A novel subtilisin-like protein, PC8, was identified by PCR using degenerate primers to conserved amino acid residues in the catalytic region of members of the prohormone convertase family. PC8 was predicted to be 785 residues long and was structurally related to the mammalian convertases furin, PACE4, PC1 and PC2, sharing more than 50% amino acid identity over the catalytic region with these family members. PC8 possessed the catalytically important Asp, His, Asn and Ser amino acids, the homo B domain of this family of enzymes and a C-terminal hydrophobic sequence indicative of a transmembrane domain. Structurally, PC8 is more related to furin and PACE4 than to PC1 or PC2. Like furin and PACE4, PC8 mRNA was found to be widely expressed; this is in contrast with PC1 and PC2, which have a restricted distribution. Two transcripts, of 4.5 and 3.5 kb, were detected in both human cell lines and rat tissues. Unlike furin and PACE4, both of which map to chromosome 15, PC8 maps to chromosome 11q23–11q24, suggesting that this gene may have resulted from an ancient gene duplication event from either furin or PACE4, or conversely that these genes arose from PC8.

Specific high-affinity receptors for 1,25-dihydroxycholecalciferol [1,25-(OH) 2 D 3 ] have been described recently in broken-cell preparations of several cultured human breast cancer cell lines including the T47 D line. It was necessary to determine whether intact breast cancer cells in culture would bind 1,25-(OH) 2 D 3 specifically and whether the next step in the proposed scheme of action, i.e. nuclear translocation, occurred. The following results were obtained. (1) Specific uptake of 1,25-(OH) 2 D 3 by T47 D cells occurs in intact cells in culture. (2) The rate of uptake is proportional to medium 1,25-(OH) 2 D 3 concentration but is slow compared with that of other steroid hormones, e.g., oestradiol, under identical conditions. Even at 0.5n m -1,25-(OH) 2 D 3 in the medium, at least 4h are required to reach maximum compared with less than 1h for oestradiol binding. (3) Estimation of binding characteristics by Scatchard analysis indicates a single class of binding sites with K d of 68p m and 11800 binding sites/cell, which are similar results to those obtained with broken-cell preparations. (4) Inclusion of various vitamin D metabolites in the incubation medium decreased specific binding of 1,25-(OH) 2 D 3 by the intact cells in a manner identical with their effects in the broken-cell preparation and with potencies similar to their potency on Ca 2+ transport and bone resorption in vivo . Order of potency was 1,25-(OH) 2 D 3 >(24 R )-1,24,25-trihydroxycholecalciferol »25-hydroxycholecalciferol>(25 R )-24,25-dihydroxycholecalciferol »(25 R )-25,26-dihydroxycholecalciferol. (5) In the 1,25-(OH) 2 D 3 -depleted state, 80% of the 1,25(OH) 2 D 3 receptor is found in the cytosol fraction of the cells even when the subcellular fractionation is performed under low-salt conditions. By contrast after incubation with [ 3 H]1,25-(OH) 2 D 3 , 59% of the specific 1,25-(OH) 2 D 3 binding is found in the partially purified nuclei fraction. These data indicate that nuclear translocation of the receptor–hormone complex takes place in the intact T47 D cell. The results also support the hypothesis that the 1,25-(OH) 2 D 3 receptor is functional in this cultured breast cancer cell line, which may provide a useful model for further study of the early biochemical events in 1,25-(OH) 2 D 3 action.