Hormones, Neuronal Phosphoproteins and Behavior

描述（由申请人提供）：卵巢类固醇激素，雌二醇和孕酮，调节中枢神经系统的功能，导致生理和生殖行为的改变。雌二醇和孕酮对生殖行为发挥其神经效应的一种方式是通过它们与作为配体依赖性转录因子的同源细胞内受体的相互作用。我实验室的研究表明，除了类固醇激素外，神经递质多巴胺还可以以不依赖配体的方式激活孕激素受体。多巴胺促进雌性大鼠和小鼠的生殖行为是通过多巴胺的膜受体和细胞内孕酮受体之间的串扰发生的。 由多巴胺和孕酮引发的信号转导级联导致下丘脑中cAMP水平、PKA活性和DARPP-32磷酸化对苏氨酸34的增加。DARPP-32除了在苏氨酸34上磷酸化外，还可以在苏氨酸75以及两个丝氨酸残基Ser 102和Ser 137上磷酸化。这种多位点磷酸化通过将DARPP-32转化为PKA磷酸化的更好底物并增强其对下游蛋白磷酸酶-1（PP-1）级联的抑制作用来放大DARPP-32的作用。虽然孕酮和多巴胺都能增强下丘脑中PKA的活性，但这种增加调节DARPP-32抑制PP-1的机制尚未得到评估。该提案的重点是孕酮和多巴胺对DARPP-32多位点磷酸化的机制及其对女性生殖行为的影响。具体目标1将评估孕酮和多巴胺对下丘脑中Thr 34和Thr 75处DARPP-32磷酸化的影响以及由此产生的PP-1在介导生物效应中的调节。具体目标2将确定多巴胺和孕酮对下丘脑中DARPP-32在Ser 102和Ser 137上的磷酸化状态的调节作用，并检查它们对DARPP-32/PP-1级联放大的影响。具体目标3将使用在DARPP-32的Thr 34、Thr 75、Ser 102和Ser 137处具有点突变的突变小鼠来检查孕酮和多巴胺对生殖行为的影响。这些研究将提供孕酮和多巴胺介导生殖行为的PKA/DARPP-32/PP-1级联调节的信息。

DESCRIPTION (provided by applicant): The ovarian steroid hormones, estradiol and progesterone, regulate functions in the central nervous system resulting in alterations in physiology and reproductive behavior. One means by which estradiol and progesterone exert their neural effects on reproductive behavior is via their interaction with cognate, intracellular receptors functioning as ligand-dependent transcription factors. Studies from my laboratory have shown that in addition to steroid hormones, neurotransmitter dopamine can activate progestin receptors in a ligand-independent manner. Dopamine facilitation of reproductive behavior in female rats and mice occurs by means of cross-talk between membrane receptors for dopamine and intracellular progestin receptors. Signal transduction cascades initiated by dopamine and progesterone, cause an increase in the level of cAMP, PKA activity and phosphorylation of DARPP-32, on Threonine34 in the hypothalamus. In addition to its phosphorylation on Threonine34, DARPP-32 can also be phosphorylated on Threonine75, as well as two Serine residues Ser102 and Ser137. This multisite phosphorylation amplifies the effects of DARPP-32 by converting it into a better substrate for PKA phosphorylation and potentiating its inhibitory effects on the downstream protein phosphatase-1 (PP-1) cascade. While both progesterone and dopamine enhance PKA activity in the hypothalamus, the mechanism(s) by which this increase regulates DARPP-32 inhibition of PP-1 have not been evaluated. This proposal focuses on the mechanisms underlying this multi-site phosphorylation of DARPP-32 by progesterone and dopamine and their effects on female reproductive behavior. Specific aim 1 will evaluate the effects of progesterone and dopamine on DARPP-32 phosphorylation at Thr34 and Thr75 in the hypothalamus and the resultant regulation of PP-1 in mediating the biological effects. Specific aim 2 will determine the modulatory role of dopamine and progesterone on the state of phosphorylation of DARPP-32 on Ser102 and Ser 137 in the hypothalamus and examine their effects on the amplification of DARPP-32/PP-1 cascade. Specific aim 3 will examine the effects of progesterone and dopamine on reproductive behavior using mutant mice with point mutations at Thr34, Thr75, Ser102 and Ser137 of DARPP-32. These studies will provide information on the PKA/DARPP-32/PP-1 cascade regulation by progesterone and dopamine in mediating reproductive behavior.