ROLE OF BETA ARRESTIN IN LH/CG RECEPTOR DESENSITIZATION

与七螺旋黄体生成素/绒毛膜促性腺激素结合的激动剂

Agonist binding to the heptahelical luteinizing hormone/choriogonadotropin receptor (LH/CG R) activates the stimulatory guanine nucleotide binding protein (Gs) and downstream adenylyl cyclase (AC). The LH/CG R subsequently exhibits reduced hormone-dependent AC activity or desensitization in response to saturating agonist. Unlike desensitization of many G-protein coupled R's, the in vivo desensitization response of the LH/CG R in ovarian follicles to the preovulatory LH surge can be mimicked under cell-free conditions. Because LH/CG R desensitization in follicular membranes exhibits physiologically relevant kinetics without the addition of any exogenous proteins, the investigators can study the mechanisms involved in LH/CG R desensitization in an unperturbed membrane without complications associated with R internalization and recycling. Using this model the investigators have shown that desensitization of LH/CG R-stimulated AC activity requires GTP and is reversed by a GDP analog that prevents G protein activation, and that LH/CG R phosphorylation is not obligatory for LH/CG R desensitization. The investigators demonstrated that purified follicular membranes contain beta-arrestin and that neutralizing anti-arrestin antibodies specifically block development of desensitization. Addition of recombinant purified beta-arrestin mimicked hCG to promote LH/CG R desensitization. A synthetic peptide corresponding to the third intracellular (3i) loop of the LH/CG R specifically prevented desensitization and saturation of this peptide with exogenous beta-arrestin revived desensitization. These results lead the investigators to hypothesize that beta-arrestin participates in agonist-dependent desensitization of the LH/CG R in an apparent phosphorylation-independent manner by binding to the 3i loop of the LH/CG R to prevent activation of Gs. The aims test this central hypothesis. Aim 1 will test the hypothesis that beta-arrestin binds tightly and uniquely to the plasma membrane of ovarian follicles, perhaps in an FSH dependent manner. Aim 2 will test the hypothesis that the binding of beta-arrestin to the active but unphosphorylated LH/CG R is necessary and sufficient to mediate LH/CG R desensitization. Aim 3 will test the hypothesis that desensitization of the LH/CG R is associated with beta-arrestin-dependent LH/CG R oligomerization. Aim 4 will test the hypothesis that the GTP-dependent step in LH/CG R desensitization consists of the release of beta-arrestin from a membrane docking site. Results from these studies will provide precise knowledge of the cellular mechanisms by which signaling from the LH/CG R is quenched to allow the reproductive cycle to advance and will likely permit development of reagents to halt the reproductive cycle or to reduce the activity of overactive R's.