The Biological Role of the Carboxyl-Terminal Extension of Human Chorionic Gonadotroin β-Subunit

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Abstract
HCG is a member of a family of glycoprotein hormones which share a common .alpha.-subunit, but differ in their hormone-specific .beta.-subunits. The CG .beta.-subunit is unique in that it contains a hydrophilic carboxyl-terminal extension with four serine O-linked oligosaccharides. To examine the role of the O-linked oligosaccharides and the carboxyl-terminal extension of hCG.beta. on receptor binding, steroidogenesis in vitro, and ovulation induction in vivo, site-directed mutagenesis and gene transfer methods were used. Wild-type hCG.alpha. and hCG.beta. expression vectors were transfected into an O-linked glycosylation mutant Chinese hamster ovary cell line to produce intact dimer hCG lacking the .beta.-subunit O-linked oligosaccharides unit. In addition, a mutant hCG.beta. gene (CG.beta..DELTA.T) was generated which contained a premature termination signal at codon 115. This gene was cotransfected with the hCG.alpha. gene into Chinese hamster ovary cells to produce hCG dimer which lacked the carboxyl-terminal amino acids 115-145 of hCG.beta. (truncated hCG). The O-linked oligosaccharide deficient or truncated hCG derivatives were examined for their ability to bind the mouse LH/hCG receptor and stimulate cAMP and steroidogenesis in vitro. These studies show the O-linked oligosaccharides and carboxyl-terminal extension play a minor role in the receptor binding and signal transduction. In contrast, comparison of the stimulatory effects of truncated and wild-type hCG in a rat ovulation assay in vivo via either intrabursal or iv injection revealed that the truncated derivative was approximately 3-fold less active than wild-type hCG. These findings indicate that the carboxyl-terminal extension of hCG.beta. and associated O-linked oligosaccharides are not important for receptor binding or in vitro signal transduction, but are critical for in vivo biological responses.

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