P. Michael Conn

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.

The Molecular Mechanism of Action of Gonadotropin Releasing Hormone (GnRH) in the Pituitary

Publisher Summary Gonadotropin-releasing hormone (GnRH) is released by the hypothalamus and travels, via a portal system, to the pituitary. Here it stimulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins enter the general circulation and regulate steroidogenesis and gamete maturation in the gonads of both sexes. Because of its unique position as a molecule that transduces signals from the neural system to the endocrine systems, GnRH has proven to be a useful compound both experimentally and clinically. GnRH and its analogs can be used to promote or inhibit fertility in men and women, treat steroid-dependent neoplasia, cryptorchidism, precocious puberty, and other diseases. In addition, GnRH has proven to be useful in veterinary medicine and for synchronized breeding of fish for food. This chapter focuses on the molecular basis of GnRH action, regulation of gonadotropic responsiveness, and stimulus-LH release coupling at the gonadotropin. Binding of GnRH to its plasma membrane receptor results in regulation of receptors and of cellular responsiveness, as well as release of the gonadotropins, LH and FSH. Because the ability to regulate gonadotropin release is important for human and veterinary medicine, there has been a great deal of interest in understanding the molecular basis of GnRH action.

The Molecular Basis of Gonadotropin-Releasing Hormone Action*

Introduction WE HAVE known for more than 15 yr that release of the pituitary gonadotropins (LH and FSH) is regulated by a decapeptide from the hypothalamus, GnRH. This releasing hormone is active in vivo, as well as in pituitary explants, and in cell cultures. Accordingly, it has been possible to obtain a great deal of data which have been used to develop an understanding of GnRH action at the cellular level. Models of GnRH action have been useful to understand and predict the actions of GnRH in vivo and to design means for using the releasing hormone in the treatment of human disorders. The present review will describe our current understanding of GnRH action and serve to update our previous review in this series (1).

Gonadotropin-Releasing Hormone and Its Receptors in Rat Brain

Since the chemical identification of gonadotropin-releasing hormone (GnRH) in 1971, significant progress has been made in understanding the mechanisms by which GnRH action is mediated in the anterior pituitary. In contrast, relatively little information is available which identifies the intracerebral sites and mechanisms of action of GnRH in the brain. Early immunohistochemical studies of GnRH distribution in the central nervous system, together with behavioral and electrophysiological experiments, suggested that GnRH functioned as a neurotransmitter and was, possibly, involved in the expression of reproductive behaviors. The subsequent identification and characterization of GnRH receptors in the brain further strengthened the view that GnRH caused specific effects in select regions of the brain known to be involved in the neuroendocrine regulation of the anterior pituitary and in the generation of reproductive behaviors. After the mouse pituitary GnRH receptor was cloned it became possible to identify brain neurons which contained the GnRH receptor mRNA. Comparison of the locations of the GnRH peptide, the GnRH receptor protein, and the neurons which contain the GnRH receptor mRNA suggests that the GnRH neuronal system itself can potentially provide a direct link between the neuroendocrine regulation of anterior pituitary function and the intracerebral regulation of reproductive behaviors; furthermore, it is possible that the GnRH neuronal system takes an active part in intracerebral feedback loop systems between the mediobasal hypothalamus and the septum-diagonal band which regulate GnRH release from the median eminence.

Distribution of gonadrotropin releasing hormone agonist binding sites in the rat central nervous system

Specific binding sites for gonadotropin releasing hormone (GnRH) in the central nervous system of the rat were studied with in vitro autoradiography and with radioligand assays. The results show that GnRH binding sites are present in the lamina glomerulosa and plexiformis externa, the nucleus olfactorius anterior pars externa, and the frontal cortex at the sulcus rhinalis. In the septum, only a few GnRH binding sites are detected in the lateral and dorsal portions of the nucleus septi lateralis. In addition, a small number of GnRH receptors are seen in the mediobasal hypothalamus and amygdala while substantial binding is apparent in the interpeduncular nucleus, central gray and superior collicle. In the hippocampal formation the GnRH agonists bind to the dorsal and ventral subiculum as well as to receptors in the areas CA1 through CA4. The highest concentration of GnRH receptors is found in the parasubiculum. Competitive binding assays with membrane preparations from the hippocampus and interpeduncular nucleus indicate that the binding of the GnRH agonists is reversible and has a binding affinity of 1 X 10(9) M-1. Injections of radioactive GnRH agonist Buserelin into the lateral ventricle results in selective and reversible labeling of the hippocampal areas CA1 through CA4 as well as the interpeduncular nucleus, central gray and the parasubiculum. The results of the present study indicate that GnRH binds to specific receptors in select areas of the central nervous system of the rat where the peptide may regulate sensory, behavioral and endocrine events.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Use of lithium ion in measurement of stimulated pituitary inositol phospholipid turnover

Publisher Summary This chapter discusses some practical considerations in the design of experiments using Li + , based on experience with pituitary gonadotrope cells in culture. These cells respond to the neuroendocrine peptide, gonadotropin-releasing hormone (GnRH), by the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Experimental procedure is provided in the chapter. Determination of optimal [Li + ] and effects on inositol phosphate (IP) accumulation is also presented. Most investigators using whole-cell preparations have been treated with Li + at 5–10 m M. These concentrations are in severalfold excess of the K i (0.8 m M) reported by Hallcher and Sherman for the Li + inhibition of inositol-l-phosphate phosphatase (I-1-Pase) and are generally found to provide maximal IP signal stabilization. Li + concentration dependence of GnRH-stimulated IP accumulation is decsribed in the chapter. It is important that available physiological end points in an experimental system be evaluated with respect to the possible extraneous effects of Li + .

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.