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This Article Full Text Full Text (PDF) [Supplemental Data] All Versions of this Article: 78/5/869    most recent biolreprod.107.063925v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Uribe, A. Articles by Ulloa-Aguirre, A. PubMed PubMed Citation Articles by Uribe, A. Articles by Ulloa-Aguirre, A. Agricola Articles by Uribe, A. Articles by Ulloa-Aguirre, A.

Functional and Structural Roles of Conserved Cysteine Residues in the Carboxyl-Terminal Domain of the Follicle-Stimulating Hormone Receptor in Human Embryonic Kidney 293 Cells¹

Wadsworth Center,⁶ David Axelrod Institute for Public Health, New York State Department of Health, and Department of Biomedical Sciences, State University of New York at Albany, Albany, New York 12208 Research Unit in Reproductive Medicine,⁴ Hospital de Ginecobstetricia "Luis Castelazo Ayala," Instituto Mexicano del Seguro Social, México 10101 D.F., México Depto de Fisicoquímica,⁵ Facultad de Química, Universidad Nacional Autónoma de México (UNAM), México 04510 D.F., México

ABSTRACT

The carboxyl-terminal segment of G protein-coupled receptors has one or more conserved cysteine residues that are potential sites for palmitoylation. This posttranslational 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 (nonconserved). 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 G_s but, when absent at position 629, does significantly impair 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 on agonist stimulation. Molecular dynamics simulations at a time scale of 100 nsec 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.

biosynthesis, expression, follicle-stimulating hormone, follicle-stimulating hormone receptor, palmitoylation

¹Supported by grants 2005/1/I/002 from the FOFOI-IMSS, México (to A.U.-A.); grants 45991 (to A.U.-A.) and J-49811Q (to Á.P.) from CONACyT, México; and grant HD18407 from the NIH, Bethesda, MD (to J.A.D.). A.U.-A. is recipient of a Research Career Development Award from the Fundación IMSS, México. Presented in part at the 88th Annual Meeting of the Endocrine Society, Boston, Massachusetts, June 2006, Abstract P3–280.

Correspondence: ²Alfredo Ulloa-Aguirre, Research Unit in Reproductive Medicine, IMSS, Apdo. Postal 99–065, Unidad Independencia, México 10101 D.F., México. FAX: 52 55 5616 2278; e-mail: aulloaa{at}servidor.unam.mx

Correspondence: ³These authors contributed equally to this work.