We are proposing to continue our long term studies towards understanding the biochemistry and physiology of the plasma sex steroid-binding protein, SBP. The project is divided into two parts: The first will comprise biochemical studies describing the chemical environment of steroid-binding sites to explain how steroids are bound to protein surfaces. We will attempt to identify amino acid residues in the steroid binding sites of both human and rabbit SBP by affinity labeling, and to determine the adjacent amino acid sequence. Since human SBP binds both E2 and T whereas rabbit SBP only binds T, a comparison should reveal the structural elements responsible for binding specificity. We will also undertake an x-ray diffraction analysis on SBP crystals and elucidate the entire amino acid sequence of SBP in an attempt to describe the three-dimensional structure of a steroid-binding protein. The second objective is to understand the role of SBP in the mechanism of action of steroid hormones in relation to reproductive biology. We will test a new hypothesis implicating SBP as a specific carrier of sex steroids across membranes. To this end, we will attempt to identify a SBP receptor on the plasma membrane by incubating 125I-hSBP with MCF-7 and HEC-50 cells in culture, and by in vivo infusion of 125I-rSBP into rabbits followed by autoradiographic examination of reproductive tissues in the electron microscope. We will also induce a long-term SBP withdrawal in the rhesus monkey by infusing F(ab)2 fragments of SBP antibodies and study the effects on the menstrual cycle and pregnancy (parturition).

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD013956-05
Application #
3312396
Study Section
Endocrinology Study Section (END)
Project Start
1981-04-01
Project End
1988-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
5
Fiscal Year
1985
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
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
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
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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