Structure-function relationships of human choriogonadotropin beta using site-directed mutagenesis
Date of Award
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 are joined noncovalently. hCG $\beta$ is unique in that it contains a C-terminal extension not present in the other subunits. This study has focused on delineation of the minimum length of hCG $\beta$ required for hormonal activity and the identification of several charged residues in two putative receptor binding regions on hCG $\beta$. Site-directed mutagenesis was performed on hCG $\beta$ wild type cDNA to make the desired mutant cDNAs. Both wild type and mutant cDNAs were expressed in CHO cells which contain a stably integrated gene for bovine $\alpha$. The concentrations of expressed $\beta$, total and heterodimeric, were determined through radioimmunoassays. The expressed hormones were assayed in vitro using competitive receptor inhibition binding, cAMP production and progesterone production in MA-10 cells, a transformed mouse Leydig cell line.Results on two C-terminal deletion mutants of hCG $\beta$, des (101-145) and des (93-145), demonstrate that the 1-100 N-terminal fragment of $\beta$ is capable of associating with $\alpha$ to form an active hormone, albeit of reduced potency compared to that with full length $\beta$. In contrast, des (93-145) $\beta$ failed to associate with $\alpha$, thus showing that the determinant loop region 93-100, a suggested receptor binding sequence, is critical in subunit assembly. Within this region, Arg 94 and Asp 99 were also shown to be important since the single replacements, Arg 94 to Asp and Asp 99 to Arg, and the interchange, Arg 94 to Asp/Asp to Arg, yielded heterodimers with reduced (Asp 94) or zero (Arg 99) activity. In another suggested receptor binding region, residues 38-57, Arg 43 was replaced with Leu, and the resulting heterodimer was partially active. The dual replacement, however, Arg 43 to Leu and Arg 94 to Asp, yielded essentially an inactive heterodimer. Normal potencies were obtained if Arg 43 and Arg 94 were each replaced with Lys; thus, positive charge is important at these two positions. Replacing Leu 92 with Lys did not alter the potency of the heterodimer, a finding that reinforces the suggestion of charge and positional specificity at residue 94.This study has identified a minimum length of hCG $\beta$ necessary for subunit assembly and receptor binding/activation, as well as several charged amino acid residues that contribute to receptor binding.
Chen, Fang, "Structure-function relationships of human choriogonadotropin beta using site-directed mutagenesis" (1991). Dissertations from ProQuest. 2932.