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.
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