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 members of the glycoprotein hormone family, LH, FSH, and TSH, and a unique beta-subunit responsible for receptor specificity. Biologically active single chain analogs of these hormones have been engineered in which the C-terminus of 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 hCGbeta C-terminal peptide (CTP). This tandem order of subunits was chosen based on studies suggesting that the N-terminal region of hCGbeta and particularly the C-terminal region of the alpha-subunit are important in receptor binding and activation. Single chain hCG (YhCG1) can, in turn, be fused to the LH receptor to yield a hormone-receptor complex that is biologically active in transfected cells. Herein, we report the construction of a new single chain hCG analog (YhCG3) in which the C-terminus of the alpha-subunit is fused to the N-terminus of hCGbeta via a CTP (N-alpha-CTP-beta-C). Compared with YhCG1, this analog binds receptor with a 25- to 30-fold lower affinity, but, surprisingly, is capable of stimulating intracellular cAMP levels to the same extent. Furthermore, YhCG3 can be covalently linked to its receptor to produce a biologically active complex that results in elevated levels of basal cAMP in transfected cells. These results suggest that free N- and C-termini of hCGbeta and the alpha-subunit, respectively, are not essential for receptor binding and activation and that YhCG3 is in a more efficacious conformation for receptor activation than YhCG1.
Narayan
P
P
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602-7229, USA. narayan@bchiris.bmb.uga.edu
Gray
J
J
Puett
D
D
D015153:Blotting, Western; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000096:biosynthesis; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D016133:Polymerase Chain Reaction; D011974:Receptors, LH / Q000378:metabolism*; D014162:Transfection
D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D000242:Cyclic AMP; D015951:Factor Xa