Genetic fusion of an alpha-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-beta subunit genes: Production of a bifunctional protein

The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a c ommon alpha-subunit assembled to a hormone-specific beta-subunit. The subun its combine noncovalently early in the secretory pathway and exist as heter odimers but not as multimers. LH/FSH are synthesized in the pituitary gonad otrophs, and several of the alpha-subunit sequences required for associatio n with either the LH beta or FSH beta subunits are different. Thus, it is i ntriguing that no ternary complexes are observed for LH and FSH in vivo (e. g. two different beta-assembled to a single alpha-subunit). To examine whet her the alpha-subunit can interact with more than one beta-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged beta-subunits fused to a single alpha-subunit gene (FSH beta-CG beta-alpha. This approach perm itted structure-function analyses of alpha/beta domain complexes without th e possibility of subunit dissociation. We reported previously that the CG b eta or FSH beta subunit gene can be genetically fused to the alpha-gene and the resulting single chains (CG beta alpha and FSH beta alpha, respectivel y) were biologically active. Here we demonstrate that a triple-domain singl e chain bearing the configuration FSH beta-CG beta-alpha is efficiently sec reted from transfected Chinese hamster ovary (CHO) cells and exhibits high- affinity receptor binding to both FSH and LH/hCG receptors, comparable to t he native heterodimers. These results indicate that the cu-subunit can inte ract with each beta-subunit in the same complex and that an alpha-domain fu sed to a beta-domain can still interact with an additional beta-subunit. Th e data also demonstrate the remarkable flexibility of the receptor to accom modate the increased bulkiness of the triple-domain ligand. In addition, th e formation of intrachain FSH- and CG-like complexes observed in a triple-d omain single chain suggests that the alpha-subunit can resonate, i.e. shutt le between alpha-beta heterodimeric intermediates during the early stages o f synthesis and accumulation in the endoplasmic reticulum. Such model compo unds could be useful as substrates to generate a new class of analogs in wh ich the ratio of the LH/FSH activity is varied. This could aid in the desig n of analogs that could be used to mimic the in vivo hormonal profiles.