Signal Transduction of Constitutive Activating and Inactivating Eel Lutropin/Choriogonadotropin Receptor (Eel LH/CGR) Mutants by Recombinant Equine Chorionic Gonadotropin (Rec-eCG).

Abstract

Lutropin/choriogonadotropin receptor (LH/CGR) is a member of the G protein-coupled receptor superfamily. LH/CGRs in fish and mammalian species have been reported to contain naturally occurring, constitutively activating, and inactivating mutations in highly conserved regions. The present study was designed to determine the functional aspect of eel LH/CGR signal transduction. Biochemical analysis was performed using cells transfected with wild-type eel LH/CG (eel LH/CGR-wt) or with activating (designated eel LH/CGR-M410T, L469R, and D590Y) and inactivating (eel LH/CGR-D 417N and Y558F) mutants. We also generated a mutant (eel LH/CGR-t651) in which the C-terminal cytoplasmic tail was truncated at residue 651. Activating mutant cells expressing eel LH/CGR-M410T, L469R, and D590Y exhibited 1.4-, 8.7-, and 4.0-fold increases in the basal cAMP response, respectively, without recombinant equine chorionic gonadotropin (rec-eCG) agonist treatment. In inactivating mutants (eel LH/CGR-D417N and Y558F), the cyclic adenosine monophosphate (cAMP) response did not result in completely impaired signal transduction. However, the eel LH/CGR-t651 mutant did not exhibit any cAMP signaling following high-agonist treatment. Rmax values did not increase with further rec-eCG agonist stimulation. Our results suggest that constitutively activating and inactivating eel LH/CGR mutants with highly conserved amino acids exhibit a significant signal transduction pathway for glycoprotein hormone receptors. Eel LH/CGRs in activating and inactivating mutants are usually processed by receptor-mediated signaling following rec-eCG agonist stimulation.