A single-chain bifunctional gonadotropin analog is secreted from Chinese hamster ovary cells as two distinct bioactive species.
One of the major developments in exploring structure activity relationships of the glycoprotein hormone family was the genetic engineering of single chains comprised of the common alpha subunit and one or more of the hormone-specific beta subunits tandemly arranged. These studies indicate that there is a structural permissiveness in the quaternary relationships between the subunits and biological activity. However, the conformational relationships between the ligand and the receptor are unclear. Bifunctional triple-domain analogs represent an ideal model to address this issue. Does a single molecule possess the ability to simultaneously interact with both specific receptors or are there two functionally distinct species in the chimeric population? Here we show, using a preadsorption protocol comprised of Chinese hamster ovary cells expressing either the luteinizing hormone (LH)/chorionic gonadotropin (CG) or follicle-stimulating hormone (FSH) receptor, that at least two distinct bioactive populations of the dually active triple-domain chimera FSHbeta-CGbeta-alpha are synthesized, each corresponding to a single activity (CG or FSH). Furthermore, we show that these bioactive populations form distinct stable heterodimer-like contacts. That there is not a single biologically active species formed during synthesis of the chimera implies that in vivo the heterodimer exists in multiple conformations and is not a static rigid molecule.
Department of Molecular Biology & Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Vicenta
V
Garcia-Campayo
Albina
A
Jablonka-Shariff
Irving
I
Boime
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015153:Blotting, Western; D016466:CHO Cells; D006063:Chorionic Gonadotropin / Q000378:metabolism; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006062:Gonadotropins / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011863:Radioimmunoassay; D011962:Receptors, FSH / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D017077:Culture Media, Conditioned; D006062:Gonadotropins; D008024:Ligands; D011962:Receptors, FSH; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone