Role of palmitoylation of conserved cysteine residues of luteinizing hormone/human choriogonadotropin receptors in receptor down-regulation1
Introduction
The biological actions of luteinizing hormone (LH) or human chorionic gonadotropin (hCG) are mediated by the specific interaction of the ligand with the Gs-protein coupled receptor to increase the intracellular levels of cyclic AMP (cAMP), which in turn, stimulates gonadal steroidogenesis [1]. The LH/hCG receptor belongs to the family of G-protein coupled receptors which is composed of three regions: an extracelluar amino-terminal region, a seven helical membrane spanning region and an intracellular carboxyl terminal region 2, 3. The interaction of LH/hCG with receptor has also been shown to activate phosphatidylinositol breakdown, although the physiological consequence of this pathway in ovarian function has yet to be determined 4, 5.
Recent studies from our laboratory have shown that the cysteine residues 621 and 622 of the LH/hCG receptor form thioester linkages with palmitic acid [6]. These findings were independently confirmed in a subsequent report [7]. This situation is analogous to that seen for rhodopsin 8, 9and the β2-adrenergic receptor [10]where the carboxy terminal cysteine residues form covalent linkages with palmitic acid. In the case of β2 adrenergic receptor, the presence of palmitoylated cysteine residue on the carboxy terminal region is reported to be critical for the efficiency of coupling to Gs protein [10]. In the case of α2A adrenergic receptor, mutation of the conserved cysteine residue had no apparent effect on its coupling to G protein [11]. Furthermore, mutation of the cys 341 residue to glycine in the β2 adrenergic receptor has been shown to increase the phosphorylation state of the receptor which leads to the uncoupling of the receptor from Gs protein [12].
In the present study we have examined the functional consequences of palmitoylation of the LH/hCG receptor in transfected human embryonic kidney cells. Our results show that mutation of conserved cysteine residues 621 and 622 to serine or glycine residues leads to an increase in ligand-induced receptor down-regulation. Furthermore, abolition of palmitoylation has no effect on the coupling of the receptor to Gs protein.
Section snippets
Chemicals and reagents
HCG (CR-127) was a gift from the Center for Population Research, National Institutes of Child Health and Human Development, (Bethesda, MD). The sodium salt of [125I] was purchased from ICN Biomedicals (Irvine, CA). Dulbecco's modified Eagle's medium (DMEM), Waymouth's MB752/1 medium, and fetal bovine serum (FBS) were purchased from Life Technologies, (Grand Island, NY). Bovine serum albumin (BSA, Fraction V) was purchased from Sigma (St. Louis, MO). The Altered Sites In Vitro Mutagenesis System
Effect of mutation of conserved cysteine residues on G protein coupling
In order to determine the role of palmitoylation of the two conserved cysteine residues on the efficiency of Gs protein coupling, 293T cells were transfected with wild type and mutant receptors and the effect of exogenously added hCG on cAMP production was examined. Since the dose response characteristics for cAMP production may be affected by the receptor density, initial experiments were carried out to determine the relationship between the receptor numbers and the responsiveness of cells to
Discussion
In the present study, we show that palmitoylation-deficient mutants of the LH/hCG receptor undergo down-regulation more rapidly than the wild type receptor, where both cysteine residues are palmitoylated. Down-regulation is defined as the loss of cell surface receptors due to prolonged exposure to homologous ligand. Down-regulation might result from increased endocytosis since palmitoylation deficient mutant receptors are internalized more rapidly than the wild type receptor. Since there are no
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Restricted lateral diffusion of luteinizing hormone receptors in membrane microdomains
2011, Journal of Biological ChemistryCitation Excerpt :Interestingly, individual LHR-C621S,C622S were generally not confined in small compartments. Nevertheless, their diffusion coefficients were slower than those of either untreated FLAG-LH receptors or FLAG-LH receptors on cells treated with cytochalasin D. Although palmitoylation-deficient mutant LH receptors are not found in rafts (12), they retain their ability to signal via cAMP (32, 33) as do dopamine D1 (51) and serotonin 4a (52) receptors with similar mutations. These results suggest that either prolonged LH receptor retention in small membrane compartments is not required for LH receptor-mediated signal transduction or, alternatively, that interactions of mutant receptors with the molecular contents of small compartments are more short-lived than for wild type receptors but still sufficient to initiate downstream signaling events.
Chimeric GnRH-LH receptors and LH receptors lacking C-terminus palmitoylation sites do not localize to plasma membrane rafts
2005, Biochemical and Biophysical Research CommunicationsCitation Excerpt :To determine whether palmitoylation of the receptor affected receptor localization in membrane microdomains, we constructed receptors, originally described by Menon and coworkers, that contained mutations to both of the two possible palmitoylation sites on the LHR C-terminus [17,18]. We confirmed that receptors with these mutations retained the ability to signal via cAMP, as previously reported [17,18], and, in fact, produced levels of cAMP that were comparably to wild-type receptors (data not shown). Nevertheless, FLAG-tagged LHR-C621,622S did not translocate into membrane rafts following binding of hormone as shown in Fig. 1.
Functional Consequences of Mutations and Polymorphisms in Gonadotropin and Gonadotropin Receptor Genes
2004, The Ovary: Second EditionRole of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function
2003, Pharmacology and TherapeuticsCitation Excerpt :Effects of mutating palmitoylated cysteines on signaling were not universally observed for all the GPCRs tested, and the list of cases where palmitoylation is believed not to significantly influence coupling to G-proteins is considerable. Examples include the α2A-adrenergic receptor (Kennedy & Limbird, 1993), the LH/hCG receptor (Kawate & Menon, 1994; Kawate et al., 1997), the dopamine D1 receptor (Jin et al., 1997), the human A1 adenosine receptor (Gao et al., 1999), and the human thyrotropin receptor (Tanaka et al., 1998). The fact that different effects were observed for different GPCRs upon mutation of palmitoylated cysteines could reflect true individual characteristics of the receptor studied.
Palmitoylation of CCR5 Is Critical for Receptor Trafficking and Efficient Activation of Intracellular Signaling Pathways
2001, Journal of Biological ChemistryCitation 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.
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Supported by NIH grant HD 06656.