RUI: Production and Characterization of Membrane Steroid Receptors

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Steroid hormones play important roles in vertebrate physiology and disease processes such as sexual behavior, reproduction, and cancer. Steroid hormones are generally known to modulate gene transcription through interactions with classical nuclear receptors. Steroids also initiate transcription-independent signaling events (nongenomic) at the cell surface through interactions with an unknown class of membrane receptors. Studies of these nongenomic steroid actions have been impaired for many years due to the lack of structure and sequence information for the membrane steroid receptors. Recently, a novel membrane steroid receptor family with high affinity to progestins and similar structure to G protein-coupled receptors has been cloned and characterized in fishes and other vertebrates. However, the recombinant proteins produced by the E. coli in previous studies have several disadvantages, such as their inability to bind its purported natural ligand and thus to allow functional and structural analyses of the receptors. The present research will produce functional zebrafish membrane receptor proteins using insect and mammalian cell lines, determine the steroid binding characteristics of the recombinant proteins, and investigate the involvement of the proteins in cell-surface signaling pathways. In addition, the proposed study will characterize and compare the function of the membrane steroid receptor subtypes that have been characterized in zebrafish, thus providing the molecular basis for future research and applications of the membrane steroid receptors. The long-term goal of the proposed study is to establish the functions of the membrane steroid receptor family in vertebrates. The study will be executed with the assistance from graduate and undergraduate students from rural areas of North Carolina. This grant will enable the training of these students in the latest knowledge and techniques in the fields of endocrinology and molecular biology.