Three single chain gonadotropins were designed based on the three-dimensional-structure of human choriogonadotropin and structure/activity relationships of the glycoprotein hormones. In each single chain, the C-terminal end of the human choriogonadotropin /? subunit is connected via Ser-Gly repeats to the N-terminal end of the a subunit. In addition, two of the single chains have truncated subunits. The three mutants were expressed in CHO cells. In vitro binding of two of the three mutants to the human lutropinl choriogonadotropin receptor was found to be comparable to wild-type lutropin. In contrast, both the receptor binding and the in vitro bioactivity of the mutant with truncated (I and /? subunits in which the p:26-110 disulphide bond cannot be formed, are lowered relative to wild-type lutropin. The fact that this mutant still displays biological activity shows that the seat-belt arrangement proposed by Isaacs and coworkers [Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. Keywords: gonadotropin ; single chain ; structure-based design; spacer; truncation.The glycoprotein hormones 11 1 form a family of structurally related proteins. Members include choriogonadotropin, follitropin, lutropin and thyrotropin. Follitropin, lutropin and thyrotropin are present in most vertebrate species and are synthesized and secreted by the pituitary. Choriogonadotropin has so far been found only in primates, including humans, and in horses, and is synthesized by placental tissue. The glycoprotein hormones are heterodimers composed of two dissimilar subunits, named 0: and /?, which are associated by non-covalent bonds.Within a species, the a subunit is the same for each member of the gonadotropin family. The , / 3 subunits are different for each member, i.e. choriogonadotropin, follitropin, thyrotropin and lutropin, and confer receptor-binding specificity on the hormones.The association of the (x and the p subunit is a very important step in the biosynthesis of the glycoprotein hormones, since only the intact dimers are biologically active. The correct assembly of the heterodimer is essential for efficient secretion, receptor binding, and signal transduction of the hormone. Free a and / 3 subunits are biologically inactive 121. Numerous mutation studies have shown that the dimerization is a highly specific process and can easily be prohibited by single point mutations. Structure/function analysis of the glycoprotein hormones is often hampered by unwanted secondary effects on the assembly of the subunits.Therefore, we and others [3 -71 have pursued approaches to determine whether the active conformation of the glycoprotein hormones can also be formed when the two subunits are synthesized in tandem on a single polypeptide chain. It has been dem- onstrated that covalently linked u and /j subunits are efficiently secreted and able to fold in a conformation that binds the lutropin/choriogonadotropin receptor with high affinity and stimulates adenylate cyclase. Thus, it can be concluded th...