Addition of an N-terminal dimerization domain promotes assembly of hCG analogs: implications for subunit combination and structure-function analysis

Human chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotei n hormone that acts through ovarian lutropin receptors (LHR) to maintain ea rly pregnancy. Its ability to distinguish LHR and follitropin receptors (FS HR) is controlled by 20 beta-subunit 'seatbelt' residues that surround alph a-subunit loop 2. Positively charged amino acids between residues 93-100, a small loop within the seatbelt, have been postulated to make essential LH receptor contacts. Previous studies showed that analogs containing negative ly charged amino acids in this small loop had 5-10% the activity of hCG and 1-10% the lutropin activities of hCC/hFSH chimeric analogs capable of bind ing LHR and FSHR. These effects might be due to the influence of these resi dues on receptor contacts or on hormone conformation. During efforts to dis tinguish these possibilities, we increased and decreased the number of resi dues in this loop, mutations we anticipated would distort its conformation. Consistent with this supposition, these changes inhibited dimer formation, precluding assessment of these mutations on hormone activity. Addition of Fos and Jun dimerization domains to the N-termini of hCG alpha- and hCG/hFS H beta-subunit chimeras overcame the effects of the seatbelt mutations on s ubunit combination and enabled preparation of heterodimers containing six, seven, or nine residues in their seatbelt loops. These had 0.1-10% the lutr opin and 3-60% the follitropin activities of bifunctional chimeras containi ng 8 residues derived from hCG in the seatbelt loop. The abilities of N-ter minal dimerization domains to promote subunit combination may permit struct ure/function analysis of other residues that influence heterodimer formatio n. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.