receptor. A pool of these fractions from the DEAE-Sephacel column inhibited 1251-IGF-II binding to pure receptor by 80% with the concentration required for half-maximal inhibition being 25 nM. The inhibition of binding by P-galactosidase was completely blocked by simultaneous incubation with Man6P. Inhibition of the enzyinatic activity of P-galactosidase with Dgalactonic acid y-lactone did not affect the ability of P-gdactosidase to inhibit the binding of 1251-IGF-II to the receptor. Scatchard analysis of IGF-I1 binding to pure receptor in the presence and absence of P-galactosidase showed that B-gdlactosidase decreased the binding affinity for IGF-I1 (Kd 0.26 nM versus 1 .0 nM in the presence of 57 nM P-galactosidase). We confirmed the observations of others that Man6P alone actually increases the binding of 1251-IGF-II to the IGF-IIIMan6P receptor, but we found that this phenomenon was dependent upon the method of preparation of the IGF-IIIMan6P receptor. Microsomal membrane preparations, solubilized membranes, and receptors purified on an IGF-II-Sepharose column all exhibited stimulation of 1251-IGF-II binding by Man6P, whereas receptors purified on lysosomal enzyme affinity columns showed little or no stimulation of '251-IGF-II binding by Man6P. We conclude that P-galactosidase decreases the binding affinity of the IGF-IIIMan-6-P receptor for IGF-I1 by binding with high affinity to the Man6P-recognition site.

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The process of liver regeneration involves the concerted action of certain growth factors, which stimulate hepatocyte proliferation, and other antiproliferative factors, which prevent uncontrolled growth of this organ. Some of the biological actions of insulin-like growth factor-II (IGF-II), a mitogenic polypeptide closely related to insulin, may be mediated by the IGF-II receptor. This receptor consists of a single chain extracellular domain and a very small cytoplasmic domain, and can bind lysosomal enzymes that contain mannose-6-phosphate (M-6-P) residues. Since these enzymes may be involved in remodelling processes in certain tissues, we measured the expression of the IGF-II/M-6-P receptor in the liver after subtotal hepatectomy. Binding of [125I]IGF-II to crude plasma membranes from regenerating liver was maximal 2 days after hepatectomy (4.9% specific binding/60 micrograms protein) and subsequently decreased. Both control livers (livers removed at the time of operation) and sham-operated control livers demonstrated specific [125I]IGF-II binding of 1.1% throughout the experimental period. This increase in binding in regenerating liver was shown to be associated with an increase in the concentration of IGF-II receptor protein by means of Western blot analysis using a polyclonal anti-IGF-II/M-6-P receptor antiserum (3637). Similarly, steady state levels of IGF-II/M-6-P receptor mRNA, measured by solution hybridization/RNase protection assays, were significantly increased in the regenerating liver (2.0-fold over the control value 2 days after hepatectomy). Five and 10 days postsurgery, the levels of IGF-II receptor mRNA were markedly reduced, and they were even lower than the levels in control livers.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the developmental pattern of the insulin-like growth factor-II (IGF-II)/mannose 6-phosphate (M6P) receptor mRNA in various rat tissues from 20-day gestation fetuses and 20-day postnatal animals by Northern blotting and solution hybridization/RNase protection assays. The major mRNA species in all fetal and postnatal tissues was 9.0 kilobases. The rank order of receptor mRNA concentrations among the fetal tissues was heart greater than limb/muscle, lung, intestine, kidney, liver greater than brain, which agrees with the previously reported rank order of the tissue concentrations of receptor protein. The concentration of IGF-II/M6P receptor mRNA was significantly lower in postnatal tissues, again reflecting the relative levels of receptor protein in fetal and postnatal tissues. We measured IGF-II/M6P receptor mRNA copy number in fetal heart, the tissue with the highest concentration of receptor protein and mRNA, by including in the solution hybridization/RNase protection assay known amounts of a sense strand transcript of the receptor cDNA. This sense strand standard was quantitated by incorporating a tracer amount of [32P]UTP into the transcript and measuring the radioactivity in the product purified by gel electrophoresis. The receptor mRNA copy number in fetal heart was 74 molecules/cell. We conclude that the IGF-II/M6P receptor mRNA concentration is an important determinant of the level of receptor protein in most tissues.

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