Cysteine-699, a Possible Palmitoylation Site of the Thyrotropin Receptor, Is Not Crucial for cAMP or Phosphoinositide Signaling but Is Necessary for Full Surface Expression

doi:10.1006/bbrc.1996.0648

Biochemical and Biophysical Research Communications

Volume 221, Issue 3, 25 April 1996, Pages 636-640

https://doi.org/10.1006/bbrc.1996.0648 Get rights and content

Abstract

We investigated the role of the cysteine residues in the cytoplasmic tail of the thyrotropin receptor (TSHR) in TSH binding and signal transduction by individually mutating two cysteine residues to serine. Neither mutant exhibited changes in basal, TSH- or Graves' IgG-stimulated cAMP or inositol phosphate responses. However, mutation of Cys-699 significantly decreased TSH binding B_maxwithout significantly changing binding affinity, amount or sizes of TSHR forms on Western blots. These findings suggest that Cys-699, which is a potential site for lipid modification and whose equivalent in other receptors was shown to be palmitoylated, is important for efficiency of proper membrane insertion and/or stability of the receptor.

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Cited by (21)

The Thyroid-Stimulating Hormone Receptor: Impact of Thyroid-Stimulating Hormone and Thyroid-Stimulating Hormone Receptor Antibodies on Multimerization, Cleavage, and Signaling
2009, Endocrinology and Metabolism Clinics of North America
Citation Excerpt :
Cysteine 699 in the cytoplasmic domain of the receptor is palmitoylated. Mutation of this site leads to delay in the trafficking of functionally normal receptors to the cell surface.59 It is well known that in addition to acylation of the protein, palmitoylation is another modification that is important in targeting some receptors into lipid rafts.53
The thyroid-stimulating hormone receptor (TSHR) has a central role in thyrocyte function and is also one of the major autoantigens for the autoimmune thyroid diseases. We review the post-translational processing, multimerization, and intramolecular cleavage of TSHR, all of which may modulate its signal transduction. The recent characterization of monoclonal antibodies to the TSHR, including stimulating, blocking, and neutral antibodies, have also revealed unique biologic insights into receptor activation and the variety of these TSHR antibodies may help explain the multiple clinical phenotypes seen in autoimmune thyroid diseases. Knowledge of the structure/function relationship of the TSHR is beginning to provide a greater understanding of thyroid physiology and thyroid autoimmunity.
Modulation of TSHR signaling by posttranslational modifications
2007, Trends in Endocrinology and Metabolism
Citation Excerpt :
The mature TSHR is palmitoylated on Cys699 in the cytoplasmatic domain of the receptor. Mutation of Cys699 to alanine (C699A) delays the cell-surface expression of the TSHR, but with respect to TSH binding and signaling the mutant is completely active [32]. Moreover, palmitoylation of the TSHR serves as a label for the entrance into sphingolipid-cholesterol domains (lipid rafts) [33] (Table 1).
Posttranslational modifications of seven transmembrane receptors (7TMRs) affect their function to a large extent. Many studies of glycosylation or phosphorylation of 7TMRs have shown that these modifications influence the cell-surface expression or signaling of the receptor. Recently, other types of posttranslational modifications of the thyrotropin-stimulating hormone receptor (TSHR) have been characterized, including sialylation and dimerization. Increased TSHR sialylation results in increased TSHR cell-surface expression. Furthermore, TSHR oligomerization and the probable modification of TSHR signaling in lipid rafts require further clarification with regard to their functional consequences. In addition to its known coupling to Gαs and Gαq, and possibly other G proteins, the TSHR also couples to further signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathway, which involves G-protein-coupled receptor kinases (GRKs) and arrestins. We discuss these emerging new findings and their implications for signaling of the TSHR.
Role of the intracellular domains of the human FSH receptor in G<inf>αS</inf> protein coupling and receptor expression
2007, Molecular and Cellular Endocrinology
The human (h) follicle-stimulating hormone receptor (FSHR) belongs to the superfamily of G protein-coupled receptors (GPCRs). This receptor consists of 695 amino acid residues and is preferentially coupled to the G_s protein. This receptor is highly conserved among species (overall homology, 85%), with a 25–69% homology drop when compared to the human LH and TSH receptors. Although studies in prototypical rhodopsin/β-adrenergic receptors suggest that multiple domains in the intracellular loops (iL) and the carboxyl-terminus (Ctail) of these receptors contribute to G protein coupling and receptor expression, there is a paucity of structure/function data on the role of these domains in FSHR function. Employing point mutations we have found that several residues present in the iL2 of the hFSHR are important for both coupling the receptor to the G_s protein and maintaining the receptor molecule in an inactive conformation. In fact, HEK-293 cells expressing several hFSHR mutants with substitutions at R⁴⁵⁰ (central to the highly conserved ERW triplet motif) and T⁴⁵³ (a potential target for phosphorylation) failed to mediate ligand-provoked G_s protein activation but not agonist binding, whereas substitutions at the hydrophobic L⁴⁶⁰ (a conserved residue present in all glycoprotein hormone receptors) conferred elevated basal cAMP to the transfected cells. Thus, this particular loop apparently acts as a conformational switch for allowing the receptor to adopt an active conformation upon agonist stimulation. Residues in both ends of the iL3 are important for signal transduction in a number of GPCRs, including the FSHR. We have recently explored the importance of the reversed BBXXB motif (BXXBB; where B represents a basic residue and X a non-basic residue) present in the juxtamembrane region of the hFSHR iL3. A hFSHR mutant with all basic amino acids present in the iL3 BXXBB motif replaced by alanine failed to bind agonist and activate effector, and was expressed as an immature ≤62 kDa form of the receptor. Individual substitutions of basic residues resulted in mutants that bound agonist normally but failed to activate effector when replaced at R⁵⁵² or R⁵⁵⁶. Triple mutations in the same motif located in the NH₂-end of the Ctail resulted in a complete inability of the receptor to bind agonist and activate effector, whereas individual substitutions resulted in decreased or virtually abolished agonist binding and cAMP accumulation, with both functions correlating with the detected levels of mature (80 kDa) forms of the receptor. Thus, the BXXBB motif at the iL3 of the FSHR is essential for coupling the activated receptor to the G_s protein, whereas the same motif in the Ctail is apparently more important for membrane expression. The role of cysteine residues present in the Ctail of the FSHR is an enigma since there are no conserved cysteines amongst LHR, FSHR and TSHR. C⁶²⁹ and C⁶⁵⁵ are conserved in the gonadotropin receptors but not in the TSHR. Alanine replacement of C⁶²⁷ had no effect on hFSHR expression and function, whereas the same mutation at C⁶²⁹ altered membrane expression and signal transduction. Serine or threonine substitutions of C⁶⁵⁵ did not modify any of the parameters analyzed. In the hFSHR, C⁶²⁹ may be a target for palmitoylation, and apparently it is the only cysteine residue in the Ctail domain that might play an important role in receptor function.
Palmitoylation of CCR5 Is Critical for Receptor Trafficking and Efficient Activation of Intracellular Signaling Pathways
2001, Journal of Biological Chemistry
Citation Excerpt :
The role of palmitoylation on GPCR endocytosis varies according to the specific receptor studied. Preventing receptor palmitoylation has been reported to increase (50, 66, 67), decrease (38, 39, 68-70), or not affect (40,51, 52) internalization of specific GPCRs. Finally, we have investigated the influence of palmitoylation onto the HIV coreceptor function of CCR5.
CCR5 is a CC chemokine receptor expressed on memory lymphocytes, macrophages, and dendritic cells and also constitutes the main coreceptor for macrophage-tropic (or R5) strains of human immunodeficiency viruses. In the present study, we investigated whether CCR5 was palmitoylated in its carboxyl-terminal domain by generating alanine substitution mutants for the three cysteine residues present in this region, individually or in combination. We found that wild-type CCR5 was palmitoylated, but a mutant lacking all three Cys residues was not. Through the use of green fluorescent fusion proteins and immunofluorescence studies, we found that the absence of receptor palmitoylation resulted in sequestration of CCR5 in intracellular biosynthetic compartments. By using the fluorescence recovery after photobleaching technique, we showed that the non-palmitoylated mutant had impaired diffusion properties within the endoplasmic reticulum. We next studied the ability of the mutants to bind and signal in response to chemokines. Chemokines binding and activation of G_i-mediated signaling pathways, such as calcium mobilization and inhibition of adenylate cyclase, were not affected. However, the duration of the functional response, as measured by a microphysiometer, and the ability to increase [³⁵S]guanosine 5′-3-O-(thio)triphosphate binding to membranes were severely affected for the non-palmitoylated mutant. The ability of RANTES (regulated on activation normal T cell expressed and secreted) and aminooxypentane-RANTES to promote CCR5 endocytosis was not altered by cysteine replacements. Finally, we found that the absence of receptor palmitoylation reduced the human immunodeficiency viruses coreceptor function of CCR5, but this effect was secondary to the reduction in surface expression. In conclusion, we found that palmitoylated cysteines play an important role in the intracellular trafficking of CCR5 and are likely necessary for efficient coupling of the receptor to part of its repertoire of signaling cascades.
The proximal portion of the COOH terminus of the oxytocin receptor is required for coupling to G(q), but not G(i). Independent mechanisms for elevating intracellular calcium concentrations from intracellular stores
1999, Journal of Biological Chemistry
As the oxytocin receptor plays a key role in parturition and lactation, there is considerable interest in defining its structure/functional relationships. We previously showed that the rat oxytocin receptor transfected into Chinese hamster ovary cells was coupled to both G_q/11 and G_i/o, and that oxytocin stimulated ERK-2 phosphorylation and prostaglandin E₂ synthesis via protein kinase C activity. In this study, we show that deletion of 51 amino acid residues from the carboxyl terminus resulted in reduced affinity for oxytocin and a corresponding rightward shift in the dose-response curve for oxytocin-stimulated [Ca²⁺]_i. However, oxytocin-stimulated ERK-2 phosphorylation and prostaglandin E₂ synthesis did not occur in cells expressing the truncated receptor. Oxytocin also failed to increase phospholipase A activity or activate protein kinase C, indicating that the mutant receptor is uncoupled from G_q-mediated pathways. The Δ51 receptor is coupled to G_i, as oxytocin-stimulated Ca²⁺ transients were inhibited by pertussis toxin, and a Gβγ sequestrant. Preincubation of Δ51 cells with the tyrosine kinase inhibitor, genistein, also blocked the oxytocin effect. A Δ39 mutant had all the activities of the wild type oxytocin receptor. These results show that the portion between 39 and 51 residues from the COOH terminus of the rat oxytocin receptor is required for interaction with G_q/11, but not G_i/o. Furthermore, an increase in intracellular calcium was generated via a G_iβγ-tyrosine kinase pathway from intracellular stores that are distinct from G_q-mediated inositol trisphosphate-regulated stores.
Mutant isoforms of the anti-Mullerian hormone type II receptor are not expressed at the cell membrane
1996, Journal of Biological Chemistry
Anti-Müllerian hormone, a member of the transforming growth factor β superfamily, produces early regression of Müllerian ducts in the male fetus through binding to a serine/threonine kinase receptor, homologous to type II receptors of the transforming growth factor β (TGF-β) family. A splice mutation of this receptor, described in a patient with abnormal retention of Müllerian derivatives, generates two mutant isoforms, one lacking the second exon and the other bearing an insertion of 12 bases between exons 2 and 3. Using hemagglutinin-tagged recombinant receptors, we have visualized wild type and mutant receptors in COS cells by Western blotting and immunoprecipitation. The 82-kDa, endoglycosidase H-insensitive, mature form of the wild type receptor is reduced to 68 kDa by N-glycosidase F treatment. Mutant receptor isoforms, 73 and 63 kDa for the long and short form, respectively, are sensitive to endoglycosidase H, suggesting that they are retained in the endoplasmic reticulum. Indeed, only the wild type receptor was expressed on the cell surface and bound iodinated anti-Müllerian hormone. These results provide a biological explanation for the failure of the mutant receptor to induce Müllerian regression.

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Regular ArticleCysteine-699, a Possible Palmitoylation Site of the Thyrotropin Receptor, Is Not Crucial for cAMP or Phosphoinositide Signaling but Is Necessary for Full Surface Expression

Abstract

Regular Article
Cysteine-699, a Possible Palmitoylation Site of the Thyrotropin Receptor, Is Not Crucial for cAMP or Phosphoinositide Signaling but Is Necessary for Full Surface Expression