Research in Dr. Petra's group is directed at understanding steroid-protein interaction as it relates to the mechanism of action of steroid hormones and, more specifically, to the growth of normal steroid-dependent cells and cancer cells of the human prostate and breast. A long-range goal is to understand the role of the sex steroid-binding protein, SBP (also called sex hormone binding globulin, SHBG), a protein that specifically binds dihydrotestosterone DHT), testosterone (T), and 17ß-estradiol (E2) in the plasma of many species, including humans. Human SBP is known to control the metabolic clearance rates of T and E2, but two additional roles have also been proposed. One is to assist in the cell uptake of these hormones by interacting with a specific receptor on the plasma membrane of target cells forming a ternary complex with the steroid. This complex is thought either to endocytose the hormone into the cell, or to accelerate its diffusion by concentrating it onto the cell surface. Another role is the formation of the ternary E2/SBP/SBP-receptor complex that may function in signal transduction. These interesting but controversial proposals suggest that sex steroid hormones could control gene expression in two ways: First, through the classic steroid receptor mechanism, and second, by regulating the activity of steroid receptors through signal transduction.

Our strategy has been to determine the structure of the human protein to lead towards an understanding of its physiological function. The protein was cloned and expressed in BHK, COS-7, and insect cells, and in yeast. The steroid-binding site was mapped by affinity groupeling and site-directed mutagenesis. The group has sequenced human and rabbit SBPs by both protein sequencing methods and cDNA cloning. Our results indicate that human SBP is a 95K homodimeric glycoprotein that binds one molecule of steroid per dimer. Protein modeling based on hydrodynamic properties and electron microscopy reveals a rod-shaped molecule with a length and diameter of about 23 and 3 nm, respectively. The human monomer is a single-chain glycoprotein composed of 373 amino acids with oligosaccharide side-chains N-linked to Asn-351 and Asn-367, and O-linked to Thr-6. Circular dichroism measurements indicate that human SBP contains about 32 % ß-structure, 13 % alpha-helix, and 15 % ß-turns. Met-139, Met-109, Lys-134, and Tyr-57 have been identified as functional residues in steroid binding. Rabbit SBP is also a homodimer but each monomer is shorter by 6 amino acid residues at the aminoterminus and lacks the O-linked oligosaccharide side-chain, the two N-linked oligosaccharide side-chains are located at the same homologous positions as those in the human protein. Rabbit SBP binds both DHT and T but, in contrast to the human protein, has a markedly reduced affinity for E2. The two proteins are homologous with 79 % identity in amino acid sequence. Recently, the crystal structure of an approximately 50% truncated version of the human protein (residues 13-188) has been solved (Grishkovskaya et al. EMBO J. 19:504, 2000). The results confirmed the presence of Met-107, Met-139, and Tyr-57 in the steroid binding site, but revealed a dimer having two steroid binding sites instead of one, as is the case for the native protein. More recently, we have determined that R140 and I141 are key determinants for the specific binding of 17ß-estradiol. These two residues are required for sustaining the right proximity of the phenyl ring of F56 to ring A of 17ß-estradiol thus optimizing its affinity to human SBP (this is indicated in the adjoining stereo Figure where purple is the native protein and green is the mutant M107I/R141K/I141L, yellow represents E2 bound to native SBP and red represents E2 bound to the mutant. The A ring of E2 is located near F56. The sole replacement M107I does not affect E2 binding). The mutant binds T as tightly as rabbit SBP and has a greatly reduced E2 binding affinity.