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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD038060-05
Application #
6733505
Study Section
Reproductive Biology Study Section (REB)
Program Officer
Yoshinaga, Koji
Project Start
2000-05-01
Project End
2006-04-30
Budget Start
2004-05-01
Budget End
2006-04-30
Support Year
5
Fiscal Year
2004
Total Cost
$280,967
Indirect Cost
Name
Northwestern University at Chicago
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Hunzicker-Dunn, Mary; Barisas, George; Song, Jinming et al. (2003) Membrane organization of luteinizing hormone receptors differs between actively signaling and desensitized receptors. J Biol Chem 278:42744-9
Mukherjee, Sutapa; Gurevich, Vsevolod V; Preninger, Anita et al. (2002) Aspartic acid 564 in the third cytoplasmic loop of the luteinizing hormone/choriogonadotropin receptor is crucial for phosphorylation-independent interaction with arrestin2. J Biol Chem 277:17916-27
Hunzicker-Dunn, Mary; Birnbaumer, Lutz (2002) Cell-free membrane desensitization assay for G protein-coupled receptors. Methods Enzymol 343:394-404
Salvador, Lisa M; Maizels, Evelyn; Hales, Dale Buchanan et al. (2002) Acute signaling by the LH receptor is independent of protein kinase C activation. Endocrinology 143:2986-94
Mukherjee, S; Casanova, J E; Hunzicker-Dunn, M (2001) Desensitization of the luteinizing hormone/choriogonadotropin receptor in ovarian follicular membranes is inhibited by catalytically inactive ARNO(+). J Biol Chem 276:6524-8
Salvador, L M; Mukherjee, S; Kahn, R A et al. (2001) Activation of the luteinizing hormone/choriogonadotropin hormone receptor promotes ADP ribosylation factor 6 activation in porcine ovarian follicular membranes. J Biol Chem 276:33773-81
Mukherjee, S; Gurevich, V V; Jones, J C et al. (2000) The ADP ribosylation factor nucleotide exchange factor ARNO promotes beta-arrestin release necessary for luteinizing hormone/choriogonadotropin receptor desensitization. Proc Natl Acad Sci U S A 97:5901-6