Role of palmitoylation of conserved cysteine residues of luteinizing hormone/human choriogonadotropin receptors in receptor down-regulation1

https://doi.org/10.1016/S0303-7207(97)04010-0Get rights and content

Abstract

The conserved cysteine residues 621 and 622 of luteinizing hormone/human chorionic gonadotropin receptors were converted to serine (C621S, C622S, C621/622S) and glycine residues (C621/C622G) by site directed mutagenesis. The wild type and mutant receptor cDNAs were cloned into the mammalian expression vector (PCMV4) and human embryonic kidney cells (293 cells) were transiently transfected with these constructs. Equilibrium binding studies with [125I]hCG (human chorionic gonadotropin) showed that the mutant and wild type receptors expressed on the cell surface exhibited similar Kd. The effect of mutation of the conserved cysteine residues on the ability of the receptors to undergo ligand-induced down-regulation was then tested. In vitro exposure of cells expressing the wild type receptor to a saturating concentration of human chorionic gonadotropin (100 ng/ml) for 24 h resulted in modest down-regulation of receptors. The palmitoylation deficient mutants, C621S, C622S, C621/622S and C621/622G, showed increased down-regulation compared with the wild type receptor. The extent of down-regulation of the mutant receptors correlated with increased internalization of the receptor. Additionally, the G protein coupling efficiency of the palmitoylation deficient mutants was not different from the wild type since the EC50s for cyclic AMP (cAMP) production were identical in both groups. These studies demonstrate that palmitoylation deficient mutants are more prone to ligand-induced receptor down-regulation. Furthermore, abrogation of palmitoylation by mutagenesis showed no effect on the efficiency of the palmitoylation deficient mutants to couple to Gs protein.

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

Unlinked list

AUTHOR, PLEASE CITE REFERENCE [19]THE TEXT

References (25)

  • Menon, K.M.J. and Gunaga, K.P. (1974) The role of cyclic AMP in reproductive processes. Fertil. Steril. 25,...
  • McFarland, K.C., Sprengel, R., Phillips, H.S., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff, D.L. and Seeburg,...
  • Loosfelt, H., Misrahi, M., Atger, M., Salesse, R., Vu Hai-Luu Thi, M.T., Jolivet, A., Guiochon-Mantel, A., Sar, S.,...
  • Davis, J.S., Weakland, L.L., Farese, R.V. and West, L.A. (1987) Luteinizing hormone increases inositol triphosphate and...
  • Gudermann, T., Nichols, C., Levy, F.O., Birnbaumer, M. and Birnbaumer, L. (1992) Ca2+ mobilization by the LH receptor...
  • Kawate, N., and Menon, K.M.J. (1994) Palmitoylation of luteinizing hormone/human chorionic gonadotropin receptors in...
  • Zhu, H., Wang, H. and Ascoli, M. (1995) The lutropin/choriogonadotropin receptor is palmitoylated at intracellular...
  • Ovchinnikov, Y.A., Abdulaev, N.G. and Bogachuk, A.S. (1988) Two adjacent cysteine residues in the C-terminal...
  • Karnik, S.S., Ridge, K.D., Bhattacharya, S. and Khorana, H.G. (1993) Palmitoylation of bovine opsin and its cysteine...
  • O'Dowd, B.F., Hnatowich, M., Caron, M.G., Lefkowitz, R.J. and Bouvier, M. (1989) Palmitoylation of the human beta...
  • Kennedy, M.E. and Limbird, L.E. (1993) Mutations of the α2A-adrenergic receptor that eliminate detectable...
  • Moffett, S., Mouillac, B., Bonin, H. and Bouvier, M. (1993) Altered phosphorylation and desensitization patterns of a...
  • Cited by (27)

    • Receptors for neuronal or endocrine signalling molecules as potential targets for the control of insect pests

      2014, Advances in Insect Physiology
      Citation Excerpt :

      The palmitoylation state of the vasopressin V2 receptor affected the MAPK pathway, but not the AC pathway (Charest and Bouvier, 2003). However, the effects of palmitoylation on signalling are not universally observed for all GPCRs (Gao et al., 1999; Kawate and Menon, 1994; Kawate et al., 1997; Kennedy and Limbird, 1993; Ponimaskin et al., 2002; Tanaka et al., 1998). Many GPCRs are capable of exhibiting some degree of spontaneous activity (Bond and Ijzerman, 2006; Milligan, 2003).

    • Restricted lateral diffusion of luteinizing hormone receptors in membrane microdomains

      2011, Journal of Biological Chemistry
      Citation 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 Communications
      Citation 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.

    • Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function

      2003, Pharmacology and Therapeutics
      Citation 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 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.

    View all citing articles on Scopus
    1

    Supported by NIH grant HD 06656.

    View full text