Reproduction and thyroid function are controlled by the glycoprotein hormones, heterodimeric proteins composed of an alpha-subunit common to all and a hormone-specific beta-subunit. These subunits are structurally similar and the heterodimer is stabilized by a portion of the beta-subunit that is wrapped around the alpha-subunit and latched by a disulfide to the beta-subunit core. While this arrangement has obvious implications for the stabilities of these proteins, it raises questions as to how they are synthesized. Some studies in vivo suggest the subunits might combine before the seatbelt is latched. However, it is clear that subunit combination in vitro occurs efficiently even when the seatbelt remains latched. Glycoprotein hormone synthesis remains poorly understood in spite of its potential importance as a source of hCG metabolites observed during ectopic pregnancy, Down's syndrome, and even some cancers. Here we describe studies designed to learn how hCG is folded and assembled in vivo and to determine how the subunits combine in vitro. During the latter studies we will test a novel model of subunit combination that explains how one part of the alpha-subunit can pass through the small opening created by attachment of the beta-subunit seatbelt to the subunit core. The following specific aims are planned.
In Aim 1 we will identify parts of the beta-subunit that influence its rate of folding in transfected CHO cells. These studies rely on the technique of homolog scanning and will employ analogs created by combining parts of the alpha- and beta-subunits and by combining parts of the hCG and hFSH beta-subunits.
In Aim 2 we will determine if subunit combination occurs before or after the seatbelt is latched in cells and learn if this process is influenced by the alpha-subunit. These studies will employ an antibody that can separate heterodimers in which the seatbelt is latched from those in which it yet to form.
In Aim 3 we will determine the mechanism of subunit combination in vitro, a process that occurs while the seatbelt latch remains closed. These studies will involve measurements of the distance between the seatbelt and other parts of the free beta-subunit. They will also involve careful measurement of the rates of association and dissociation of subunit analogs, some of which lack seatbelts or contain seatbelts that are unable to be latched.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
1R01HD038547-01
Application #
6042777
Study Section
Biochemical Endocrinology Study Section (BCE)
Program Officer
Yoshinaga, Koji
Project Start
2000-02-15
Project End
2005-01-31
Budget Start
2000-02-15
Budget End
2001-01-31
Support Year
1
Fiscal Year
2000
Total Cost
$183,982
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
622146454
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Moyle, William R; Lin, Win; Myers, Rebecca V et al. (2005) Models of glycoprotein hormone receptor interaction. Endocrine 26:189-205
Bernard, Michael P; Lin, Win; Myers, Rebecca et al. (2005) Crosslinked bifunctional gonadotropin analogs with reduced efficacy. Mol Cell Endocrinol 233:25-31
Xing, Yongna; Myers, Rebecca V; Cao, Donghui et al. (2004) Glycoprotein hormone assembly in the endoplasmic reticulum: IV. Probable mechanism of subunit docking and completion of assembly. J Biol Chem 279:35458-68
Xing, Yongna; Myers, Rebecca V; Cao, Donghui et al. (2004) Glycoprotein hormone assembly in the endoplasmic reticulum: I. The glycosylated end of human alpha-subunit loop 2 is threaded through a beta-subunit hole. J Biol Chem 279:35426-36
Xing, Yongna; Myers, Rebecca V; Cao, Donghui et al. (2004) Glycoprotein hormone assembly in the endoplasmic reticulum: III. The seatbelt and its latch site determine the assembly pathway. J Biol Chem 279:35449-57
Xing, Yongna; Lin, Win; Jiang, Mei et al. (2004) Use of protein knobs to characterize the position of conserved alpha-subunit regions in lutropin receptor complexes. J Biol Chem 279:44427-37
Xing, Yongna; Myers, Rebecca V; Cao, Donghui et al. (2004) Glycoprotein hormone assembly in the endoplasmic reticulum: II. Multiple roles of a redox sensitive beta-subunit disulfide switch. J Biol Chem 279:35437-48
Bernard, Michael P; Cao, Donghui; Myers, Rebecca V et al. (2004) Tight attachment of chitin-binding-domain-tagged proteins to surfaces coated with acetylated chitosan. Anal Biochem 327:278-83
Moyle, William R; Xing, Yongna; Lin, Win et al. (2004) Model of glycoprotein hormone receptor ligand binding and signaling. J Biol Chem 279:44442-59
Birken, S; Yershova, Oksana; Myers, Rebecca V et al. (2003) Analysis of human choriogonadotropin core 2 o-glycan isoforms. Mol Cell Endocrinol 204:21-30

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