Pleiotropic effects of substitutions of a highly conserved leucine in transmembrane helix III of the human lutropin/choriogonadotropin receptor with respect to constitutive activation and hormone responsiveness.
It has been shown previously that a naturally occurring mutation of the human LH/CG receptor (hLHR), which replaces L457 in helix III with arginine, results in a receptor that constitutively elevates basal cAMP but does not respond to human CG (hCG) with further cAMP production. In the present study, substitutions of L457 with several amino acids were examined. The constitutive activation of cAMP production was observed only when L457 was replaced with a positively charged residue. Although constitutive activation of the inositol phosphate pathway could not be detected when measuring inositol phosphate production, the use of a more sensitive reporter gene assay for protein kinase C activation revealed the constitutive activation of this pathway by the R- and K-substituted mutants. Therefore, L457 of the hLHR plays a key role in stabilizing the receptor in an inactive conformation. Molecular modeling shows that the insertion of R, K, or H at position 457 triggers the receptor transition toward an active state due to the proximity of an anionic amino acid, D578, in helix VI. These substitutions cause perturbations in helix III-helix VI and helix III-helix VII interactions that culminate in the opening of a solvent-accessible site in the cytosolic domains potentially involved in Gs recognition. Interestingly, L457R was completely unresponsive and the K- and H-substituted L457 hLHR mutants were significantly blunted in their cAMP responses to hCG stimulation. Cells expressing L457R were also unresponsive to hCG with regards to increased inositol phosphate production. Other substitutions of L457 were identified, though, that selectively permit the hormonal stimulation of only one of the two signaling pathways. These results suggest a pivotal role for L457 in hormone-stimulated signal transduction by the hLHR.
Shinozaki
H
H
Department of Physiology and Biophysics, The University of Iowa, Iowa City, IA 52242, USA.
Fanelli
F
F
Liu
X
X
Jaquette
J
J
Nakamura
K
K
Segaloff
D L
DL
D000262:Adenylyl Cyclases / Q000378:metabolism; D019943:Amino Acid Substitution; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011974:Receptors, LH / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D014162:Transfection
D006063:Chorionic Gonadotropin; D007295:Inositol Phosphates; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D000262:Adenylyl Cyclases