Protein engineering of a novel constitutively active hormone-receptor complex.
Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone consisting of an alpha and a beta subunit that stimulates intracellular levels of cAMP via a G protein-coupled receptor. Herein we report the engineering and characterization of a novel molecule in which the receptor and its heterodimeric ligand were covalently linked in a single polypeptide chain. The hormone-receptor complex was expressed in cells transfected with this construct, but the cells were unable to bind significant amounts of exogenous hCG. However, cleavage of the hormone with a site-specific protease rendered the receptor accessible to exogenously added hormone. Cells transfected with the hCG-receptor construct contained elevated basal levels of cAMP; moreover, addition of hormone had no significant effect. These results are consistent with a strong and stable interaction between the single-chain hormone and its covalently linked receptor that results in a constitutively active complex.
Wu
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Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229, USA.
Narayan
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Puett
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D000818:Animals; D019556:COS Cells; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000737:chemistry*; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D011487:Protein Conformation; D015202:Protein Engineering; D011967:Receptors, Gonadotropin / Q000378:metabolism; D011974:Receptors, LH / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins; D015183:Restriction Mapping; D014162:Transfection
D006063:Chorionic Gonadotropin; D011967:Receptors, Gonadotropin; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; C102587:yoked hormone receptor, recombinant; D000242:Cyclic AMP