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 glycoprotein hormone that acts through ovarian lutropin receptors (LHR) to maintain early pregnancy. Its ability to distinguish LHR and follitropin receptors (FSHR) is controlled by 20 beta-subunit 'seatbelt' residues that surround alpha-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 negatively charged amino acids in this small loop had 5-10% the activity of hCG and 1-10% the lutropin activities of hCG/hFSH chimeric analogs capable of binding LHR and FSHR. These effects might be due to the influence of these residues on receptor contacts or on hormone conformation. During efforts to distinguish these possibilities, we increased and decreased the number of residues 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 hCGalpha- and hCG/hFSHbeta-subunit chimeras overcame the effects of the seatbelt mutations on subunit combination and enabled preparation of heterodimers containing six, seven, or nine residues in their seatbelt loops. These had 0.1-10% the lutropin and 3-60% the follitropin activities of bifunctional chimeras containing 8 residues derived from hCG in the seatbelt loop. The abilities of N-terminal dimerization domains to promote subunit combination may permit structure/function analysis of other residues that influence heterodimer formation.
Lin
W
W
Department of Obstetrics and Gynecology, UMDNJ-Robert Wood Johnson (Rutgers) Medical School, Piscataway, NJ 08854, USA.
Ransom
M X
MX
Myers
R V
RV
Bernard
M P
MP
Moyle
W R
WR
D000595:Amino Acid Sequence; D001665:Binding Sites; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D011247:Pregnancy; D011485:Protein Binding; D011487:Protein Conformation; D011974:Receptors, LH / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins