Mutagenesis of cysteine residues in the human gonadotropin α subunit. Roles of individual disulfide bonds in secretion, assembly, and biologic activity

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common α subunit but differ in their hormone-specific β subunits. Site-directed mutagenesis was used to examine the role of the five disulfide bonds in the α subunit on the folding, assembly with the hCGβ subunit, and in cases where dimer formation occurred, receptor binding and signal transduction. Cysteine residues in the disulfide bonds formed by cysteines 7-31, 10-60, 28-82, 59-87, and 32-84 (Lapthorn, A., Harris, D. Littlejohn, A., Lustbader, J. Canfield, R., Machin, K., Morgan, F., and Isaacs, N. (1994) Nature 369, 455-461) were converted to alanine, and these mutants were transfected alone or together with the wild- type hCGβ gene into Chinese hamster ovary cells. The α Cys-10, 28, 60, 82, and 84 mutants were not secreted and in most cases were degraded at a faster rate than the native subunit. In addition, these mutants failed to assemble with the hCGβ subunit. Mutants with alterations at α Cys-7, 31, 32, 59, or 87 were secreted and combined with the β subunit. Heterodimers containing a 7-31 double mutant bound to human lutropin-chorionic gonadotropin receptor expressed in transfected human fetal kidney cells, and stimulated cAMP comparable to wild-type hCG. Dimers containing the β subunit with either single mutant α59, α87, α32, or the α59-87 double mutant showed much lower affinity for the receptor than wild-type hCG. These results suggest that disulfide bonds associated with α7, α31, α59, α87, and α32 are not essential for the α subunit to fold into a form that will combine with the hCGβ subunit and to produce a biologically active dimer. This contrasts with observations of the hCGβ subunit where all the disulfide bonds are required for efficient combination and folding (Suganuma, N., Matzuk, M., and Boime, I. (1989) J. Biol. Chem. 264, 19302-19307). In addition, the lack of secretion of some mutants reflects previous observations that proteins which do not fold correctly are rapidly degraded. Thus, α subunit mutants which fold properly are secreted and can form heterodimers.