Annexin VI is a mannose-6-phosphate-independent endocytic receptor for bovine ß-glucuronidase.

Endocytosis and transport of bovine liver ß-glucuronidase to lysosomes in human fibroblasts are mediated by two receptors: the well-characterized cation-independent mannose 6-phosphate receptor (IGF-II/Man6PR) and an IGF-II/Man6PR-independent receptor, which recognizes a Ser-Trp*-Ser sequence present on the ligand. The latter receptor was detergent extracted from bovine liver membranes and purified. LC/ESI-MS/MS analysis revealed that this endocytic receptor was annexin VI (AnxA6). Several approaches were used to confirm this finding. First, the binding of bovine ß-glucuronidase to the purified receptor from bovine liver membranes and His-tagged recombinant human AnxA6 protein was confirmed using ligand-blotting assays. Second, western blot analysis using antibodies raised against IGF-II/Man6PR-independent receptor as well as commercial antibodies against AnxA6 confirmed that the receptor and AnxA6 were indeed the same protein. Third, double immunofluorescence experiments in human fibroblasts confirmed a complete colocalization of the bovine ß-glucuronidase and the AnxA6 receptor on the plasma membrane. Lastly, two cell lines were stably transfected with a plasmid containing the cDNA for human AnxA6. In both transfected cell lines, an increase in cell surface AnxA6 and in mannose 6-phosphate-independent endocytosis of bovine ß-glucuronidase was detected. These results indicate that AnxA6 is a novel receptor that mediates the endocytosis of the bovine ß-glucuronidase.

Involvement of insulin-like growth factors-I and -II and their receptors in medroxyprogesterone acetate-induced growth of mouse mammary adenocarcinomas.

The role of the insulin-like growth factors (IGFs) system was investigated in hormone-dependent (HD) and -independent (HI) in vivo lines of the medroxyprogesterone acetate (MPA)-induced mammary tumor model in Balb/c mice. IGF-II protein and message showed a three- to four-fold increase in HD lines growing in MPA-treated mice, as compared with HD tumors growing in untreated mice. Progression to a hormone-independent phenotype in all these lines was accompanied by a high constitutive expression of IGF-II. Similar IGF-I mRNA levels were detected in HD and HI lines. Both IGF-I and -II messages arose from the malignant epithelial cells, as shown by in situ hybridization studies. A significant decrease in Man-6P/type II IGF-R content was detected in HD tumors growing in MPA-treated mice as compared with HD lines growing in untreated mice. On the other hand, in HI tumors, notwithstanding high IGF-II synthesis, the levels of Man-6P/type II IGF-R remain high. Competitive inhibition and affinity labeling studies showed an almost exclusive binding of IGF-II to Man-6P/type II IGF-R on tumor membranes. The involvement of IGFs in the growth of epithelial primary cultures of the C4-HD line was evaluated. Exogenous IGF-I potentiated MPA stimulatory effect at concentrations of 50-100 ng/ml. Treatment of C4-HD cells with antisense oligodeoxynucleotides (ASODNs) to type I IGF-R and to IGF-II RNA resulted in a dose-dependent inhibition of MPA-mediated cell proliferation. The inhibition caused by IGF-II ASODNs could not be overcome by the addition of IGF-II up to 150 ng/ml. ASODNs to type I IGF-R at 40 microg/ml reduced by 75% the number of type I IGF-R; ASODNs to IGF-II at 1 microM decreased by 83% the levels of IGF-II protein. Our results provide support for the involvement of IGF-I and -II in MPA-induced mammary tumor growth by autocrine pathways.

Role of receptors for epidermal growth factor and insulin-like growth factors I and II in the differentiation of rat mammary glands from lactogenesis I to lactogenesis II.

In addition to ovarian steroids and lactogenic hormones from the placenta and pituitary, growth factors control the growth and differentiation of mammary glands. Lactogenesis II at the end of pregnancy is under the control of progesterone. Ovariectomy results in a significant decrease in the number of receptors for epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) and an increase in IGF-II binding sites in mammary gland acini of rats, without affecting the affinity for their respective ligand. Although concentrations of EGF, IGF-I and IGF-II receptors are regulated by oestradiol and progesterone, replacement treatment with ovarian steroids after ovariectomy showed that receptor concentrations do not mediate the restraint on lactogenesis. Progesterone treatment, which inhibits the onset of lactogenesis II, did not restore EGF receptor concentrations to control values, and the presence of oestradiol was required to reverse the effect of ovariectomy. Oestradiol, which potentiates the effect of ovariectomy on milk synthesis, increases IGF-I receptor concentrations. IGF-II receptor concentrations, after the different steroid treatments, were consistent with the steroid effect on milk synthesis. The changes observed in the concentrations of these growth factor receptors at the onset of mammary gland secretion are not considered to affect the progesterone block to lactogenesis II, but rather are a consequence of the shift of the hormonal and, hence, physiological status of the gland.