Rat androgen-binding protein (ABP), a secretory product of the Sertoli cell, is released primarily into seminiferous tubular lumen and transported to the epididymis where a portion of it is internalized. It is now known to belong to a family of extracellular androgen-binding proteins, which are synthesized in various organs under different regulation, and were originally thought to be distinct, but are, within a species, the product of a single gene. The high affinity and specificity of steroid binding lead to the view that it is central to the biologic function of these molecules. The recent finding that they can also bind to selected cells and, depending on the target tissue, stimulate cAMP accumulation or be internalized or both, suggests they may play a role in androgen delivery or subserve a signaling function. The observation that binding of steroid to these proteins can mediate their interaction with cellular receptors suggests a coupling of these two events, possibly through a conformational change in the protein upon steroid binding. Although these protein are known to be homodimers binding one mole of steroid per mole of native dimer, and the primary amino acid sequences for several of them have been determined, little is known about their physical structure or the nature of the steroid- or cell-recognition sites or how they may be coupled. Our goal is to relate these known actions to structure using site-directed or how they may be coupled. Our goal is to relate these known actions to structure using site-directed mutagenesis to probe the steroid binding site, the cell recognition domain, and the influence of glycosylation on biological activity. To accomplish this we will construct appropriate expression vectors for production of native and mutated ABPs in transient transfection studies and in stably transformed cells and assess their ability to bind steroid and to bind and stimulate target cells. While this approach will further delineate sequences involved in steroid and cellular binding, an understanding of how these regions interact and are related to each other spatially depends on elucidation of the tertiary and quaternary structure of ABP. To this end we will produce non-glycosylated ABP in quantities sufficient to do crystallization studies with the ultimate aim of determining its three-dimensional structure.
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