Human lutropin (hLH) and choriogonadotropin (CG) are assembled by different pathways: a model of hLH assembly.

The glycoprotein hormones are all structurally related heterodimers consisting of an α-subunit and a ligand-specific β-subunit that confers their unique biological activity. Crystal structures showed how the β-subunit surrounds a part of the α-subunit, and we showed the existence of the two mechanisms responsible for that assembly. In human choriogonadotropin, the β-subunit is folded before the subunits dock, and the α-subunit becomes incorporated into the dimer by a mechanism we termed "threading," passing between parts of the preassembled β-subunit.

Follitropin receptors contain cryptic ligand binding sites.

Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with different regions of an FSHR/LHR chimera having only two unique LHR residues and that binds both hormones with high affinity. hCG and hFSH analogs dock with this receptor chimera in a manner similar to that in which they bind LHR and FSHR, respectively.

Models of glycoprotein hormone receptor interaction.

The glycoprotein hormones regulate reproduction and development through their interactions with receptors in ovarian, testicular, and thyroid tissues. Efforts to design hormone agonists and antagonists useful for treat-ing infertility and hyperthyroidism would benefit from a molecular understanding of hormone-receptor interaction. The structure of a complex containing FSH bound to a fragment of its receptor has been determined at 2.

Crosslinked bifunctional gonadotropin analogs with reduced efficacy.

The N-linked oligosaccharides on human choriogonadotropin (hCG) and follitropin (hFSH) alpha-subunit loop 2 (alpha2) have a dominant influence on hormone efficacy. hCG analogs lacking this oligosaccharide retain approximately 40% the efficacy of the fully glycosylated hormone in cyclic AMP accumulation assays. Previous efforts to reduce efficacy further have involved removing the other N-linked oligosaccharides.

Only a portion of the small seatbelt loop in human choriogonadotropin appears capable of contacting the lutropin receptor.

Twenty residues of the human choriogonadotropin (hCG) beta-subunit that are wrapped around alpha-subunit loop 2 like a "seatbelt" stabilize the heterodimer and enable the hormone to distinguish lutropin (LHR), follitropin, and thyrotropin receptors. The N-terminal portion of the seatbelt contains a small disulfide-stabilized loop needed for heterodimer assembly and is thought to mediate hCG-LHR interactions. To test the latter notion, we compared the LHR binding and signal transduction activities of hCG analogs in which the alpha-subunit C terminus (alphaCT) was cross-linked to residues in the small seatbelt loop.

Model of glycoprotein hormone receptor ligand binding and signaling.

Studies described here were initiated to develop a model of glycoprotein hormone receptor structure and function. We found that the region that links the lutropin receptor leucine-rich repeat domain (LRD) to its transmembrane domain (TMD) has substantial roles in ligand binding and signaling, hence we term it the signaling specificity domain (SSD). Theoretical considerations indicated the short SSDs in marmoset lutropin and salmon follitropin receptors have KH domain folds.

Use of protein knobs to characterize the position of conserved alpha-subunit regions in lutropin receptor complexes.

Efforts to identify the manner in which human choriogonadotropin (hCG) contacts lutropin receptors (LHR) have been stymied by the complex structure of the hormone and the likelihood that it contacts the receptor at multiple sites. During studies of hCG assembly in mammalian cells, we found that addition of a cysteine to the long disordered beta-subunit COOH terminus (betaCT) enabled it to become cross-linked by a disulfide to cysteines that are substituted for residues in loop alpha2 or in the alpha-subunit COOH terminus (alphaCT). This created a "knob" on the alpha-subunit at the location of the cysteine.

Glycoprotein hormone assembly in the endoplasmic reticulum: IV. Probable mechanism of subunit docking and completion of assembly.

The unique structures of human choriogonadotropin (hCG) and related glycoprotein hormones make them well suited for studies of protein folding in the endoplasmic reticulum. hCG is stabilized by a strand of its beta-subunit that has been likened to a "seatbelt" because it surrounds alpha-subunit loop 2 and its end is "latched" by an intrasubunit disulfide bond to the beta-subunit core. As shown here, assembly begins when parts of the NH(2) terminus, cysteine knot, and loops 1 and 3 of the alpha-subunit dock reversibly with parts of the NH(2) terminus, cystine knot, and loop 2 of the hCG beta-subunit.

Glycoprotein hormone assembly in the endoplasmic reticulum: I. The glycosylated end of human alpha-subunit loop 2 is threaded through a beta-subunit hole.

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.

Glycoprotein hormone assembly in the endoplasmic reticulum: III. The seatbelt and its latch site determine the assembly pathway.

Vertebrate glycoprotein hormone heterodimers are stabilized by a strand of their beta-subunits known as the "seatbelt" that is wrapped around loop 2 of their alpha-subunits (alpha2). The cysteine that terminates the seatbelt is "latched" by a disulfide to a cysteine in beta-subunit loop 1 (beta1) of all vertebrate hormones except some teleost follitropins (teFSH), wherein it is latched to a cysteine in the beta-subunit NH(2) terminus. As reported here, teFSH analogs of human choriogonadotropin (hCG) are assembled by a pathway in which the subunits dock before the seatbelt is latched; assembly is completed by wrapping the seatbelt around loop alpha2 and latching it to the NH(2) terminus.

Glycoprotein hormone assembly in the endoplasmic reticulum: II. Multiple roles of a redox sensitive beta-subunit disulfide switch.

All three human glycoprotein hormone heterodimers are assembled in the endoplasmic reticulum by threading the glycosylated end of alpha-subunit loop two (alpha2) beneath a disulfide "latched" strand of the beta-subunit known as the "seatbelt." This remarkable event occurs efficiently even though the seatbelt effectively blocks the reverse process, thereby stabilizing each heterodimer. Studies described here show that assembly is facilitated by the formation, disruption, and reformation of a loop within the seatbelt that is stabilized by the most easily reduced disulfide in the free beta-subunit.

Tight attachment of chitin-binding-domain-tagged proteins to surfaces coated with acetylated chitosan.

Several excellent procedures for trapping tagged proteins have been devised, but many of these are expensive, cannot be used outside a limited pH range, fail to work in the presence of chaotropic agents, or are difficult to use. The chitin binding domain (CBD) of Bacillus circulans chitinase, which binds to chitin matrices prepared from inexpensive reagents isolated from crab shells, is an alternative tag that can be used under a variety of pH and denaturing conditions. Kits based on the interaction between the CBD and the chitin beads are available commercially.

Efficient preparation of glycoprotein hormones lacking an alpha-subunit oligosaccharide.

The oligosaccharide on alpha-subunit loop 2 (alpha 2) is needed for full glycoprotein hormone efficacy. Efforts to prepare glycoprotein hormone antagonists usually involve removing the alpha 2 oligosaccharide and are hampered by its requirement for efficient heterodimer secretion from mammalian cells. Here we show that hormones lacking this oligosaccharide can be produced by treating them at low pH to dissociate the heterodimer and permitting the subunits to re-associate in the presence of peptide N-glycosidase F (PNGase F).