Laboratoria di Endocrinologia Oncologica and Endocrinologia, Dipartimento di Oncologia, Azienda Ospedaliera San Giovanni Battista, Via Genova 3, 10126, Turin, Italy.
Human sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein, and each SHBG monomer may have an O-linked oligosaccharide at Thr(7) and up to two N-linked oligosaccharides at Asn(351) and Asn(367). In addition, a common genetic variant of SHBG exists with an extra site for N-glycosylation at residue 327. In the present study, we isolated MCF-7 derived cell lines expressing human SHBG cDNAs encoding the wild type protein or various glycosylation mutants. Estradiol (1 nM) treatment of parental (untransfected) MCF-7 cells or MCF-7 cells transfected with control expression vectors resulted in an increase in proliferation which was fully abrogated by co-incubation with an equimolar amount of human SHBG. In contrast, the same amount of purified SHBG added to MCF-7 cells expressing wild type SHBG partially inhibited the estradiol-induced cell proliferation. A high affinity binding site for SHBG was detectable on untransfected and control cells, but not on MCF-7 cells expressing wild type SHBG. Moreover, the treatment of MCF-7 cells with the conditioned medium containing wild type SHBG caused the disappearance of the SHBG plasma membrane-binding site. Media containing SHBG N-glycosylation mutants exerted the same effect, but mutants lacking the O-linked oligosaccharide at Thr(7) failed to do so. Estradiol-induced proliferation of parental MCF-7 cells was also inhibited by treatment with conditioned medium containing wild type SHBG or SHBG mutants lacking N-linked oligosaccharides, or containing an additional N-linked oligosaccharide at residue 327. However, MCF-7 conditioned medium containing SHBG mutants lacking an O-linked oligosaccharide at Thr(7) failed to exert this effect. These data suggest that O-glycosylation of SHBG is essential for SHBG binding to a membrane receptor that is responsible for inhibiting the estradiol-induced proliferation of MCF-7 breast cancer cells.