Submitted July 3, 2007
Returned for revision August 16, 2007
Accepted January 8, 2008
Neuroendocrinology
Functional and Structural Roles of Conserved Cysteine Residues in the Carboxyl-Terminal Domain of the Follicle-Stimulating Hormone Receptor in Human Embryonic Kidney 293 Cells
Aída Uribe ,
Teresa Zariñán ,
Marco A. Pérez-Solis ,
Rubén Gutiérrez-Sagal ,
Eduardo Jardón-Valadez ,
Ángel Piñeiro ,
James A. Dias ,
and
Alfredo Ulloa-Aguirre *
* To whom correspondence should be addressed. E-mail: aulloaa{at}servidor.unam.mx.
Abstract
The carboxyl-terminal segment of G protein-coupled receptors has one or more conserved cysteine residues which are potential sites for palmitoylation. This post-translational modification contributes to membrane association, internalization, and membrane targeting of proteins. In contrast to other members of the glycoprotein hormone receptor family (the LH and thyroid-stimulating hormone receptors), it is not known whether the follicle-stimulating hormone receptor (FSHR) is palmitoylated and what are the effects of abolishing its potential palmitoylation sites. In the present study, a functional analysis of the FSHR carboxyl-terminal segment cysteine residues was carried out. We constructed a series of mutant FSHRs by substituting cysteine residues with alanine, serine or threonine individually and together at positions 629 and 655 (conserved cysteines) and 627 (non-conserved). The results showed that all three cysteine residues are palmitoylated but that only modification at Cys629 is functionally relevant. The lack of palmitoylation does not appear to greatly impair coupling to Gs, but when absent at position 629 significantly impairs cell surface membrane expression of the partially palmitoylated receptor. All FSHR Cys mutants were capable of binding agonist with the same affinity as the wild-type receptor and internalizing upon agonist stimulation. Molecular dynamics simulations at a time scale of ~100 ns revealed that replacement of Cys629 resulted in structures that differed significantly from that of the wild-type receptor. Thus deviations from wild type conformation may potentially contribute to the severe impairment in plasma membrane expression and the modest effects on signaling exhibited by the receptors modified in this particular position.
Key words:
Follicle-stimulating hormone •
Follicle-stimulating hormone receptor •
biosynthesis •
expression •
palmitoylation
