Primary and secondary structural determinants in the receptor binding sequence beta-(38-57) from human luteinizing hormone.
The intercysteine "loop" sequence 38-57 in the beta subunit has been shown to be a determinant for expression of biological activity in human lutropin (hLH) and choriogonadotropin (hCG) [Keutmann, H. T., Charlesworth, M. C., Mason, K. A., Ostrea, T., Johnson, L., & Ryan, R. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2038]. Together with other sequences, the 38-57 region may contribute to a multicomponent receptor binding domain in hLH/hCG. Because the structural features influencing activity in this important region are not easy to evaluate in the full-length subunit, we have used analogues of hLH beta-(38-57) prepared by solid-phase synthesis. The peptides were tested for inhibition of 125I-labeled hCG binding to rat ovarian membrane receptors. Secondary structure was analyzed by circular dichroism (CD) and by reactivity with antibodies to the native 38-57 peptide. An analogue lacking the 38-57 disulfide linkage retained 20% receptor binding and full immunoreactivity. "Far"-ultraviolet CD profiles were essentially identical with those of the disulfide-intact peptide; a transition from 10% to 30% alpha-helix in 90% trifluoroethanol was characteristic of both. The peptide thus appears not to require the disulfide bridge to retain a looped conformation with amphipathic secondary structure. An essential positive charge at position 43 was shown by complete loss of activity upon substitution of Asp or Ala for the Arg found in all known species of LH. Other analogues showed a requirement for a neutral residue at position 47, also highly conserved.(ABSTRACT TRUNCATED AT 250 WORDS)
Keutmann
H T
HT
Endocrine Unit, Massachusetts General Hospital, Boston 02114.
Charlesworth
M C
MC
Kitzmann
K
K
Mason
K A
KA
Johnson
L
L
Ryan
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D004220:Disulfides; D005260:Female; D006801:Humans; D007986:Luteinizing Hormone / Q000378:metabolism*; D008969:Molecular Sequence Data; D010053:Ovary / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011974:Receptors, LH / Q000378:metabolism*; D013329:Structure-Activity Relationship
D004220:Disulfides; D010446:Peptide Fragments; D011974:Receptors, LH; D007986:Luteinizing Hormone