Abstract
Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the alpha-subunit straddles a hole in the beta-subunit. This hole is formed when a cysteine at the end of a beta-subunit strand known as the "seatbelt" becomes "latched" by a disulfide to a cysteine in the beta-subunit core. The heterodimer is stabilized in part by the difficulty of threading the glycosylated end of the alpha-subunit loop 2 through this hole, a phenomenon required for subunit dissociation. Subunit combination in vitro, which occurs by the reverse process, can be accelerated by removing the alpha-subunit oligosaccharide. In cells, heterodimer assembly was thought to occur primarily by a mechanism in which the seatbelt is wrapped around the alpha-subunit after the subunits dock. Here we show that this "wraparound" process can be used to assemble disulfide cross-linked human choriogonadotropin analogs that contain an additional alpha-subunit cysteine, but only if the normal beta-subunit latch site has been removed. Normally, the seatbelt is latched before the subunits dock and assembly is completed when the glycosylated end of alpha-subunit loop 2 is threaded beneath the seatbelt. The unexpected finding that most assembly of human choriogonadotropin, human follitropin, and human thyrotropin heterodimers occurs in this fashion, indicates that threading may be an important phenomenon during protein folding and macromolecule assembly in the endoplasmic reticulum. We suggest that the unusual structures of the glycoprotein hormones makes them useful for identifying factors that influence this process in living cells.
Highlights
Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the ␣-subunit straddles a hole in the -subunit
We suggest that the unusual structures of the glycoprotein hormones makes them useful for identifying factors that influence this process in living cells
Pulse-chase analyses of hCG assembly in cells led to the suggestion that the seatbelt remains unlatched until after the subunits dock with one another [7]. In this pathway, which we term “wraparound” (Fig. 1, upper pathway), formation of the seatbelt latch disulfide is the final step in heterodimer assembly and occurs after the seatbelt has been wrapped around loop ␣2. This pathway circumvents the need for the glycosylated end of loop ␣2 to pass through the hole in the -subunit and explains the abilities of cells to make cross-linked hormone analogs in which the seatbelt is latched to cysteines added to the ␣-subunit [8]
Summary
Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the ␣-subunit straddles a hole in the -subunit. Pulse-chase analyses of hCG assembly in cells led to the suggestion that the seatbelt remains unlatched until after the subunits dock with one another [7] In this pathway, which we term “wraparound” (Fig. 1, upper pathway), formation of the seatbelt latch disulfide is the final step in heterodimer assembly and occurs after the seatbelt has been wrapped around loop ␣2. This pathway circumvents the need for the glycosylated end of loop ␣2 to pass through the hole in the -subunit and explains the abilities of cells to make cross-linked hormone analogs in which the seatbelt is latched to cysteines added to the ␣-subunit [8]. It appears as if these human glycoproteins are assembled in the ER primarily by a threading route and that the wraparound pathway is used sparingly, if at all
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