Jo Ann Janovick

Texas Tech University Health Sciences Center, El Paso, Texas, United States

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Publications (94)422.99 Total impact

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    ABSTRACT: Abstract We describe a phenotypic high throughput screening (HTS) calcium flux assay designed to identify pharmacoperones for the gonadotropin releasing hormone receptor (GnRHR). Pharmacoperones are target-specific, small molecules that diffuse into cells, rescue misfolded protein mutants, and restore them to function. Rescue is based on correcting the trafficking of mutants that would otherwise be retained in the endoplasmic reticulum and unable to function correctly. This approach identifies drugs with a significant degree of novelty, relying on cellular mechanisms that are not currently exploited. Development of such assays is important, since the extensive use of agonist/antagonist screens alone means that useful chemical structures may be present in existing libraries but have not been previously identified using existing methods. Our assay utilizes cell lines stably expressing a GnRHR mutant under the control of a tetracycline (OFF) transactivator. This allows us to quantitate the level of functional and properly trafficked G protein coupled receptors present in each test well. Furthermore, since we are able to turn receptor expression on and off, we can rapidly eliminate the majority of false positives from our screening results. Our data show that this approach is likely to be successful in identifying hits from large chemical libraries.
    Assay and Drug Development Technologies 05/2014; 12(4):238-46. · 1.90 Impact Factor
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    ABSTRACT: Molecular chaperones triage misfolded proteins via action as substrate selectors for quality control (QC) machines that fold or degrade clients. Herein, the endoplasmic reticulum (ER)-associated Hsp40 JB12 is reported to participate in partitioning mutant conformers of gonadotropin-releasing hormone receptor (GnRHR), a G protein-coupled receptor, between ER-associated degradation (ERAD) and an ERQC autophagy pathway. ERQC autophagy degrades E90K-GnRHR because pools of its partially folded and detergent-soluble degradation intermediates are resistant to ERAD. S168R-GnRHR is globally misfolded and disposed of via ERAD, but inhibition of p97, the protein retrotranslocation motor, shunts S168R-GnRHR from ERAD to ERQC autophagy. Partially folded and grossly misfolded forms of GnRHR associate with JB12 and Hsp70. Elevation of JB12 promotes ERAD of S168R-GnRHR, with E90K-GnRHR being resistant. E90K-GnRHR elicits association of the Vps34 autophagy initiation complex with JB12. Interaction between ER-associated Hsp40s and the Vps34 complex permits the selective degradation of ERAD-resistant membrane proteins via ERQC autophagy.
    Molecular cell 03/2014; · 14.61 Impact Factor
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    ABSTRACT: A pharmacoperone (from "pharmacological chaperone") is a small molecule that enters cells and serves as molecular scaffolding in order to cause otherwise-misfolded mutant proteins to fold and route correctly within the cell. Pharmacoperones have broad therapeutic applicability since a large number of diseases have their genesis in the misfolding of proteins and resultant misrouting within the cell. Misrouting may result in loss-of-function and, potentially, the accumulation of defective mutants in cellular compartments. Most known pharmacoperones were initially derived from receptor antagonist screens and, for this reason, present a complex pharmacology, although these are highly target specific. In this summary, we describe efforts to produce high throughput screens that identify these molecules from chemical libraries as well as a mouse model which provides proof-of-principle for in vivo protein rescue using existing pharmacoperones.
    Pharmacological Research 12/2013; · 4.35 Impact Factor
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    ABSTRACT: Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E(90)K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E(90)K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.
    Proceedings of the National Academy of Sciences 12/2013; · 9.81 Impact Factor
  • Pharmacological Research 11/2013; · 4.35 Impact Factor
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    ABSTRACT: Plasma membrane expression (PME) of the human GnRHR (hGnRHR) is regulated by a primate-specific Lys(191) which destabilizes a Cys(14)-Cys(200) bridge required by the cellular quality control system (QCS). A 4-amino, non-contiguous "motif" (Leu(112), Gln(208), Leu(300), Asp(302)) is required for this effect. The hGnRHR sequence, with or without Lys(191), decreases PME and inositol phosphate (IP) production when co-expressed with calnexin, a QCS chaperone. WT rat GnRHR, decreases PME and IP production, when co-expressed with calnexin, but to a lesser degree than hGnRH. When the human sequence contains the rat motif, IP production is closer to that of rat GnRHR. When Lys(191) is deleted from hGnRHR and co-expressed with calnexin, IP production is similar to the rat sequence. When rat GnRHR containing Lys(191) and the human motif is co-expressed with calnexin, IP production is similar to cells expressing the hGnRHR. The motif sequence appears to be a determinant of calnexin recognition.
    Molecular and Cellular Endocrinology 07/2013; · 4.04 Impact Factor
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    ABSTRACT: Pharmacoperone drugs correct the folding of misfolded protein mutants and restore function (i.e., "rescue") by correcting the routing of (otherwise) misrouted mutants. Assays for pharmacoperones have not been applied to screen large libraries previously. Currently, most pharmacoperones possess intrinsic agonist or antagonist activities since these were identified using high-throughput screens aimed at discovering direct agonists or antagonists. Here we describe an ultra-high-throughput compatible no-wash assay system designed to specifically identify pharmacoperones of the vasopressin type 2 receptor (V2R). Development of such assays is important and novel since useful chemical structures with the ability to control cellular trafficking but lacking intrinsic agonist or antagonist properties have not likely been identified using existing screens. In the described assay, the level of functional human V2R (hV2R) (mutant) present in each test well is quantitated by stimulation with saturating levels of agonist followed by use of a luminescent-based cyclic adenosine monophosphate assay. This allows the assay to identify compounds that increase the trafficking of mutant hV2R[L(83)Q] in our model system.
    Journal of Biomolecular Screening 05/2013; · 2.21 Impact Factor
  • Pharmacological Research. 01/2013;
  • David C Smithson, Jo Ann Janovick, P Michael Conn
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    ABSTRACT: Mutations cause protein folding defects that result in cellular misrouting of otherwise functional proteins. Such mutations are responsible for a wide range of disease states, especially among G-protein coupled receptors. Drugs which serve as chemical templates and promote the proper folding of these proteins are valuable therapeutic molecules since they return functional proteins to the proper site of action. Small molecules have been identified that are able to function as pharmacological chaperones or "pharmacoperones" and stabilize the correct conformations of their target proteins with high specificity. Most of these are also agonists or antagonists of the proteins of interest, complicating potential therapeutic use. This is due, in part, to the fact that the majority of these were discovered during high-throughput screening campaigns using assays designed to detect agonists and antagonists, rather than compounds which improve the trafficking of misrouted mutants. The assays described in this report are designed specifically to identify compounds which result in the reactivation and correct trafficking of misfolded gonadotropin releasing hormone receptor and vasopressin type 2 receptor mutants, rather than those which act as agonists directly. The system reported is a generalizable approach amenable to use in automated (robotic) high-throughput screening efforts and can be used to identify compounds which affect protein conformation without necessarily acting as direct agonists or antagonists.
    Methods in enzymology 01/2013; 521:3-16. · 1.90 Impact Factor
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    ABSTRACT: GnRH, produced in the hypothalamus, acts on pituitary gonadotropes to stimulate release of the gonadotropins LH and FSH. Reduced responsiveness of gonadotropes to GnRH is a primary cause of hypogonadotropic hypogonadism (HH), a disease characterized by gonadal dysfunction and low blood levels of gonadotropins. Loss-of-function mutations in the gene encoding the receptor for GnRH (GNRHR) are a common cause of HH. Sequencing of the GNRHR gene in patients with HH revealed mainly point mutations producing single amino acid substitutions that cause misfolding and misrouting of this G protein-coupled receptor. To generate a mouse model that mimics the human disease, we introduced a single amino acid substitution (E90K) into the mouse Gnrhr gene, which is identical to a known human recessive mutation. In humans, E90K causes severe HH by preventing formation of the E90-K121 salt bridge, which is essential for correct folding. In cell cultures, E90K causes misfolding that leads to almost complete retention by the protein quality control system and subsequent degradation. Here we report that the primary phenotype of mice homozygous for E90K is female infertility due to ovulation failure. Mutant males are fertile despite reduced gonadotropin levels and smaller testes. These results suggest decreased GnRH receptor signaling in the mutant animal, compared with wild type. Our findings suggest that a threshold level of GnRH receptor activity is required for ovulation.
    Molecular Endocrinology 08/2012; · 4.75 Impact Factor
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    ABSTRACT: G protein-coupled receptors (GPCR) play central roles in almost all physiological functions, and mutations in GPCR are responsible for over 30 hereditary diseases associated with loss or gain of receptor function. Gain of function mutants are frequently described as having constitutive activity (CA), that is, they activate effectors in the absence of agonist occupancy. Although many GPCR have mutants with CA, the GnRH receptor (GnRHR) was not, until 2010, associated with any CA mutants. The explanation for the failure to observe CA appears to be that the quality control system of the cell recognizes CA mutants of GnRHR as misfolded and retains them in the endoplasmic reticulum. In the present study, we identified several human (h)GnRHR mutants with substitutions in transmembrane helix 6 (F(272)K, F(272)Q, Y(284)F, C(279)A, and C(279)S) that demonstrate varying levels of CA after being rescued by pharmacoperones from different chemical classes and/or deletion of residue K(191), a modification that increases trafficking to the plasma membrane. The movement of the mutants from the endoplasmic reticulum (unrescued) to the plasma membrane (after rescue) is supported by confocal microscopy. Judging from the receptor-stimulated inositol phosphate production, mutants F(272)K and F(272)Q, after rescue, display the largest level of CA, an amount that is comparable with agonist-stimulated activation. Because mutations in other GPCR are, like the hGnRHR, scrutinized by the quality control system, this general approach may reveal CA in receptor mutants from other systems. A computer model of the hGnRHR and these mutants was used to evaluate the conformation associated with CA.
    Molecular Endocrinology 05/2012; 26(7):1179-88. · 4.75 Impact Factor
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    ABSTRACT: G-protein-coupled receptors (GPCRs) are a large superfamily of plasma membrane proteins that play central roles in transducing endocrine, neural and -sensory signals. In humans, more than 30 disorders are associated with mutations in GPCRs and these proteins are common drug development targets, with 30-50% of drugs targeting them. GPCR mutants are frequently misfolded, recognized as defective by the cellular quality control system, retained in the endoplasmic reticulum and do not traffic to the plasma membrane. The use of small molecules chaperones (pharmacological chaperones or "pharmacoperones") to rescue misfolded GPCRs has provided a new approach for treatment of human diseases caused by misfolding and misrouting. This chapter provides an overview of the molecular basis of this approach using the human gonadotropin-releasing hormone receptor (hGnRHR) as model for treatment of conformational diseases provoked by -misfolded GPCRs.
    Sub-cellular biochemistry 01/2012; 63:263-89.
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    ABSTRACT: G protein-coupled receptors (GPCRs) play central roles in most physiological functions, and mutations in them cause heritable diseases. Whereas crystal structures provide details about the structure of GPCRs, there is little information that identifies structural features that permit receptors to pass the cellular quality control system or are involved in transition from the ground state to the ligand-activated state. The gonadotropin-releasing hormone receptor (GnRHR), because of its small size among GPCRs, is amenable to molecular biological approaches and to computer modeling. These techniques and interspecies comparisons are used to identify structural features that are important for both intracellular trafficking and GnRHR activation yet distinguish between these processes. Our model features two salt (Arg(38)-Asp(98) and Glu(90)-Lys(121)) and two disulfide (Cys(14)-Cys(200) and Cys(114)-Cys(196)) bridges, all of which are required for the human GnRHR to traffic to the plasma membrane. This study reveals that both constitutive and ligand-induced activation are associated with a "coincidence detector" that occurs when an agonist binds. The observed constitutive activation of receptors lacking Glu(90)-Lys(121), but not Arg(38)-Asp(98) ionic bridge, suggests that the role of the former connection is holding the receptor in the inactive conformation. Both the aromatic ring and hydroxyl group of Tyr(284) and the hydrogen bonding of Ser(217) are important for efficient receptor activation. Our modeling results, supported by the observed influence of Lys(191) from extracellular loop 2 (EL2) and a four-residue motif surrounding this loop on ligand binding and receptor activation, suggest that the positioning of EL2 within the seven-α-helical bundle regulates receptor stability, proper trafficking, and function.
    Journal of Pharmacology and Experimental Therapeutics 04/2011; 338(2):430-42. · 3.89 Impact Factor
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    P Michael Conn, Jo Ann Janovick
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    ABSTRACT: G protein-coupled receptors (GPCRs), which includes the gonadotropin releasing hormone (GnRH) receptor (GnRHR), comprises the largest family of validated drug targets-more than half of all approved drugs derive their benefits by selective targeting of GPCRs. Most drugs in this class are either agonists or antagonists of GPCRs and high throughput screens (HTSs) have typically been designed and performed with a view toward identification of such compounds as lead drug candidates. This manuscript presents the case that valuable drugs which effect the trafficking of GPCRs may have been overlooked because pharmacoperones have been selected from existing screens that identify agonists and antagonists. A "gain of activity assay" is proposed; this assay relies on the expression of a mutant of the GnRHR that is known to be rescuable by pharmacoperone drugs, and which is restored to activity in their presence. Accordingly, "hits" are identified by the appearance of activity. The gene for the mutant is under control of tetracycline and may be prevented from being expressed. This is a valuable feature since it allows false positives to be identified. Such drugs will show apparent activity whether or not the mutant is expressed. This assay will enable identification of these drugs from chemical libraries and does not rely on their activity as agonists or antagonists.
    Frontiers in Endocrinology 03/2011; 2(6).
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    Jo Ann Janovick, Byung S Park, P Michael Conn
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    ABSTRACT: Functional rescue of misfolded mutant receptors by small non-peptide molecules has been demonstrated. These small, target-specific molecules (pharmacological chaperones or "pharmacoperones") serve as molecular templates, promote correct folding and allow otherwise misfolded mutants to pass the scrutiny of the cellular quality control system (QCS) and be expressed at the plasma membrane (PM) where they function similarly to wild type (WT) proteins. In the case of the gonadotropin releasing hormone receptor (GnRHR), drugs that rescue one mutant typically rescue many mutants, even if the mutations are located at distant sites (extracellular loops, intracellular loops, transmembrane helices). This increases the value of these drugs. These drugs are typically identified, post hoc, from "hits" in screens designed to detect antagonists or agonists. The therapeutic utility of pharmacoperones has been limited due to the absence of screens that enable identification of pharmacoperones per se. We describe a generalizable primary screening approach for pharmacoperone drugs based on measurement of gain of activity in stable HeLa cells stably expressing the mutants of two different model G-protein coupled receptors (GPCRs) (hGnRHR[E(90)K] or hV2R[L(83)Q]). These cells turn off expression of the receptor mutant gene of interest in the presence of tetracycline and its analogs, which provides a convenient means to identify false positives. The methods described and characterized here provide the basis of novel primary screens for pharmacoperones that detect drugs that rescue GPCR mutants of specific receptors. This approach will identify structures that would have been missed in screens that were designed to select only agonists or antagonists. Non-antagonistic pharmacoperones have a therapeutic advantage since they will not compete for endogenous agonists and may not have to be washed out once rescue has occurred and before activation by endogenous or exogenous agonists.
    PLoS ONE 01/2011; 6(7):e22784. · 3.53 Impact Factor
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    ABSTRACT: 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.
    Molecular and Cellular Endocrinology 01/2011; 337(1-2):16-23. · 4.04 Impact Factor
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    Jo Ann Janovick, P Michael Conn
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    ABSTRACT: G protein-coupled receptors (GPCRs) play central roles in almost all physiological functions; mutations in GPCRs are responsible for more than 30 disorders. There is a great deal of information about GPCR structure but little information that directly relates structure to protein trafficking or to activation. The gonadotropin releasing hormone receptor, because of its small size among GPCRs, is amenable to preparation of mutants and was used in this study to establish the relation among a salt bridge, protein trafficking, and receptor activation. This bridge, between residues E(90) [located in transmembrane segment (TM) 2] and K(121) (TM3), is associated with correct trafficking to the plasma membrane. Agonists, but not antagonists, interact with residue K(121), and destabilize the TM2-TM3 association of the receptor in the plasma membrane. The hGnRHR mutant E(90)K has a broken salt bridge, which also destabilizes the TM2-TM3 association and is typically retained in the endoplasmic reticulum. We show that this mutant, if rescued to the plasma membrane by either of two different means, has constitutive activity and shows modified ligand specificity, revealing a role for the salt bridge in receptor activation, ligand specificity, trafficking, and structure. The data indicate that destabilizing the TM2-TM3 relation for receptor activation, while requiring an intact salt bridge for correct trafficking, provides a mechanism that protects the cell from plasma membrane expression of constitutive activity.
    Proceedings of the National Academy of Sciences 02/2010; 107(9):4454-8. · 9.81 Impact Factor
  • Jo Ann Janovick, P Michael Conn
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    ABSTRACT: The gonadotropin-releasing hormone (GnRH) receptor (GnRHR), because of its small size among G-protein-coupled receptors (GPCRs), is amenable to facile preparation of mutants. This receptor is used in our laboratory as a structural model for this super-family of protein receptors and has helped us understand the requirements for proper trafficking. We have demonstrated that pharmacoperones ("pharmacological chaperones"), small target-specific drugs that diffuse into cells, are capable of rescuing misfolded and misrouted GnRHR mutants and restoring them to function. By rescuing these proteins, these drugs enable the plasma membrane expression of such mutants in living cells and allow examination of mutants that would otherwise be retained in the endoplasmic reticulum and would not be available for ligand binding and signal transduction. As an example of the efficacy of this method, we have shown that mutant E⁹⁰K, which breaks a salt bridge (E⁹⁰-K¹²¹) normally found in the GnRHR, results in constitutive activity when rescued by pharmacoperones. A second method of rescue, involving a mutation that increases the expression of GnRHRs, is shown to have a similar effect. Normally, in the absence of rescue by either of these methods, this mutant, associated with human hypogonadotropic hypogonadism, is misrouted and this constitutive activity has gone unrecognized. This observation [Janovick, J. A., and Conn, P. M. (2010). Salt bridge integrates GPCR activation with protein trafficking. Proc. Natl. Acad. Sci. USA107, 4454-4458.] showed that the cell normally recognizes this protein as defective and prevents its routing to the plasma membrane.
    Methods in enzymology 01/2010; 485:277-92. · 1.90 Impact Factor
  • P Michael Conn, Jo Ann Janovick
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    ABSTRACT: Proteins serve in cellular roles that necessitate structural precision, a requirement overseen by the cellular quality control system (QCS). By rejecting misfolded proteins, the QCS protects against aberrant activity. Misfolding and subsequent retention by the QCS results in proteins that might maintain function but become misrouted and cause disease. Correcting the misrouting of misfolded mutant proteins often restores activity and addresses the underlying disease. Because of its small size, the gonadotropin-releasing hormone receptor has been an excellent model for G-protein-coupled receptor trafficking and has recently enabled elucidation of both the requirements to pass the QCS and the biochemical mechanism of rescue by pharmacological chaperones; this information will now enable rational design of these therapeutic agents. Here, we summarize what is known about the relation between receptor structure and interactions with the QCS with a view toward therapeutic development based on the rescue of misfolded and, consequently, misrouted mutants with drugs.
    Trends in Pharmacological Sciences 04/2009; 30(5):228-33. · 9.25 Impact Factor
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    W Lucca-Junior, J A Janovick, P M Conn
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    ABSTRACT: 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.
    Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 03/2009; 42(2):164-7. · 1.08 Impact Factor

Publication Stats

2k Citations
422.99 Total Impact Points

Institutions

  • 2013–2014
    • Texas Tech University Health Sciences Center
      • Department of Internal Medicine
      El Paso, Texas, United States
  • 1994–2013
    • Oregon Health and Science University
      • • Department of Physiology & Pharmacology
      • • Division of Reproductive Sciences
      Portland, OR, United States
  • 1999–2011
    • Mexican Institute of Social Security
      Ciudad de México, The Federal District, Mexico
  • 1996–2011
    • Wisconsin National Primate Research Center
      Madison, Wisconsin, United States
  • 2000
    • Duke University Medical Center
      • Department of Cell Biology
      Durham, NC, United States
  • 1993
    • University of Iowa
      • Department of Pharmacology
      Iowa City, IA, United States
  • 1990
    • Wright State University
      • Department of Anatomy
      Dayton, OH, United States