Fsh isoformas

Molecular and Cellular Endocrinology 223 (2004) 17–26

Functional significance of the BBXXB motif reversed present in the cytoplasmic domains of the human follicle-stimulating hormone receptor
Carlos Timossi a , Carmen Ortiz-Elizondo a , David B. Pineda a , James A. Dias b , P. Michael Conn a,c , Alfredo Ulloa-Aguirre a,c,∗
a

Research Unit in Reproductive Medicine, Hospital deGinecobstetricia “Luis Castelazo Ayala”, Instituto Mexicano del Seguro Social, Apartado Postal 99-065, Unidad Independencia, México 10101, D.F., Mexico b Wadsworth Center, New York State Department of Health, David Axelrod Institute for Public Health, Albany, NY, USA c Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, USA Received 23 April 2004; received in revisedform 31 May 2004; accepted 9 June 2004

Abstract The minimal structural motif, BBXXB (where B represents a basic amino acid residue and X a non-basic residue), located in particular regions of the intracellular domains of cell surface membrane receptors is involved in the G protein-activating activity of a number of G protein-coupled receptors. The human FSH receptor (hFSHR) exhibits a reversedBBXXB motif (BXXBB) in the juxtamembrane region of the third intracellular loop (IL3) and the carboxyl terminus (Ctail) of the receptor; however the importance of this sequence on receptor function remains unclear. In the present study, we analyzed the effects of mutations in this structural motif on hFSHR expression, receptor-mediated effector activation and agonist-provoked receptorinternalization. Human embryonic kidney 293 cells were transiently transfected with plasmids containing the cDNA of the wild-type (Wt) hFSHR or several hFSHR mutants in which basic amino acids of the minimal structural motif at the IL3 and Ctail were replaced with alanine (i.e. AXXAA, AXXBB, BXXAB and BXXBA mutants). Alanine substitution of the three basic residues present in the IL3-BXXBB (IL3-AXXAA mutant)yielded a ≤60 kDa possibly under-glycosylated form of the FSHR, whereas the same substitutions in the Ctail resulted in the immature >62 kDa form of the receptor; both AXXAA hFSHR mutants completely failed to bind agonist and activate effector. Individual substitutions resulted in different cAMP responses to agonist stimulation: the IL3-AXXBB and IL3-BXXBA mutant hFSHRs failed to evoke Gs proteinactivation, whereas agonist-stimulated cAMP production was completely normal when the IL3-BXXAB mutant was expressed. All three IL3 mutants bound [125 I]-labelled FSH in a similar fashion to the Wt hFSHR. Ligand-binding, cell surface membrane receptor expression and agonist-provoked effector activation were significantly affected by the individual substitutions at the Ctail-BXXBB motif: theCtail-AXXBB variant exhibited reduced (∼50%) maximal cAMP response and ability to bind ligand, whereas both ligand binding and effector activation was severely reduced or abolished by expression of the Ctail-BXXBA and -BXXAB hFSHR mutants; the expression levels of the 80 kDa form of the receptor correlated with the magnitude of ligand-provoked cAMP production and binding capability of the mutantreceptors. Upon stimulation by agonist, all mutants with detectable ligand-binding activity internalized following the pattern exhibited by the Wt hFSHR species. These results indicate that the BXXBB motif at the IL3 of the hFSHR is essential for coupling the activated receptor to the Gs protein, whereas the same motif in the Ctail is apparently more important for membrane expression. © 2004 ElsevierIreland Ltd. All rights reserved.
Keywords: Follicle-stimulating hormone receptor; FSHR; BBXXB motif; Receptor mutation

1. Introduction Follicle-stimulating hormone (FSH) is produced by the anterior pituitary gland. It belongs to a family of chemically
Corresponding author. Tel.: +525 55616 2278; fax: +525 55616 2278. E-mail address: aulloaa@servidor.unam.mx (A. Ulloa-Aguirre).
∗

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