Rescue of intracellularly trapped lutropin receptor exodomain by endodomain and reconstitution of a functional membrane receptor: interaction between exo- and endodomains.
The lutropin receptor consists of an extracellular N-terminal half and a membrane-associated C-terminal half. hCG initially binds the exodomain with a high affinity and the resulting complex is thought to interact with the endodomain through a secondary contact generating a hormonal signal. Therefore, the exodomain and endodomain are likely to associate directly or indirectly with each other, but lack of fruitful materials and technology has hampered knowledge about their physical relationship and contact sites. In this work, we engineered a double-recombinant (separate exodomain and endodomain) baculovirus system successfully expressing on the surface of insect cells high levels of split LH receptor, binding the hormone with high affinity and inducing cAMP synthesis. In contrast, the exodomain and endodomain expressed separately were mostly trapped in cells. Our data indicate that the exodomain and endodomain are disulfide linked in the split receptor. When the disulfide links were reduced, the split receptor still induced cAMP up to 60%, which raises the intriguing possibility of a residual induction activity of the endodomain in the absence of high-affinity ligand binding. Our results also underscore that the targeting and transport of the LH receptor to plasma membrane require both domains, whereas each domain is independently sufficient for folding. The expression level of functional lutropin receptors is the highest ever reported. Our system may also be useful for future studies requiring a high amount of soluble secreted exodomain.
Bozon
Véronique
V
Laboratoire de Physiologie des Cellules Cardiaques et Vasculaires, CNRS-UMR 6542, Tours, France.
Couture
Laurence
L
Pajot-Augy
Edith
E
Richard
Fabien
F
Remy
Jean-Jacques
JJ
Salesse
Roland
R
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015151:Immunoblotting; D007313:Insecta; D007477:Ions; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011974:Receptors, LH / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D013552:Swine; D013997:Time Factors
D018076:DNA, Complementary; D004220:Disulfides; D007477:Ions; D008024:Ligands; D011974:Receptors, LH; D011994:Recombinant Proteins; D012965:Sodium Chloride; D000242:Cyclic AMP; D004229:Dithiothreitol