Refolding of misfolded mutant GPCR: Post-translational pharmacoperone action in vitro

https://doi.org/10.1016/j.mce.2007.04.012Get rights and content

Abstract

All reported GnRH receptor mutants (causing human hypogonadotropic hypogonadism) are misfolded proteins that cannot traffic to the plasma membrane. Pharmacoperones correct misfolding and rescue mutants, routing them to the plasma membrane where they regain function. Because pharmacoperones are often peptidomimetic antagonists, these must be removed for receptor function after rescue; in vivo this necessitates pulsatile pharmacoperone administration. As an antecedent to in vivo studies, we determined whether pharmacoperones need to be present at the time of synthesis or whether previously misfolded proteins could be refolded and rescued. Accordingly, we blocked either protein synthesis or intra-cellular transport. Biochemical and morphological studies using 12 mutants and 10 pharmacoperones representing three different chemical classes show that previously synthesized mutant proteins, retained by the quality control system (QCS), are rescued by pharmacoperones, showing that pharmacoperone administration in vivo likely need not consider whether the target protein is being synthesized at the time of drug administration.

Introduction

Because of interest in the GnRHR as a therapeutic target (Conn and Crowley, 1991, Ulloa-Aguirre et al., 2003) and the small size of this receptor (328 amino acids in the human sequence, which makes site-directed mutation more facile than for larger proteins), it has become an important model for understanding the folding of G protein coupled receptors (Conn et al., 2002, Ulloa-Aguirre et al., 2004, Castro-Fernandez et al., 2005, Janovick et al., 2002). Seventeen GnRHR point mutants have been identified from patients with human hypogonadotropic hypogonadism (HH; Leaños-Miranda et al., 2002, Leaños-Miranda et al., 2003, Ulloa-Aguirre et al., 2003, Ulloa-Aguirre et al., 2004) and all appear to exert their effect (i.e. loss or diminution of GnRH efficacy) by producing misfolded and misrouted receptors rather than by loss of the ability to bind ligand or couple to effector. Accordingly, 15 of the 17 HH-associated mutants can be rescued to some degree by pharmacological chaperones (“pharmacoperones”), small molecules (usually peptidomimetic antagonists) that enter cells and correct folding errors (Conn et al., 2002, Bernier et al., 2004a, Bernier et al., 2004b). In the two remaining cases (hGnRHR(Ser168Arg) and hGnRHR(Ser217Arg)), thermodynamically unfavorable substitutions cause severe twisting of transmembrane segment 4 (TMS4) or TMS5, respectively, irreversibly preventing the alignment of extracellular loop 2 (ECL2) and the amino terminal, needed for proper positioning of amino acids Cys14 and Cys200, a bridge which is requisite for creation of a properly folded human GnRHR that passes the cellular quality control system (Knollman et al., 2005, Janovick et al., 2006).

When coexpressed with wild type (WT) hGnRHR, many of the mutants isolated from HH patients, show a dominant-negative effect on WT expression, resulting in loss of WT plasma membrane binding and effector coupling (Leaños-Miranda et al., 2002, Leaños-Miranda et al., 2003), an effect that is explained by recognition of the WT-mutant oligomer by the QCS and retention of the oligomer in the endoplasmic reticulum (ER; Brothers et al., 2004). As in the case of mutants expressed alone, pharmacoperones rescue both the mutant and the WT receptor from ER retention of this oligomer by this dominant-negative effect.

In making the jump from cell culture to an in vivo model, it is necessary to address the fact that many pharmacoperones are actually antagonists of the receptors that they rescue. Accordingly, this will likely mean that drugs based on these agents will have to be administered in a pulsatile fashion to enable them to be washed out and avoid blocking the receptor from activation by agonist; this is independent of the route of administration. In principle, the need to administer pharmacoperones periodically could conflict with the need to maintain their presence if they are required at the time of synthesis of the target molecule. A key question for moving from cell culture to in vivo work is then, “can a misfolded/misrouted protein that is already retained by the QCS be rescued” or, alternatively, “is it necessary that the pharmacoperone be present at the time of synthesis?” For this reason experiments were designed to determine if it is necessary for pharmacoperones to be present at the time of (mutant) receptor synthesis in order to be functional.

Section snippets

Materials

The GnRH analog, d-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH (Buserelin, Hoechst-Roussel Pharmaceuticals, Somerville, NJ), myo-[2-3H(N)]-inositol (Perkin-Elmer, Boston, MA; NET-114A), competent cells (Promega, Madison, WI), PCR primers, DMEM, OPTI-MEM, lipofectamine, phosphate buffered saline and pcDNA3.1 (Invitrogen, San Diego, CA), endofree maxi-prep kits (Qiagen, Valencia, CA), were obtained as indicated. The following chemical structures (collectively referenced as “pharmacoperones”)

Results

Fig. 1 shows the effect of varying the time of rescue and time of preloading with tritiated inositol on total IP production (CPM) following stimulation with a saturating concentration of the GnRH agonist, Buserelin (100 nM). The number of CPM recovered is a linear function of the amount of the time allowed for radiolabeled inositol incorporation into the cells. Presumably, this reflects an increase in specific activity of the intra-cellular inositol pool over time.

More surprisingly however, was

Discussion

Our prior cell culture studies have relied on transient transfection in Cos-7 cells in order to show that pharmacoperones rescue many misfolded mutants of the GnRHR. While Cos-7 cells cannot be stably transfected, they are well known for their ability to aggressively synthesize proteins. In the current studies, we used both Cos-7 cells and a stably transfected HeLa cell line expressing hGnRHR (E90K) mutant. The stable cells were included in these studies because the transcription and

Acknowledgements

This work has been supported by NIH grants HD-19899, RR-00163 and HD-18185.

References (38)

  • J.A. Janovick et al.

    Regulation of G protein-coupled receptor trafficking by inefficient plasma membrane expression: molecular basis of an evolved strategy

    J. Biol. Chem.

    (2006)
  • B. Kleizen et al.

    Protein folding and quality control in the endoplasmic reticulum

    Curr. Opin. Cell. Biol.

    (2004)
  • P.E. Knollman et al.

    Parallel regulation of membrane trafficking and dominant-negative effects by misrouted gonadotropin-releasing hormone receptor mutants

    J. Biol. Chem.

    (2005)
  • N. Kresge et al.

    The thermodynamic hypothesis of protein folding: the work of Christian Anfinsen

    J. Biol. Chem.

    (2006)
  • J.D. Schrag et al.

    Lectin control of protein folding and sorting in the secretory pathway

    Trends Biochem. Sci.

    (2003)
  • T.F. Walsh et al.

    Potent antagnists of gonadotropin releasing hormone receptors derived from quinolone-6-carboxamides

    Bioorg. Med. Chem. Lett.

    (2000)
  • S. Wuller et al.

    Pharmacochaperones post-translationally enhance cell surface espression by increasing conformational stability of wild-type and mutant vasopressin V2 receptors

    J. Biol. Chem.

    (2004)
  • V. Bernier et al.

    Pharmacologic chaperones as a potential treatment for X-linked nephrogenic diabetes insipidus

    J. Am. Soc. Nephrol.

    (2006)
  • S.P. Brothers et al.

    Human loss-of-function gonadotropin-releasing hormone receptor mutants retain wild-type receptors in the endoplasmic reticulum: molecular basis of the dominant-negative effect

    Mol. Endocrinol.

    (2004)
  • Cited by (0)

    View full text