A biologically active single chain human chorionic gonadotropin analog with altered receptor binding properties

hCG is a heterodimer consisting of an alpha-subunit common among all member s of the glycoprotein hormone family, LH, FSH, and TSH, and a unique beta-s ubunit responsible for receptor specificity. Biologically active single cha in analogs of these hormones have been engineered in which the C-terminus o f the beta-subunit was fused to the N-terminus of the alpha-subunit (N-beta -alpha-C) either with or without a linker such as the hCG beta C-terminal p eptide (CTP). This tandem order of subunits was chosen based on studies sug gesting that the N-terminal region of I hCG beta and particularly the C-ter minal region of the alpha-subunit are important in receptor binding and act ivation. Single chain hCG (YhCG1) can, in turn, be fused to the LH receptor to yield a hormone-receptor complex that is biologically active in transfe cted cells. Herein, we report the construction of a new single chain hCG an alog (YhCG3) in which the C-terminus of the alpha-subunit is fused to the N -terminus of hCG beta via a CTP (N-alpha-CTP-beta-C). Compared with YhCG1, this analog binds receptor with a 25- to 30-fold lower affinity, but, surpr isingly, is capable of stimulating intracellular cAMP levels to the same ex tent. Furthermore, YhCG3 can be covalently linked to its receptor to produc e a biologically active complex that results in elevated levels of basal cA MP in transfected cells. These results suggest that free N- and C-termini o f hCG beta and the alpha-subunit, respectively, are not essential for recep tor binding and activation and that YhCG3 is in a more efficacious conforma tion for receptor activation than YhCG1.