Specific receptors for lutropin (luteinizing hormone; LH) and follitropin (follicle-stimulating hormone; FSH) mediate the actions of human chorionic gonadotropin (hCG) and FSH5 on the gonads. Here we report that short independent sequences of the beta-subunit enable hCG to distinguish between the receptors for FSH and LH. Residues between the 11th and 12th cysteines restrict FSH receptor binding; residues between the 10th and 11th cysteines and, to a much lesser extent, residues carboxy-terminal to the 12th cysteine also affect LH receptor binding. CF101-109, an hCG analogue containing hFSH beta residues between the 11th and 12th cysteines, had high affinity for both LH and FSH receptors. Modifications to CF101-109 that reduce binding to either LH or FSH receptors yield gonadotropin analogues having differing ratios of LH:FSH activity. Ligand-binding specificity of the LH receptor is determined by residues encoded by parts of exons 2-4 and 7-9 which prevent hFSH binding but have little effect on hCG binding. FSH receptor specificity is controlled primarily by residues encoded by exons 5 and 6 that prevent hCG binding but have little effect on hFSH binding. These determinants can be interchanged to create receptor analogues that bind hCG and hFSH. Our observations support a model in which distinct negative determinants restrict ligand-receptor interaction. This explains coevolution of binding specificity in families of homologous ligands and their receptors. Natural or designed manipulation of these determinants leads to the 'evolution' of new, specific protein-protein interactions.

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. We assembled models of lutropin, follitropin, and thyrotropin receptors by aligning models of their LRD, TMD, and shortened SSD in a manner that explains how substitutions in follitropin and thyrotropin receptors distant from their apparent ligand binding sites enable them to recognize lutropins. In these models, the SSD is parallel to the concave surface of the LRD and makes extensive contacts with TMD outer loops 1 and 2. The LRD appears to contact TMD outer loop 3 and a few residues in helices 1, 5, 6, and 7. We propose that signaling results from contacts of the ligands with the SSD and LRD that alter the LRD, which then moves TMD helices 6 and 7. The positions of the LRD and SSD support the notion that the receptor can be activated by hormones that dock with these domains in either of two different orientations. This would account for the abilities of some ligands and ligand chimeras to bind multiple receptors and for some receptors to bind multiple ligands. This property of the receptor may have contributed significantly to ligand-receptor co-evolution.

All three human glycoprotein hormone heterodimers are assembled in the endoplasmic reticulum by threading the glycosylated end of ␣-subunit loop two (␣2) beneath a disulfide "latched" strand of the ␤-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 ␤-subunit. We refer to this disulfide as the "tensor" because it shortens the seatbelt, thereby securing the heterodimer. Formation of the tensor disulfide appears to precede and facilitate seatbelt latching in most human choriogonadotropin ␤-subunit molecules. Subsequent disruption of the tensor disulfide elongates the seatbelt, thereby increasing the space beneath the seatbelt and the ␤-subunit core. This permits the formation of hydrogen bonds between backbone atoms of the ␤-subunit cystine knot and the tensor loop with backbone atoms in loop ␣2, a process that causes the glycosylated end of loop ␣2 to be threaded between the seatbelt and the ␤-subunit core. Contacts between the tensor loop and loop ␣2 promote reformation of the tensor disulfide, which explains why it is more stable in the heterodimer than in the uncombined ␤-subunit. These findings unravel the puzzling nature of how a threading mechanism can be used in the endoplasmic reticulum to assemble glycoprotein hormones that have essential roles in vertebrate reproduction and thyroid function.The glycoprotein hormones are heterodimers of two cystine knot proteins (1-3) in which a glycosylated loop of one subunit (loop ␣2) 1 is surrounded by a strand of the other "like a seatbelt" (1). This topology raises questions as to how these heterodimers might be assembled. We have found that the human glycoprotein hormone subunits combine by a process in which the glycosylated end of loop ␣2 is threaded beneath the seatbelt while it is latched (22). Although the hCG heterodimer can be assembled by a mechanism in which the seatbelt is wrapped around loop ␣2 after the subunits dock (4, 5), this appears to be a minor pathway that can be used to form some hormone analogs that are unable to latch their seatbelts to ␤-subunit loop 1. This "salvage" pathway may have had a role in the evolution of glycoprotein hormones in some teleost fish (23).Purified glycoprotein hormone subunits have long been known to recombine slowly in vitro in oxidizing conditions (6), a phenomenon that occurs while all the disulfides in both subunits remain intact (7). This showed that assembly can occur by a mechanism in which the glycosylated end of loop ␣2 is threaded beneath the seatbelt. hCG assembly is accelerated substantially by protein-disulfide isomerase (8) and low concentrations of reducing agents, however (7). Furthermore, ␤-mercaptoethanol-catalyzed assembly is blocked by agents that react with thiols, e.g. iodo...