CYSTINE KNOT OF THE GONADOTROPIN ALPHA-SUBUNIT IS CRITICAL FOR INTRACELLULAR BEHAVIOR BUT NOT FOR IN-VITRO BIOLOGICAL-ACTIVITY

The common alpha subunit of glycoprotein hormones contains five disulf ide bonds. Based on the published crystal structure, the assignments a re 7-31, 59-87, 10-60, 25-82, and 32-84; the last three comprise the c ystine knot, a structure also seen in a variety of growth factors, Pre viously, we demonstrated that the efficiency of secretion and the abil ity to form heterodimers by alpha subunits hearing single cysteine res idue mutants in the cystine knot were significantly reduced. These res ults suggested that the cystine knot is critical for the intracellular integrity of the subunit, To assess if the presence of the free thiol . affected the secretion kinetics, we constructed paired cysteine muta nts of each disulfide bond of the alpha subunit. The secretion rate fo r these monomers was comparable with mild type except for the alpha-10 -60 mutant, which was 40% lower. The recovery of the alpha 7-31 and al pha 59-87 mutants was greater than 95%, whereas for the cystine knot m utants, it was 20-40%. Co-expression of the wild-type chorionic gonado tropin beta subunit with double cysteine mutants did not enhance the r ecovery of alpha mutants in the media, Moreover, compared with wild-ty pe, the efficiency of heterodimer formation of the alpha 10-60 or alph a 32-84 mutants was less than 5%. Because subunit assembly is required for biological activity, steadies oil the role of these disulfide bon ds in signal transduction mere not possible. To bypass the assembly st ep, we exploited the single chain model, where the alpha and beta subu nits are genetically fused. The recovery of secreted tethered gonadotr opins bearing mutations in the cystine knot was increased significantl y. Although dimer-specific monoclonal antibodies discriminated the con formation of single chain alpha 10-60 and alpha 32-84 mutants from the native heterodimer, these mutants were nevertheless biologically acti ve. Thus, individual bonds of cystine knot are important for secretion and heteradimer formation but not for in vitro bioactivity. Moreover, the data suggest that the native heterodimer configuration is not a p rerequisite for receptor binding or signal transduction.