Expression of biologically active fusion genes encoding the common alpha subunit and either the CG beta or FSH beta subunits: role of a linker sequence.
The gonadotropin/thyrotropin hormone family is characterized by a heterodimeric structure composed of a common alpha subunit non-covalently linked to a hormone-specific beta subunit. The conformation of the heterodimer is essential for controlling secretion, hormone-specific post-translational modifications and signal transduction. Structure-function studies of FSH and the other glycoprotein hormones are often hampered by mutagenesis induced defects in subunit combination. Thus, the ability to overcome the limitation of subunit assembly would expand the range of structure activity relationships that can be performed on these hormones. Here we converted the FSH heterodimer to a single chain by genetically fusing the carboxyl end of the FSH beta subunit to the amino end of the alpha subunit in the presence or absence of a natural linker sequence. In the absence of the CTP linker, the secretion rate was decreased over three fold. (The CTP sequence is the last 28 amino acids of the CG beta sequence and contains four serine-linked oligosaccharides). Unexpectedly however receptor binding/signal transduction was unaffected by absence of the linker. Molecular modelling of the tethers lacking the linker sequence show that the alignment of the alpha/beta domains in the single chain differ substantially from that seen in the heterodimer. These data show that the single chain FSH was secreted efficiently and is biologically active and that the conformation determinants required for secretion and biologic activity are not the same.
Sugahara
T
T
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Grootenhuis
P D
PD
Sato
A
A
Kudo
M
M
Ben-Menahem
D
D
Pixley
M R
MR
Hsueh
A J
AJ
Boime
I
I
D000818:Animals; D016466:CHO Cells; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000235:genetics; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry / Q000235:genetics*; D006224:Cricetinae; D005640:Follicle Stimulating Hormone / Q000737:chemistry / Q000235:genetics*; D037201:Follicle Stimulating Hormone, beta Subunit; D015870:Gene Expression; D006062:Gonadotropins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008958:Models, Molecular; D011962:Receptors, FSH / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014961:X-Ray Diffraction
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D037201:Follicle Stimulating Hormone, beta Subunit; D006062:Gonadotropins; D011962:Receptors, FSH; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone