Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit.
Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common alpha-subunit and distinct beta-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGbeta have been separately fused to the N-terminus of the alpha-subunit to give N-des[1-91]hCGbeta-alpha-C and N-des[1-91,110-114]hCGbeta-alpha-C, i.e. fusion proteins of the hCGbeta determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the alpha-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-beta-alpha cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-beta-alpha was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-beta-alpha fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGbeta in addition to the determinant loop/seat belt.
Schubert
R L
RL
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Puett
D
D
D001665:Binding Sites; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011974:Receptors, LH / Q000378:metabolism; D014162:Transfection
D006063:Chorionic Gonadotropin; D021122:Protein Subunits; D011974:Receptors, LH; D000242:Cyclic AMP