Cyclic-AMP-dependent protein kinase (PKA) phosphorylates serine and threonine residues of substrate proteins to modify cellular functions such as gene expression and cell cycle progression. The RIIalpha regulatory subunit of PKA is expressed at elevated levels in differentiating haploid germinal cells and occupies a central role in cAMP signaling during mammalian spermatogenesis. In spermatozoa, RIIalpha is associated with the flagella and regulates cAMP-dependent activation of sperm cell motility. In earlier steps of germ cell development, non-flagellar RIIalpha is associated with the nucleus and will regulate PKAdependent phosphorylation of haploid specific transcription factors such as CREMtau. Activation of nuclear PKA could be the genetic switch which leads to broad expression of genes in haploid cells. Two germ cell specific mRNA isoforms for RIIalpha are observed to be differentially regulated in stage- synchronized testis. We hypothesize that the RIIalpha mRNA isoforms represent flagellar and cytoplasmic/nuclear protein isoforms required for appropriate sub cellular localization and propose to (i) characterize the structural (sequence) differences between the two RIIalpha isoforms and to identify corresponding germ cell specific RIIalpha A-kinase anchoring proteins (AKAPs) and (ii) to confirm the precise stage specific regulation of RIIalpha isoforms in testis. Using the 5' promoter region of the RIIalpha gene and transient transfection assays, we have identified a DNA regulatory region which is responsible for androgen activation and transcriptional feedback inhibition by PKA. This novel region of DNA also interacts with germ cell nuclear proteins. We propose to study signal transduction crosstalk between the cAMP and androgen signaling pathways through (iii) transient transfection of transcriptional reporter genes, PKA expression vectors, and cloned transcription factors into permissive cell lines, and to characterize the germ cell nuclear proteins which bind to promoter sequences. We also propose to (iv) analyze RIIalpha promoter regulation in developing germ cells using transgenic mice. The long term goal of this research is to provide a foundation for understanding male infertility problems related to motility or abnormal germ cell development, and to provide basic information on androgen and cAMP signal transduction crosstalk mechanisms used by normal and neoplastic tissues.
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