RESPONSIVENESS OF GNRH RECEPTOR SIGNALING

拟议研究的长期目标是调查对

The long-term aim of the proposed studies is to investigate regulation of the responsiveness of GnRH receptor signaling. The health importance of these studies is that the mechanism of GnRH's use as a treatment of prostate cancer is based on its propensity to desensitize LH secretion which, in turn, reduced testosterone secretion. Intracellular proteins that have come under scrutiny has potential regulators of the responsiveness of GnRH receptor signaling are members of the GRK family (G protein coupled receptor kinases) and beta arrestins 1 and 2. GRKs 2, 3, and 5 and beta arrestin 1 and 2 are expressed naturally in the rat anterior pituitary gland, and their experimental expression in GnRH receptor expressing COS-1 cells suppresses GnRH-stimulated production of IP3, a member of the second messenger cascade evoke by GnRH. Development of an innovative adenoviral approach for expression of GRKs in rat anterior pituitary cells has shown that they are strongly inhibitory. Therefore, in the proposed studies, this innovative adenoviral expression approach will be used to probe the extent to which the GRKs and beta arrestins participate in setting the responsiveness level of GnRH receptor signaling. The Specific Aims of the proposal are: Specific Aim 1: Determine the effects of GRK2, GRK3, beta arrestin 1 and 2 alone and in combination on GnRH-stimulated LH secretion using adenoviral-mediated gene expression in rat pituitary cells. Specific Aim 2: Investigate ablation or neutralization of endogenous GRK2/3 on responsiveness of GnRH receptor signaling using adenoviral-mediated gene transfer in pituitary gonadotropes of: a) a peptide GRK inhibitor that competitively inhibits GRKs), and; b) a GRK 2/3 antisense construct that suppresses synthesis of GRKs. Specific Aim 3: Determine if the external factors (estradiol, GnRH, etc.) that set responsiveness of GnRH receptor signaling do so by regulating the level of expression of GRK 2, 3, and 6 and beta arrestins 1 and 2. Specific Aim 4: Investigate GRKs' site and mode of action and inhibiting GnRH receptor signaling: a) Measure IP3-responsiveness of gonadotropes during sensitized and desensitized states to determine if GRK's site of action occurs before or after phospholipase C. b) Determine if LH secretion in gonadotropes infected with adeno-GRK2 and beta arrestin 2 remains stimulable to NaF treatment, a reagent that directly activated G proteins; 3) Look for phosphorylation of the epitope-tagged GnRH receptor in GRK2/beta arrestin 2 expressing COS-1 cells using epitope- tagged angiotensin II receptor as a positive control.