Pharmacological Chaperones for Misfolded Gonadotropin-Releasing Hormone Receptors
Introduction
The heptahelical G protein-coupled receptors (GPCRs) constitute a large and functionally diverse superfamily of membrane proteins. Their primary function is to transduce extracellular stimuli into the intracellular environment through the activation of one or more signal transduction pathways mediated by G proteins and other interacting proteins. This system of cellular communication is so effective that the ligands which recognize and activate these receptors are highly variable in chemical structure and include photons, odorants, pheromones, hormones, lipids, and neurotransmitters that vary in size from small biogenic amines to peptides to large proteins (Rosenbaum et al., 2009, Ulloa-Aguirre and Conn, 1998, Ulloa-Aguirre and Conn, 2009, Ulloa-Aguirre et al., 1999). Estimates suggest that 1–5% of the genome in mammals encodes for this superfamily of receptors, for which at least 50% are thought to be the target for endogenous ligands and the other half are sensory (odorant and taste) receptors. These receptors currently constitute the single most important source of therapeutic targets for many diseases; in fact, 60–70% of all approved drugs derived their benefits by selective targeting to these proteins (Lagerstrom and Schioth, 2008, Overington et al., 2006, Schlyer and Horuk, 2006).
As with other protein molecules, structural alterations provoked by mutations or genetic variations in the gene sequence of GPCRs may provoke abnormal function of the receptor leading to disease. Mutations in these receptors are known to be responsible for a large number of disorders, including cancers, heritable obesity, and endocrine disease, which underline their importance as therapeutic targets. Structural alterations may provoke either gain- or loss-of-function of the affected receptor (Milligan, 2003, Ulloa-Aguirre and Conn, 1998). Loss-of-function mutations usually alter domains involved in specific functions of the receptor, such as agonist binding, receptor activation, interaction with accessory/scaffold proteins or with coupled effectors, or sequences that dictate proper folding and intracellular trafficking of the receptor to the cell surface plasma membrane (PM). It is becoming well recognized that mutations of GPCRs frequently lead to misfolding and subsequent retention/degradation by the quality control system (QCS) of the cell. Further, misfolding can result in protein molecules that retain intrinsic function yet become misrouted and, for reasons of mislocation only, cease to function normally and result in disease. Recognition of this latter concept immediately presents the therapeutic opportunity to correct misrouting and rescue mutants, thereby restoring function and, potentially, curing disease. In fact, there is now a wealth of information demonstrating that complete functional rescue of misfolded mutant receptors both in vitro and in vivo is possible by using small nonpeptide molecules called pharmacological chaperones or pharmacoperones. These compounds, often designed originally to serve as receptor antagonists, have proved to serve as effective molecular templates, promoting correct folding and allowing the mutants to pass the scrutiny of the cellular QCS and be expressed at the PM (Bernier et al., 2004a, Bernier et al., 2006, Conn and Janovick, 2009a, Loo and Clarke, 2007, Nakamura et al., 2010, Ulloa-Aguirre et al., 2003, Ulloa-Aguirre et al., 2004a). Thus, the endoplasmic reticulum (ER) QCS represents a potential site for a variety of therapeutic interventions in an array of diseases characterized by conformationally aberrant proteins. This chapter summarizes updated information on the gonadotropin-releasing hormone (GnRH) receptor type I (GnRHR) misfolding, which has proved to be a valuable paradigm for the development of drugs potentially useful in regulating GPCR trafficking in health and disease.
Section snippets
The Endoplasmic Reticulum Quality Control System
Synthesis and processing of secretory and membrane proteins occur in the ER and Golgi apparatus. Similar to other proteins synthesized by the cell, GPCRs are subjected to a stringent QCS that checks the integrity and correct folding of the newly synthesized protein into a three-dimensional protein structure determined by its amino acid sequence. The three-dimensional protein structure is stabilized by several noncovalent interactions involving hydrogen bonds, electrostatic interactions, and
Misfolding of GPCRs and Disease
As mentioned previously, mutations in GPCRs may cause misrouting of otherwise functional proteins and lead to disease (Table I). Mutations in the V2R gene cause X-linked nephrogenic diabetes insipidus, a disease characterized by an inability to concentrate urine despite normal or elevated plasma concentrations of the antidiuretic hormone arginine vasopressin. A number (nearly 70%) of V2R mutants causing X-linked diabetes insipidus are unable to reach the cell surface membrane and respond to
Structural Features of the GnRHR
The GnRHR is a GPCR that has already been a focus of drug development. This receptor belongs to the rhodopsin/β-adrenergic-like family of GPCRs (family A) (Millar et al., 2004, Ulloa-Aguirre and Conn, 1998). Its natural ligand is GnRH, a decapeptide produced by the hypothalamus and released in synchronized pulses to the anterior pituitary to regulate pubertal development, sexual maturation, and reproductive competence (Conn and Crowley, 1994, Knobil, 1974, Krsmanovic et al., 2009, Krsmanovic et
Rescue of Misfolded hGnRHR Mutants with Pharmacoperones
Understanding the structure and mechanism of GnRH action has already led to pharmaceutical development of useful drugs for the treatment of cancer and disorders of reproduction. Beyond this, however, the GnRHR-ligand system is a particularly good model to understand both protein routing and the mechanism of rescue by pharmacoperones. Among the reasons for these views are the following:
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The GnRHR is one of the smallest GPCRs (328 amino acids in the human and most nonrodent mammals; 327 in rat and
Mechanism of Action of Pharmacoperones
Desirable characteristics of molecules that could potentially function as pharmacoperones for misfolded proteins include (i) ability to reach physiological concentrations, (ii) capacity to permeate the cell surface membrane, (iii) ability to localize and intervene at the ER and/or post-ER compartments where the misfolded protein is synthesized or retained, (iv) ability to remain undegraded long enough to stabilize the target mutant, (v) specificity for the target protein, and (vi) ability to
The Dominant-Negative Effect of hGnRHR Mutants and Receptor Rescue
Although the GnRHR was one of the first GPCRs shown to oligomerize upon agonist activation at the PM as part of normal receptor function, the finding of oligomerization in the ER–Golgi complex at the time of protein synthesis and routing to the cell surface has emerged as a new and important concept for GPCRs function (Angers et al., 2002, Bouvier, 2001, Bulenger et al., 2005, Cornea et al., 2001, Milligan, 2007). Constitutive oligomerization at the ER has been demonstrated for a number of
Conclusion
In this chapter, we review the conceptual and developmental history of pharmacoperone drugs. Among the reasons that mutations result in disease is that conformationally defective proteins are misrouted and do not reach their site of physiological action. Some mutants exacerbate mutational disease by binding nascent WT proteins and causing them, also, to become misrouted as part of a mutant–WT complex. Pharmacoperones (from pharmacological chaperones) are small, target-specific, cell-permeating
Acknowledgments
This work was supported by National Institutes of Health Grants DK85040, RR030229, TW/HD-00668, and P51RR000163 (P. M. C.), and grant 86881 from CONACyT, Mexico (A. U.-A). A. U.-A. is a recipient of a Research Career Development Award from the Fundación IMSS, México.
Conflict of Interest: The authors have no conflicts of interest to declare.
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