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

Janovick, Jo Ann; Brothers, Shaun P.; Cornea, Anda; Bush, Eugene N.; Goulet, Mark T.; Ashton, Wallace T.; Sauer, Daryl R.; Haviv, Fortuna; Greer, Jonathan; Conn, P. Michael

doi:10.1016/j.mce.2007.04.012

Cited by 45 publications

(40 citation statements)

References 43 publications

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“…The cell surface expression levels of the mutants with greatly reduced R exp (less than one-fourth of the wild type: L175A, F178L, and R179A mutant receptors) were rescued by a non-peptide antagonist, NBI-42902 (26). We propose that this cell-permeant small molecule may also act as a pharmacological chaperone to stabilize the receptor structure, increasing trafficking through the ER, preventing degradation of misfolded receptors in a fashion similar to that of IN3 (8,9) and other non-peptide GnRH antagonists (38). After rescue, the cell surface expression levels of the mutant receptors were about 3-fold higher relative to their non-rescued mutants (Table 1).…”

Section: Comparative Modeling Of the Human Gnrh Receptor And MDmentioning

confidence: 99%

Role of the Transmembrane Domain 4/Extracellular Loop 2 Junction of the Human Gonadotropin-releasing Hormone Receptor in Ligand Binding and Receptor Conformational Selection

Forfar

2011

Journal of Biological Chemistry

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Recent crystal structures of G protein-coupled receptors (GPCRsG protein-coupled receptors (GPCRs), 2 which comprise the largest family of cell surface receptors, are characterized by seven-transmembrane (7-TM) domains joined by three extracellular loops (ECLs) and three intracellular loops. It is of fundamental importance to understand how these receptors, which share a common architecture, are activated by a variety of structurally diverse ligands. In this way, therapeutics that are better targeted to mimic or block these molecules can be designed. The crystal structure of bovine rhodopsin provided the first structural insight into this family of receptors (1) and aided much of our understanding of the molecular mechanisms of GPCR activation, in combination with a number of biochemical and biophysical studies.The gonadotropin-releasing hormone (GnRH) receptor is a member of the rhodopsin-like subfamily of GPCRs. GnRH I is released in pulses from the hypothalamus into the portal blood system where it is transported to the anterior pituitary gland. Here it binds to GnRH receptors expressed on gonadotrope cells, which predominantly causes G q/11 activation. The resultant signaling pathway brings about the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) to regulate steroidogenesis and gametogenesis. As such, GnRH analogues are used extensively to treat many hormone-dependent diseases, including infertility, endometriosis, benign prostatic hyperplasia, and breast cancer (2). However, it has become evident that GnRH also acts on many cells outside of the pituitary gland. There are two endogenous ligands, hypothalamic GnRH I and extrahypothalamic GnRH II, despite there being only one functional receptor subtype expressed in humans. Both ligands exert their effects through the same receptor subtype, although they exhibit distinct pharmacological and signaling profiles (3). Different physiological outcomes are proposed to be mediated by different receptor coupling to G s (4) or G i/o (5) in addition to the classical G q/11 pathway, but this remains controversial (6). However, we have shown that the human GnRH receptor can activate other G q/11 -independent signaling pathways such as G 12/13 (7). Hence, the divergent signaling is believed to be mediated by different receptor-active conformations induced by differential ligand-receptor interactions. Binding of GnRH I and II to the GnRH receptor may cause different intramolecular interactions to be broken, allowing the receptor to adopt varied conformations and thus enabling divergent signaling pathways. We have termed this ligand-induced selective signaling (3,8,9).Investigations into the structure of the GnRH receptor and identification of ligand binding contacts between GnRH and its receptor will enable refinement of molecular models for structure-based drug design. Previous site-directed mutagenesis studies and modeling have identified multiple contact sites of the GnRH receptor with its ligands (2, 3, 10 -12). As these pep-

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Section: Comparative Modeling Of the Human Gnrh Receptor And MDmentioning

confidence: 99%

Role of the Transmembrane Domain 4/Extracellular Loop 2 Junction of the Human Gonadotropin-releasing Hormone Receptor in Ligand Binding and Receptor Conformational Selection

Forfar

2011

Journal of Biological Chemistry

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“…6 and 7). Alternatively, folding of both WT and mutant GPCRs, including V2 vasopressin receptors (8,9), -and ␦-opioid receptors (10 -12), and gonadotropin-releasing hormone receptors (13,14), can be facilitated by membrane-permeant agonists or antagonists acting as pharmacochaperones to stabilize helix packing by binding in the transmembrane heptahelical domain.…”

Section: Biosynthesis Of G Protein-coupled Receptors (Gpcrs)mentioning

confidence: 99%

Calcium-sensing Receptor Biosynthesis Includes a Cotranslational Conformational Checkpoint and Endoplasmic Reticulum Retention

Cavanaugh

McKenna

Stepanchick

et al. 2010

Journal of Biological Chemistry

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“…E 90 K can be rescued by pharmacoperones, drugs that diffuse into cells and provide a folding template. This template enables (otherwise) misfolded mutants to fold or refold correctly (10), pass a quality control system (constituted of chemically heterogeneous chaperone proteins of the ER that either promote correct folding or retain misfolded proteins) (11), traffic to the plasma membrane, bind agonist, and produce a signal. Pharmacoperones of the hGnRHR rescue mutant E 90 K by creating a surrogate bridge between D 98 and K 121 that also stabilizes the relation between TM2 and TM3 (4).…”

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confidence: 99%

Salt bridge integrates GPCR activation with protein trafficking

Conn

2010

Proc. Natl. Acad. Sci. U.S.A.

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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.constitutive activity | hormone action | receptor | G-protein coupled receptor | receptor trafficking H undreds of gonadotropin releasing hormone receptor (GnRHR) mutants have been reported but none have constitutive activity (CA) (1). This observation is surprising, because many G protein coupled receptors (GPCRs) have mutants (2) or WT receptors (3) with CA. A highly conserved (4) structural feature of the GnRHR is a salt bridge between transmembrane segment (TM) 2 and TM3 (residues E 90 and K 121 in the human sequence) (5, 6). Gonadotropin releasing hormone (GnRH) agonists, but not peptide antagonists, bind receptor residues D 98 and K 121 (5, 6) and alter the relation between TM2 and TM3; this same relation is important for trafficking of the human GnRHR (hGnRHR) to the plasma membrane (4, 7). We considered that perturbing this relation is also a determinant of receptor activation.Another way to perturb this relation, other than ligand binding, is to alter the charge of one of its constituents; mutant E 90 K would result in charge repulsion between K 90 and K 121 . This mutant, which occurs in some cases of human hypogonadotropic hypogonadism, is typically recognized by the quality control system (QCS) as a misfolded protein (8), retained in the endoplasmic reticulum (ER) (9) and does not traffic to the plasma membrane. E 90 K can be rescued by pharmacoperones, drugs that diffuse into cells and provide a folding template. This template enables (otherwise) misfolded mutants to fold or refold correctly (10), pass a qua...

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

Cited by 45 publications

References 43 publications

Role of the Transmembrane Domain 4/Extracellular Loop 2 Junction of the Human Gonadotropin-releasing Hormone Receptor in Ligand Binding and Receptor Conformational Selection

Role of the Transmembrane Domain 4/Extracellular Loop 2 Junction of the Human Gonadotropin-releasing Hormone Receptor in Ligand Binding and Receptor Conformational Selection

Calcium-sensing Receptor Biosynthesis Includes a Cotranslational Conformational Checkpoint and Endoplasmic Reticulum Retention

Salt bridge integrates GPCR activation with protein trafficking

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