Title

Molecular basis of the human choriogonadotropin-receptor interaction

Date of Award

1995

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

David Puett, Committee Chair

Abstract

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 noncovalently bound. The hormones act through the membrane-associated G protein-coupled receptors, in which hCG binds to the LH/CG receptor. The objective of this work is to study the determinants of gonadotropin and receptor interaction by site-directed mutagenesis. A comparison of the amino acid sequences of the mammalian $\beta$ subunits of glycoprotein hormones and the N-terminal extracellular domain of the glycoprotein hormone receptors of different species shows that some amino acid residues are invariant or highly conserved, indicating an important role in protein folding, holoprotein formation, hormone-receptor binding, or signaling.hCG$\beta$ is unique in that it contains a C-terminal extension not present in other subunits. Site-directed mutagenesis was used to prepare three deletion mutants, des(111-145), des(1-7, 111-145), and des(1-7, 101-145). A minimum functional core fragment, comprising amino acid residues 8-110, was delineated. Within this region, Lys-104 was identified to participate in receptor binding either directly as a contact site or indirectly as a conformational determinant. Two conserved amino acid residues, Gln-54 and Thr-98 on the hCG$\beta$ subunit, were mapped to be involved in holoprotein formation. Our data suggest that the two determinant loops, 38-57 and 93-100, on hCG$\beta$ possess dual functionality in heterodimer formation and hormone-receptor interaction.Mutagenesis at selected sites through the extracellular domain of the LH/CG receptor generated 23 replacements of 16 invariant or highly conserved charged residues, which are: Arg-21, Arg-31, Lys-40, Lys-55, Glu-65, Glu-68, Lys-104, Arg-114, Asp-118, Lys-121, Glu-132, Asp-135, Glu-221, Lys-235, Glu-332, and Asp-333. The results demonstrated that Glu-132 and Asp-135 are important in hormone binding. They may play a direct role in hCG binding by serving as a contact site, or they may be required for the proper folding of the receptor. Glu-332 and Asp-333, which are close to the first transmembrane helix, have been identified as important in signaling. It was also noted that a number of point mutants of the LH/CG receptor greatly reduced receptor density on transiently transfected cells.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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