During the coming budget period we intend to continue our long term studies designed at understanding the phenomenon of steroid-protein interaction at the molecular level and the implication of its existence in plasma on the mechanism of action of steroid hormones. We plan to initiate experiments to study the regulation of the biosynthesis of the sex steroid-binding protein, SBP, in liver and factors influencing its expression. To accomplish that goal we will undertake two major approaches which are as follows: Firstly, we will describe the biochemistry of two specific steroid- binding proteins, i.e. the sex-steroid-binding proteins (SBP) of human and rabbit plasma, through determination and comparison of their structures and steroid-binding sites. The amino acid sequence of rSBP will be determined by cDNA cloning and compared to that of hSBP sequenced in our lab in the past two years. The steroid binding sites will be characterized by affinity labeling and group specific modification to reveal the structural parameters responsible for binding specificity. X-ray diffraction of SBP crystals will be done to reveal the three-dimensional arrangement between steroid and protein. Secondly, we will engage in molecular biology studies to clone the human SBP gene and determine the molecular basis of its regulation by analysis of its genomic structure. We will use the partial hSBP cDNA clone we have recently sequenced to isolate or construct a full-length cDNA and express it in mammalian cell lines. Site- directed mutagenesis will be done to identify amino acids in the steroid binding site. We will also establish HepG2 cell lines to study the effect of various hormones on SBP biosynthesis. The information will help clinicians interested in controlling disorders associated with abnormal levels of SBP. The molecular biology studies will help us determine the site and regulation of its biosynthesis. Finally, we will isolate and sequence a full- length cDNA coding for human ABP, the testis androgen binding protein, to establish its relationship to human SBP.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD013956-08
Application #
3312392
Study Section
Endocrinology Study Section (END)
Project Start
1981-04-01
Project End
1993-03-31
Budget Start
1988-04-15
Budget End
1989-03-31
Support Year
8
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Petra, P H; Adman, E T; Orr, W R et al. (2001) Arginine-140 and isoleucine-141 determine the 17beta-estradiol-binding specificity of the sex-steroid-binding protein (SBP, or SHBG) of human plasma. Protein Sci 10:1811-21
Petra, P H; Woodcock, K T; Orr, W R et al. (2000) The sex steroid binding protein (SBP or SHBG) of human plasma: identification of Tyr-57 and Met-107 in the steroid binding site. J Steroid Biochem Mol Biol 75:139-45
Beck, K; Gruber, T M; Ridgway, C C et al. (1997) Secondary structure and shape of plasma sex steroid-binding protein--comparison with domain G of laminin results in a structural model of plasma sex steroid-binding protein. Eur J Biochem 247:339-47
Sui, L M; Hughes, W; Hoppe, A J et al. (1996) Direct evidence for the localization of the steroid-binding site of the plasma sex steroid-binding protein (SBP or SHBG) at the interface between the subunits. Protein Sci 5:2514-20
Sui, L M; Wong, C; Petra, P H (1995) Over-expression of human sex steroid-binding protein (hSBP/hABP or hSHBG) in insect cells infected with a recombinant baculovirus. Characterization of the recombinant protein and comparison to the plasma protein. J Steroid Biochem Mol Biol 52:173-9
Petra, P H; Griffin, P R; Yates 3rd, J R et al. (1992) Complete enzymatic deglycosylation of native sex steroid-binding protein (SBP or SHBG) of human and rabbit plasma: effect on the steroid-binding activity. Protein Sci 1:902-9
Casali, E; Petra, P H; Ross, J B (1990) Fluorescence investigation of the sex steroid binding protein of rabbit serum: steroid binding and subunit dissociation. Biochemistry 29:9334-43
Namkung, P C; Kumar, S; Walsh, K A et al. (1990) Identification of lysine 134 in the steroid-binding site of the sex steroid-binding protein of human plasma. J Biol Chem 265:18345-50
Griffin, P R; Kumar, S; Shabanowitz, J et al. (1989) The amino acid sequence of the sex steroid-binding protein of rabbit serum. J Biol Chem 264:19066-75
Namkung, P C; Stanczyk, F Z; Cook, M J et al. (1989) Half-life of plasma sex steroid-binding protein (SBP) in the primate. J Steroid Biochem 32:675-80

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