Effects of removal of carboxy-terminal extension from equine luteinizing hormone (LH) beta-subunit on LH and follicle-stimulating hormone receptor-binding activities and LH steroidogenic activity in rat testicular Leydig cells.
Residues 121-149 of equine LH beta (eLH beta) were removed by a simple mild acid treatment procedure. The modified eLH beta, des(121-149)eLH beta, was isolated by gel permeation chromatography on Sephacryl S-200. Recombination of des(121-149)eLH beta with eLH alpha and ovine LH alpha (oLH alpha) produced LH derivatives as efficiently as recombination with native eLH beta. In rat testicular LH radioligand assay systems employed in this study the potencies of the resulting LH preparations were, in order of decreasing potency: des(121-149)eLH beta:eLH alpha hybrid greater than eLH greater than eLH alpha + beta greater than oLH greater than des(121-149)eLH beta:oLH alpha greater than oLH alpha + eLH beta (1:0.82:0.67:0.15:0.02:0.006, eLH tracer; 1:0.88:0.67:0.21:0.02:0.006, hCG tracer). In a horse testicular LH radioligand assay with eLH tracer, only the equine LH derivatives were active, and the order of potencies was the same: des(121-149)eLH beta:eLH alpha hybrid greater than eLH greater than eLH alpha + beta (1:0.58:0.46). In a rat testicular Leydig cell steroidogenesis assay, eLH was the most active preparation, but the relative potencies of the other preparations remained unchanged: eLH greater than des(121-149)eLH beta:eLH alpha greater than eLH alpha + beta greater than oLH greater than des(121-149)eLH beta:oLH alpha greater than oLH alpha + eLH beta (1:0.61:0.55:0.27:0.004:0.003). We have previously reported that the hybrid consisting of native eLH beta and oLH alpha was inactive (less than 1%) in LH receptor and steroidogenesis assays. The data reported herein confirm this observation and demonstrate that the absence of LH activity in eLH beta:oLH alpha cannot be attributed to the C-terminal extension on eLH beta, since the des(121-149)eLH beta:oLH alpha hybrid LH is also inactive. Examination of the intrinsic FSH activity of eLH in both rat and chicken testicular FSH radioligand assays produced the following results; eLH, recombined eLH subunits, and des(121-149)eLH beta:eLH alpha were all of the same potency (13% and 0.9% as active as eFSH in rats and chickens, respectively). We conclude that the C-terminal extension on eLH and eCG beta-subunits is not involved in subunit association, LH receptor binding, or FSH receptor binding. The derivative des(121-149)eLH beta:eLH alpha provides a model compound that may be useful in determining the role, if any, of the glycoprotein hormone C-terminal extension that appears to have arisen independently at least twice in mammalian evolution.
Bousfield
G R
GR
Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Liu
W K
WK
Ward
D N
DN
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001681:Biological Assay; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D015292:Glycoprotein Hormones, alpha Subunit / Q000502:physiology; D006736:Horses; D006868:Hydrolysis; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000302:isolation & purification / Q000494:pharmacology / Q000502:physiology*; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D010446:Peptide Fragments / Q000502:physiology*; D055503:Protein Multimerization; D051381:Rats; D011962:Receptors, FSH / Q000378:metabolism*; D011974:Receptors, LH / Q000378:metabolism*; D013256:Steroids / Q000096:biosynthesis*; D013329:Structure-Activity Relationship
D000596:Amino Acids; D015292:Glycoprotein Hormones, alpha Subunit; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011962:Receptors, FSH; D011974:Receptors, LH; D013256:Steroids; D007986:Luteinizing Hormone