The glycoprotein hormones comprise a structurally related family of proteins produced in the pituitary gland (TSH, FSH, LH) and in the placenta (CG). The hormones are heterodimers, each containing a common alpha-subunit and different beta-subunits, which confer distinct biological activities to the hormones. The overall goals of this project are to understand the mechanisms that control the expression and actions of the glycoprotein hormones. The hypotheses to be tested are that the expression of the CREB gene is positively autoregulated in the Sertoli cells of the rat testis by the activation of the cAMP-dependent signalling pathway either by itself binding to and activating the cAMP response element(s) in the promoter of the CREB gene and/or by cAMP-responsive CREB-like protein(s). It is proposed that the temporally cyclical CREB gene expression is initiated by the cyclical appearance of the FSH receptor on Sertoli cells, followed by increased cellular levels of cAMP and positive autoregulation of CREB gene transcription. Then the positive feedback loop is interrupted by the alternative splicing into the transcript of one or more exons containing blocked reading frames resulting in the formation of C-terminally truncated CREB isoforms devoid of their DNA-binding domains and encoded nuclear translocation signals. An additional study is to determine the sequences of certain CREM transcripts present in the testis and to thereby ascertain their protein- coding capabilities.
The aims are to: (1) Carry out structure-function studies of the CREB gene promoter by analyses of promoter-CAT reporter vectors with mutations in the CREs and Sp1 binding sites using Sertoli cell lines and primary Sertoli cells as host target cells for transfections and analyses of CAT activities. The 110 kD protein that binds to the GC-rich CREs of the CREB gene promoter will be cloned and characterized. (2) Test the hypothesis that FSH-mediated increases in cAMP levels sets up a positive feed-back autoregulatory loop for CREB gene expression, determining changes in CREB and FSH receptor RNAs by using segments of the rat seminiferous tubule at varying temporal stages of the spermatogenic cycle of the rat testis. This hypothesis also proposes that the FSH receptor mRNA itself undergoes cyclical regulation, thus determining the appearance of cell surface assembled receptors. (3) A new Aim of these studies is to examine a possible role for alternatively spliced exons of CREB in directing internal translation so as to convert transactivator to transrepressor forms of CREB. These studies have potential relevance to understanding the molecular mechanisms controlling spermatogenesis and fertility.
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