Expression of functional lutropin/choriogonadotropin receptor in the baculovirus system.
The LH/CG receptor (LH/CG-R) is a G protein-coupled receptor with a relatively large glycosylated extracellular domain. The complete 674 amino acid residue rat receptor was expressed in Sf9 insect cells using the baculovirus expression system. Optimal expression under the control of the polyhedrin promoter was obtained at 72 h post-infection and a multiplicity of infection of 0.1. The recombinant LH/CG-R was expressed on the cell surface (ca. 4500 receptors/cell) and exhibited saturable, high affinity binding of human CG (hCG) with a Kd of 0.4 nM. There was no evidence of intracellular trapping of the receptor. The intracellular concentration of cAMP was increased in response to hCG binding. In contrast, baculovirus-expressed recombinant hCG only weakly stimulated intracellular cAMP levels at relatively high doses. Two forms of the receptor (approximately 75 and approximately 200 kDa) were detected by Western blot analysis. These results demonstrate that the full length LH/CG-R expressed in insect cells is functional in that it binds hormone with high affinity and is able to couple to adenylate cylase.
Narayan
P
P
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30060, USA.
Gray
J
J
Puett
D
D
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051381:Rats; D011974:Receptors, LH / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera / Q000166:cytology
D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D000262:Adenylyl Cyclases