Jo Ann Janovick

Pharmacologic Rescue of Conformationally‐Defective Proteins: Implications for the Treatment of Human Disease

The process of quality control in the endoplasmic reticulum involves a variety of mechanisms which ensure that only correctly folded proteins enter the secretory pathway. Among these are conformation‐screening mechanisms performed by molecular chaperones that assist in protein folding and prevent non‐native (or misfolded) proteins from interacting with other misfolded proteins. Chaperones play a central role in the triage of newly formed proteins prior to their entry into the secretion, retention, and degradation pathways. Despite this stringent quality control mechanism, gain‐ or loss‐of‐function mutations that affect protein folding in the endoplasmic reticulum can manifest themselves as profound effects on the health of an organism. Understanding the molecular, cellular, and energetic mechanisms of protein routing could prevent or correct the structural abnormalities associated with disease‐causing misfolded proteins. Rescue of misfolded, “trafficking‐defective”, but otherwise functional, proteins is achieved by a variety of physical, chemical, genetic, and pharmacological approaches. Pharmacologic chaperones (or “pharmacoperones”) are template molecules that may potentially arrest or reverse diseases by inducing mutant proteins to adopt native‐type‐like conformations instead of improperly folded ones. Such restructuring leads to a normal pattern of cellular localization and function. This review focuses on protein misfolding and misrouting related to various disease states and describes promising approaches to overcoming such defects. Special attention is paid to the gonadotropin‐releasing hormone receptor, since there is a great deal of information about this receptor, which has recently emerged as a particularly instructive model.

G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo

G protein-coupled receptors (GPCR) comprise the largest family of drug targets. This is not surprising as many signaling systems rely on this class of receptor to convert external and internal stimuli to intracellular responses. As is the case with other membrane proteins, GPCRs are subjected to a stringentquality control mechanism at the endoplasmic reticulum, which ensures that only correctly folded proteins enter the secretory pathway. Because of this quality control system, point mutations resulting in protein sequence variations may result in the production of misfolded and disease-causing proteins that are unable to reach their functional destinations in the cell. There is now a wealth of information demonstrating the functional rescue of misfolded mutant receptors by small nonpeptide molecules originally designed to serve as receptor antagonists; these small molecules (“pharmacoperones”) serve as molecular templates, promoting correct folding and allowing the mutants to pass the scrutiny of the cellular quality control system and be expressed at the cell surface membrane. Two of these systems are especially well characterized: the gonadotropin-releasing hormone and the vasopressin type 2 receptors, which play important roles in regulating reproduction and water homeostasis, respectively. Mutations in these receptors can lead to well defined diseases that are recognized as being caused by receptor misfolding that may potentially be amenable to treatment with pharmacoperones. This review is focused on protein misfolding and misrouting related to various disease states, with special emphasis on these two receptors, which have proved to be of value for development of drugs potentially useful in regulating GPCR trafficking in healthy and disease states.

Regulation of G Protein-coupled Receptor Trafficking by Inefficient Plasma Membrane Expression MOLECULAR BASIS OF AN EVOLVED STRATEGY

Despite the prevalence of G protein-coupled receptors as transducers of signals from hormones, neurotransmitters, odorants, and light, little is known about mechanisms that regulate their plasma membrane expression (PME), although misfolded receptors are recognized and retained by a cellular quality control system (QCS). Convergent evolution of the gonadotropin-releasing hormone (GnRH) receptor (GnRHR) progressively decreases inositol phosphate production in response to agonist, validated as a measure of PME of receptor. A pharmacological chaperone that optimizes folding also increases PME of human, but not of rat or mouse, GnRHR because a higher percentage of human GnRHRs are misfolded structures due to their failure to form an apparent sulfhydryl bridge, and they are retained by the QCS. Bridge formation is increased by deleting (primate-specific) Lys191. In rat or mouse GnRHR that lacks Lys191, the bridge is non-essential and receptor is efficiently routed to the plasma membrane. Addition of Lys191 alone to the rat sequence did not diminish PME, indicating that other changes are required for its effects. A strategy, based on identification of amino acids that both 1) co-evolved with the Lys191 and 2) were thermodynamically unfavorable substitutions, identified motifs in multiple domains of the human receptor that control the destabilizing influence of Lys191 on a particular Cys bridge, resulting in diminished PME. The data show a novel and underappreciated means of posttranslational control of a G protein-coupled receptor by altering its interaction with the QCS and provide a biochemical explanation of the basis of disease-causing mutations of this receptor.

Mechanisms mediating multiple physiological responses to gonadotropin-releasing hormone

A central question in endocrinology is how a single ligand interacting with a single receptor can mediate multiple responses. GnRH interaction with receptor offers a prime example, leading to the regulation of synthesis and release of at least three molecules, regulation of target cell responsiveness and receptor number. The present study suggests a molecular model consistent with extant data that provides a mechanism by which this may occur and, further, which allows for coordinate regulation.

Quality Control Autophagy Degrades Soluble ERAD-Resistant Conformers of the Misfolded Membrane Protein GnRHR

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.

Drug development and the cellular quality control system

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.

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.

Regulation of FSHβ and GnRH receptor gene expression in activin receptor II knockout male mice

To examine in vivo, the local effects of inhibins and activins within the anterior pituitary, independent of their endocrine effects exerted from the gonad, in mediating FSH homeostasis, we used castrated knockout mice lacking either inhibin alpha or activin receptor II (ACVR2) alone or in combination. Compared to castrated wild-type (WT) mice, FSHbeta mRNA levels in the pituitaries of Acvr2 null mice were significantly downregulated in the absence of gonadal feedback. FSHbeta mRNA levels were not significantly higher in the pituitaries of castrated inhibin alpha null mice compared to those in Acvr2 null mice and remained the same in the pituitaries of castrated double mutant mice lacking both inhibin and ACVR2. In contrast to FSHbeta mRNA expression changes, pituitary FSH content was significantly reduced in Acvr2 null mice whereas it was only slightly upregulated in inhibin alpha null mice. Combined absence of both ACVR2 signaling and inhibins caused a decrease in FSH content compared to that in the absence of inhibins alone. These changes in pituitary content were in parallel to those in serum FSH levels in these three groups of castrated mice, suggesting that the unopposed actions of locally produced inhibins are dominant over those effects mediated by ACVR2 signaling to regulate FSH biosynthesis and secretion. Thus, our in vivo results demonstrate that within the pituitary, locally produced activins and inhibins exert their actions at distinct phases of FSH homeostasis. In an independent set of experiments, we tested whether in vivo signaling via ACVR2 is necessary for hypothalamic GnRH biosynthesis and for GnRH receptor expression. Our results demonstrate that in contrast to previous in vitro studies, signaling through ACVR2 is neither required for hypothalamic synthesis of GnRH peptide nor for expression of GnRH receptors in the anterior pituitary. We conclude that within the hypothalamic-pituitary short loop, ACVR2 signaling is critical only for FSH homeostasis and not for GnRH biosynthesis or induction of pituitary GnRH receptor expression. Our studies confirm the importance of using in vivo genetic models for studying regulation of the hypothalamic-pituitary-gonadal axis.

The Third Intracellular Loop of the Rat Gonadotropin-Releasing Hormone Receptor Couples the Receptor to Gs- and Gq/11-Mediated Signal Transduction Pathways: Evidence from Loop Fragment Transfection in GGH3 Cells1

The GnRH receptor (GnRH-R) belongs to the rhodopsin/β-adrenergic family of G protein-coupled receptors. The intracellular domains of these receptors, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some regions located in the membrane-proximal end of the COOH-terminus, are frequently important sites for G protein coupling and specificity determination. Although studies in mouse and human GnRH-R have identified loop 2i as a critical determinant for coupling the receptor to the Gq/11-mediated signal transduction pathway, given the functional similarity among the members of this particular G protein-coupled receptor subfamily and the fact that the GnRH-R lacks the typical intracellular COOH-terminal domain of its superfamily (a potential site for G protein coupling), we investigated the possibility that loop 3i of this receptor also participates in GnRH-R coupling to G proteins. GGH31′ cells, a pituitary-derived cell line that expresses a functional...

Restoration of testis function in hypogonadotropic hypogonadal mice harboring a misfolded GnRHR mutant by pharmacoperone drug therapy

Significance Many diseases result from genetic mutations that cause protein misfolding. Medical treatments often address the symptoms, but do not correct the underlying etiology. This study illustrates proof of principle that a disease caused by a misfolded cell surface receptor can be corrected with a pharmacoperone, a unique class of target-specific drugs that assist protein folding. 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 E90K) 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 E90K 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.