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

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 approximately 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.

Differential Regulation of Two Palmitoylation Sites in the Cytoplasmic Tail of the β1-Adrenergic Receptor

“…Mutation of the distal site reduced the rates of internalization of the FSH receptor (44), and although all three palmitoylation sites of TP␤ contributed to ligand-induced internalization, only the distal site mutants were deficient in tonic internalization. By contrast, the proximal site alone was found to promote maximal coupling to G␣ q (42).…”

“…The presence of an additional distal palmitoylation site on the tail of ␤ 1 AR places it in a third group of GPCRs. The 5-hydroxytryptamine (HT) 4(a), 5-HT 7(a) , the TP␤ isoform of thromboxane A 2 (TP␤), and the follicle-stimulating hormone (FSH) receptors have all recently been reported to have distal palmitoylation sites, in addition to palmitoylation at the proximal site (41)(42)(43)(44). Functionally, there is no obvious connection among these receptors, which have varied tissue distribution, signaling pathways, and are coupled to different G proteins.…”

Normal testicular function without detectable follicle-stimulating hormone. A novel mutation in the follicle-stimulating hormone receptor gene leading to apparent constitutive activity and impaired agonist-induced desensitization and internalization

“…Asn431 (Fig. S2 E) was replaced with Ile (FSHR N431I) using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), as previously described (Uribe et al, 2008).…”

“…SDS-PAGE (7.5%) and Western blotting of protein extracts from cells transfected with the WT or mutant N431I cDNAs were performed as described previously (Uribe et al, 2008) employing the primary antibody mAb 106.105 (Nechamen and Dias, 2003). Equal protein loading was confirmed in a stripped, washed and reprobed membrane for glyceraldehyde-3-phosphate dehydrogenase detection.…”

Molecular cloning and functional analysis of the FSH receptor gene promoter from the volcano mouse (Neotomodon alstoni alstoni)

“…of the structure-function relationship of the FSHR [3,4], but also for the improvement of our knowledge of the phylogenetic relationships and divergence of the FSHR and related receptors. In sharp contrast, the 5 0 -flanking region of the FSHR has only been cloned from human and sheep, as well as from two rodent species, the rat and mouse [5][6][7][8].…”