M1 and M3 muscarinic receptors may play a role in the neurotoxicity of anhydroecgonine methyl ester, a cocaine pyrolysis product.

The smoke of crack cocaine contains cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). AEME possesses greater neurotoxic potential than cocaine and an additive effect when they are combined. Since atropine prevented AEME-induced neurotoxicity, it has been suggested that its toxic effects may involve the muscarinic cholinergic receptors (mAChRs). Our aim is to understand the interaction between AEME and mAChRs and how it can lead to neuronal death. Using a rat primary hippocampal cell culture, AEME was shown to cause a concentration-dependent increase on both total [(3)H]inositol phosphate and intracellular calcium, and to induce DNA fragmentation after 24 hours of exposure, in line with the activation of caspase-3 previously shown. Additionally, we assessed AEME activity at rat mAChR subtypes 1-5 heterologously expressed in Chinese Hamster Ovary cells. l-[N-methyl-(3)H]scopolamine competition binding showed a preference of AEME for the M2 subtype; calcium mobilization tests revealed partial agonist effects at M1 and M3 and antagonist activity at the remaining subtypes. The selective M1 and M3 antagonists and the phospholipase C inhibitor, were able to prevent AEME-induced neurotoxicity, suggesting that the toxicity is due to the partial agonist effect at M1 and M3 mAChRs, leading to DNA fragmentation and neuronal death by apoptosis.

Pharmacoperones: targeting therapeutics toward diseases caused by protein misfolding.

Pharmacoperones are hydrophobic molecule drugs that enter cells and serve as a molecular framework to cause misfolded mutant proteins to fold properly and adopt a stable conformation compatible with proper intracellular trafficking. Pharmacoperones have successfully been used experimentally to rescue function of some misfolded proteins (enzymes, receptors, channels) that lead to disease. Identification of pharmacoperones by high-throughput screens of drug libraries will likely provide new molecules that may be potentially useful to treat diseases caused by protein misfolding.

Gonadotropin-releasing hormone receptor activates GTPase RhoA and inhibits cell invasion in the breast cancer cell line MDA-MB-231.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) and its receptor (GnRHR) are both expressed by a number of malignant tumors, including those of the breast. In the latter, both behave as potent inhibitors of invasion. Nevertheless, the signaling pathways whereby the activated GnRH/GnRHR system exerts this effect have not been clearly established. In this study, we provide experimental evidence that describes components of the mechanism(s) whereby GnRH inhibits breast cancer cell invasion. METHODS: Actin polymerization and substrate adhesion was measured in the highly invasive cell line, MDA-MB-231 transiently expressing the wild-type or mutant DesK191 GnRHR by fluorometry, flow cytometric analysis, and confocal microscopy, in the absence or presence of GnRH agonist. The effect of RhoA-GTP on stress fiber formation and focal adhesion assembly was measured in MDA-MB-231 cells co-expressing the GnRHRs and the GAP domain of human p190Rho GAP-A or the dominant negative mutant GAP-Y1284D. Cell invasion was determined by the transwell migration assay. RESULTS: Agonist-stimulated activation of the wild-type GnRHR and the highly plasma membrane expressed mutant GnRHR-DesK191 transiently transfected to MDA-MB-231 cells, favored F-actin polymerization and substrate adhesion. Confocal imaging allowed detection of an association between F-actin levels and the increase in stress fibers promoted by exposure to GnRH. Pull-down assays showed that the effects observed on actin cytoskeleton resulted from GnRH-stimulated activation of RhoA GTPase. Activation of this small G protein favored the marked increase in both cell adhesion to Collagen-I and number of focal adhesion complexes leading to inhibition of the invasion capacity of MDA-MB-231 cells as disclosed by assays in Transwell Chambers. CONCLUSIONS: We here show that GnRH inhibits invasion of highly invasive breast cancer-derived MDA-MB-231 cells. This effect is mediated through an increase in substrate adhesion promoted by activation of RhoA GTPase and formation of stress fibers and focal adhesions. These observations offer new insights into the molecular mechanisms whereby activation of overexpressed GnRHRs affects cell invasion potential of this malignant cell line, and provide opportunities for designing mechanism-based adjuvant therapies for breast cancer.

Pharmacoperones: a new therapeutic approach for diseases caused by misfolded G protein-coupled receptors.

G Protein-coupled receptors (GPCRs) are cell membrane proteins that recognize specific chemical signals such as drugs and hormones and transduce these signals into cellular responses by activating G-proteins. As is the case for all newly synthesized proteins, GPCRs are subjected to conformational scrutiny at the endoplasmic reticulum prior to processing and trafficking to the cell surface membrane. Because of this stringent quality control screening mechanism, mutations that result in protein misfolding frequently lead to retention in the endoplasmic reticulum, aggregation or other misrouting and, eventually, to disease. This article reviews some patents and new therapeutic opportunities based on the misfolding and retention of otherwise functional GPCRs that represent promising approaches to correct conformational abnormalities leading to distinct disease states.

Biochemical mechanism of pathogenesis of human gonadotropin-releasing hormone receptor mutants Thr104Ile and Tyr108Cys associated with familial hypogonadotropic hypogonadism.

The pathogenic mechanisms whereby the Thr104Ile and Tyr108Cys mutations in the gonadotropin-releasing hormone receptor (GnRHR) gene cause hypogonadotropic hypogonadism in humans are unknown. Transient expression of Thr104Ile and Tyr108Cys mutants in COS-7 cells revealed that both GnRHR mutants neither bind nor respond to agonist. Removal of Lys191 rescued function of both mutants, while addition of a carboxyl-terminal targeting sequence only rescued function of the Thr104Ile mutant. Exposure to the pharmacoperone In3 rescued almost completely Thr104Ile mutant function to wild-type levels, whereas rescue was partial for the Tyr108Cys GnRHR. Additional mutations that block formation of bridges involving Cys108 showed that a Cys108-Cys200 disulfide bridge is the predominant moiety formed in the Tyr108Cys mutant. Thr104Ile and Tyr108Cys GnRHRs are misfolded structures whose function is rescuable by genetic and/or pharmacological strategies. The Tyr108Cys mutant forms an aberrant disulfide bridge that prevents formation of the required Cys14-Cys200 bridge essential for GnRHR plasma membrane expression.

Dominant negative effects of human follicle-stimulating hormone receptor expression-deficient mutants on wild-type receptor cell surface expression. Rescue of oligomerization-dependent defective receptor expression by using cognate decoys.

Current evidence indicates that G protein-coupled receptors form dimers that may affect biogenesis and membrane targeting of the complexed receptors. We here analyzed whether expression-deficient follicle-stimulating hormone receptor (FSHR) mutants exert dominant negative actions on wild-type FSHR cell surface membrane expression. Co-transfection of constant amounts of wild-type receptor cDNA and increasing quantities of mutant (R556A or R618A) FSHR cDNAs progressively decreased agonist-stimulated cAMP accumulation, [(125)I]-FSH binding, and plasma membrane expression of the mature wild-type FSHR species. Co-transfection of wild-type FSHR fragments involving transmembrane domains 5-6, or transmembrane domain 7 and/or the carboxyl-terminus specifically rescued wild-type FSHR expression from the transdominant inhibition by the mutants. Mutant FSHRs also inhibited function of the luteinizing hormone receptor but not that of the thyrotropin receptor or non-related receptors. Defective intracellular transport and/or interference with proper maturation due to formation of misfolded mutant:wild-type receptor complexes may explain the negative effects provoked by the altered FSHRs.

Ethical research as the target of animal extremism: an international problem

Animal extremism has been increasing worldwide; frequently researchers are the targets of actions by groups with extreme animal rights agendas. Sometimes this targeting is violent and may involve assaults on family members or destruction of property. In this article, we summarize recent events and suggest steps that researchers can take to educate the public on the value of animal research both for people and animals

Ethical research as the target of animal extremism: an international problem.

Animal extremism has been increasing worldwide; frequently researchers are the targets of actions by groups with extreme animal rights agendas. Sometimes this targeting is violent and may involve assaults on family members or destruction of property. In this article, we summarize recent events and suggest steps that researchers can take to educate the public on the value of animal research both for people and animals.

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane.

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 microCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

Targeting of G protein-coupled receptors to the plasma membrane in health and disease.

G protein-coupled receptors (GPCRs) are cell surface membrane proteins that recognize specific signals (ligands) from an immense number of chemically diverse substances. These receptors act as signal transducers for messages carried by external, systemic, or local stimuli. As complex molecular structures, which must attain specific shapes, newly synthesized GPCRs are subjected to conformational scrutiny at the endoplasmic reticulum level before their passage to the plasma membrane. Such a quality control mechanism guards against aberrant protein structures and checks for proper folding, processing and structural integrity of nascent proteins. Despite this stringent quality control screening mechanism, gain- or loss-of-function mutations that result in GPCR misfolding in the endoplasmic reticulum can manifest themselves as profound effects on health. Understanding the molecular, cellular and energetic mechanisms controlling GPCR intracellular routing is essential for preventing or correcting the conformational abnormalities associated with disease-causing misfolded receptors. This article reviews the mechanisms subserving plasma membrane targeting of GPCRs and describes novel and promising approaches to correct misfolding and misrouting related to various disease states.

Conformational effects of Lys191 in the human GnRH receptor: mutagenesis and molecular dynamics simulations studies.

In the present study, we analyzed the role of Lys191 on function, structure, and dynamic behavior of the human GnRH receptor (hGnRHR) and the formation of the Cys14-Cys200 bridge, which is essential for receptor trafficking to the plasma membrane. Several mutants were studied; mutants lacked either the Cys14-Cys200 bridge, Lys191 or both. The markedly reduced expression and function of a Cys14Ser mutant lacking the 14-200 bridge, was nearly restored to wild-type/DeltaLys191 levels upon deletion of Lys191. Lys191 removal resulted in changes in the dynamic behavior of the mutants as disclosed by molecular dynamics simulations: the distance between the sulfur- (or oxygen-) sulfur groups of Cys (or Ser)14 and Cys200 was shorter and more constant, and the conformation of the NH(2)-terminus and the exoloop 2 exhibited fewer fluctuations than when Lys191 was present. These data provide novel information on the role of Lys191 in defining an optimal configuration for the hGnRHR intracellular trafficking and function.

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

In vitro coexpression and pharmacological rescue of mutant gonadotropin-releasing hormone receptors causing hypogonadotropic hypogonadism in humans expressing compound heterozygous alleles.

We analyzed the function of mutant GnRH receptor (GnRHR) pairs associated with compound heterozygous patients showing complete or partial forms of hypogonadotropic hypogonadism. We did this to examine potential interactions between misfolded mutants that may influence net receptor function and response to pharmacological rescue. Nine pairs of GnRHR mutants and an unreported combination (L314X((stop))/R262Q) were studied. Coexpression of each pair of mutants in COS-7 cells resulted in an active predominant effect (Q106R/L266R, A171T/Q106R, T32I/C200Y, and R262Q/A129D mutant GnRHR pairs), an additive effect (R262Q/Q106R, N10K/Q106R, and R262Q/Y284C human GnRHR pairs), or a dominant-negative effect (L314X((stop))/Q106R, Q106R+S217R/R262Q, and L314X((stop))/R262Q GnRHRs). For all combinations, addition of the pharmacoperone IN3 increased both agonist binding and effector coupling. The IN3 response was unpredictable because responses could be either similar, higher, or lower, compared with that exhibited by the less affected mutant. The clinical phenotype in patients expressing complex heterozygous alleles appears to be dictated by both the contribution from each mutant and a dominant-negative effect similar to that reported for mutants and wild-type receptor. Depending on the genotype, partial or full restoration of receptor function in response to pharmacological chaperones may be achievable goals in patients bearing inactivating mutations in the GnRHR gene.

Functional significance of the BBXXB motif reversed present in the cytoplasmic domains of the human follicle-stimulating hormone receptor.

The minimal structural motif, BBXXB (where B represents a basic amino acid residue and X a non-basic residue), located in particular regions of the intracellular domains of cell surface membrane receptors is involved in the G protein-activating activity of a number of G protein-coupled receptors. The human FSH receptor (hFSHR) exhibits a reversed BBXXB motif (BXXBB) in the juxtamembrane region of the third intracellular loop (IL3) and the carboxyl terminus (Ctail) of the receptor; however the importance of this sequence on receptor function remains unclear. In the present study, we analyzed the effects of mutations in this structural motif on hFSHR expression, receptor-mediated effector activation and agonist-provoked receptor internalization. Human embryonic kidney 293 cells were transiently transfected with plasmids containing the cDNA of the wild-type (Wt) hFSHR or several hFSHR mutants in which basic amino acids of the minimal structural motif at the IL3 and Ctail were replaced with alanine (i.e. AXXAA, AXXBB, BXXAB and BXXBA mutants). Alanine substitution of the three basic residues present in the IL3-BXXBB (IL3-AXXAA mutant) yielded a < or =60 kDa possibly under-glycosylated form of the FSHR, whereas the same substitutions in the Ctail resulted in the immature >62 kDa form of the receptor; both AXXAA hFSHR mutants completely failed to bind agonist and activate effector. Individual substitutions resulted in different cAMP responses to agonist stimulation: the IL3-AXXBB and IL3-BXXBA mutant hFSHRs failed to evoke Gs protein activation, whereas agonist-stimulated cAMP production was completely normal when the IL3-BXXAB mutant was expressed. All three IL3 mutants bound [125I]-labelled FSH in a similar fashion to the Wt hFSHR. Ligand-binding, cell surface membrane receptor expression and agonist-provoked effector activation were significantly affected by the individual substitutions at the Ctail-BXXBB motif: the Ctail-AXXBB variant exhibited reduced (approximately 50%) maximal cAMP response and ability to bind ligand, whereas both ligand binding and effector activation was severely reduced or abolished by expression of the Ctail-BXXBA and -BXXAB hFSHR mutants; the expression levels of the 80 kDa form of the receptor correlated with the magnitude of ligand-provoked cAMP production and binding capability of the mutant receptors. Upon stimulation by agonist, all mutants with detectable ligand-binding activity internalized following the pattern exhibited by the Wt hFSHR species. These results indicate that the BXXBB motif at the IL3 of the hFSHR is essential for coupling the activated receptor to the Gs protein, whereas the same motif in the Ctail is apparently more important for membrane expression.

Structural determinants in the second intracellular loop of the human follicle-stimulating hormone receptor are involved in G(s) protein activation.

In the present study, we analyzed the structural determinants present in the second intracellular loop (IL-2) of the human follicle-stimulating hormone (FSH) receptor (R) involved in G(s) protein-mediated signal transduction. Human embryonic kidney 293 (HEK-293) cells, stably expressing wild-type (Wt) human FSHR (HEK-293((+))), were transiently transfected with plasmids containing cDNAs encoding the entire IL-2 or several IL-2 sequences mutated in R467 (a residue located at the center of the conserved ERW motif in the glycoprotein hormone receptors), T470 (a potential site for phosphorylation by protein kinase-A and -C) or L477 (a residue conserved in all glycoprotein hormone receptors). Expression of the IL-2 Wt in HEK-293((+)) cells reduced the maximum FSH-stimulated cAMP production significantly by approximately 40%; similar results were observed with the R467A and R467K IL-2 mutants. The IL-2(R467H), IL-2(T470A), the triple R467A/T470A/L477A IL-2 mutant and the IL-2 of the oxytocin receptor (G(q/11)-coupled) had no effects on Wt FSHR-mediated intracellular signaling whereas the L477A mutation provoked a higher ( approximately 55%) inhibition of FSH-stimulated cAMP than the free, Wt IL-2. These results suggested a specific role of IL-2 residues in FSHR function. Site directed mutagenesis of the FSHR and the expression of resulting mutants in HEK-293 cells were performed in order to corroborate the effects of these substitutions. Expression of FSHR(R467H), FSHR(R467A) and FSHR(T470A) failed to mediate ligand-provoked G(s) protein activation, whereas the R467K mutant behaved as the Wt receptor. Interestingly, the expression of L477A, L477D and L477P FSHR mutants conferred elevated basal cAMP levels to HEK-293 cells. This study indicates that the IL-2 of the human FSHR possesses amino acid residues that are important for both coupling the receptor to the G(s) protein (R467 and T470) and maintaining the receptor molecule in an inactive conformation (L477). It appears that this particular intracellular domain may act as a conformational switch to produce the activation of G proteins as has been reported for the IL-2 of other G protein-coupled receptors.

Structure-activity relationships of G protein-coupled receptors.

The primary function of cell-surface receptors is to discriminate the specific signaling molecule or ligand from a large array of chemically diverse extracellular substances and to activate an effector signaling cascade that triggers an intracellular response and eventually a biological effect. G protein-coupled cell-surface receptors (GPCRs) mediate their intracellular actions through the activation of guanine nucleotide-binding signal-transducing proteins (G proteins), which form a diverse family of regulatory GTPases that, in the GTP-bound state, bind and activate downstream membrane-localized effectors. Hundreds of GPCRs signal through one or more of these G proteins in response to a large variety of stimuli including photons, neurotransmitters, and hormones of variable molecular structure. The mechanisms by which these ligands provoke activation of the receptor/G-protein system are highly complex and multifactorial. Knowledge and mapping of the structural determinants and requirements for optimal GPCR function are of paramount importance, not only for a better and more detailed understanding of the molecular basis of ligand action and receptor function in normal and abnormal conditions, but also for a rational design of early diagnostic and therapeutic tools that may allow exogenous regulation of receptor and G protein function in disease processes.