Structural determinants in the second intracellular loop of the human follicle-stimulating hormone receptor are involved in G(s) protein activation.
In the present study, we analyzed the structural determinants present in the second intracellular loop (IL-2) of the human follicle-stimulating hormone (FSH) receptor (R) involved in G(s) protein-mediated signal transduction. Human embryonic kidney 293 (HEK-293) cells, stably expressing wild-type (Wt) human FSHR (HEK-293((+))), were transiently transfected with plasmids containing cDNAs encoding the entire IL-2 or several IL-2 sequences mutated in R467 (a residue located at the center of the conserved ERW motif in the glycoprotein hormone receptors), T470 (a potential site for phosphorylation by protein kinase-A and -C) or L477 (a residue conserved in all glycoprotein hormone receptors). Expression of the IL-2 Wt in HEK-293((+)) cells reduced the maximum FSH-stimulated cAMP production significantly by approximately 40%; similar results were observed with the R467A and R467K IL-2 mutants. The IL-2(R467H), IL-2(T470A), the triple R467A/T470A/L477A IL-2 mutant and the IL-2 of the oxytocin receptor (G(q/11)-coupled) had no effects on Wt FSHR-mediated intracellular signaling whereas the L477A mutation provoked a higher ( approximately 55%) inhibition of FSH-stimulated cAMP than the free, Wt IL-2. These results suggested a specific role of IL-2 residues in FSHR function. Site directed mutagenesis of the FSHR and the expression of resulting mutants in HEK-293 cells were performed in order to corroborate the effects of these substitutions. Expression of FSHR(R467H), FSHR(R467A) and FSHR(T470A) failed to mediate ligand-provoked G(s) protein activation, whereas the R467K mutant behaved as the Wt receptor. Interestingly, the expression of L477A, L477D and L477P FSHR mutants conferred elevated basal cAMP levels to HEK-293 cells. This study indicates that the IL-2 of the human FSHR possesses amino acid residues that are important for both coupling the receptor to the G(s) protein (R467 and T470) and maintaining the receptor molecule in an inactive conformation (L477). It appears that this particular intracellular domain may act as a conformational switch to produce the activation of G proteins as has been reported for the IL-2 of other G protein-coupled receptors.
Timossi
Carlos
C
Research Unit in Reproductive Medicine, Hospital de Ginecobstetricia 'Luis Castelazo Ayala', Instituto Mexicano del Seguro Social, Mexico D.F., Mexico.
Maldonado
David
D
Vizcaíno
Andrea
A
Lindau-Shepard
Barbara
B
Conn
P Michael
PM
Ulloa-Aguirre
Alfredo
A
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D011962:Receptors, FSH / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018045:Receptors, Oxytocin / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D011962:Receptors, FSH; D018045:Receptors, Oxytocin; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs