Structure-function relationships of human chorionic gonadotropin using site-directed mutagenesis: Contact sites for holoprotein formation and receptor binding

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology

First Committee Member

David Puett, Committee Chair


Human choriogonadotropin (hCG) is a member of the glycoprotein hormone family. These hormones form heterodimers in which the common $\alpha$ subunit and the hormone-specific $\beta$ subunit associate noncovalently. A comparison of the amino acid sequences of the mammalian $\beta$ subunits of glycoprotein hormones shows that some amino acid residues are invariant or highly conserved. Such stringent conservation suggests an important role for those residues in chain folding, holoprotein formation, or receptor binding/activation.Site-directed mutagenesis was used to prepare and characterize 23 replacements of 17 invariant or highly conserved amino acid residues in hCG $\beta$ for which there is little, if any, information available: Lys-2, Pro-7, Thr-32, Tyr-37, Thr-40, Val-56, Tyr-59, Phe-64, Arg-68, Pro-70, Gly 71, Ser-81, Tyr-82, Val-84, Ala-85, Ser-87, and Arg-95. In addition, a double mutant, in which Thr-40 and Val-56 were each replaced with Arg, and a multiple mutant, in which Arg-68-Leu-69-Pro-70-Gly-71 was replaced with Ala-Ala-Ala-Ala were prepared and characterized.The results demonstrated that Tyr-37, Thr-40, Tyr-59, and Ala-85 participate in holoprotein formation, directly as contact sites or indirectly as conformational determinants. Lys-2 and Arg-95 are important in receptor binding. Replacements of Arg-68, Ser-81, Tyr-82, and perhaps Leu-69 interfered with secretion, perhaps via conformational effects such that the mutant subunits are degraded more rapidly than wild-type $\beta$.A previous study suggested that the middle portion (amino acid residues 36-51) of human $\alpha$ is a subunit determinant in holoprotein formation. Site-directed mutagenesis was used to demonstrate that Ala-36 and Pro-38 of human $\alpha$ participate in subunit association.This work has identified several amino acid residues in the $\alpha$ and $\beta$ subunits of hCG that are important in holoprotein formation and others on the $\beta$ subunit that function in receptor binding. Also, a region in hCG $\beta$ was delineated that appears to be important in proper chain folding and secretion. These results, in conjunction with other findings, have led to a "duality model" in which neighboring amino acid residues on $\alpha$ and $\beta$ subunits function both in subunit assembly and receptor recognition.


Biology, Molecular

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