Trafficking of G-protein-coupled receptors to the plasma membrane: insights for pharmacoperone drugs

Abstract

G protein-coupled receptors (GPCRs) are among the most common potential targets for pharmacological design. Synthesized in the endoplasmic reticulum, they interact with endogenous chaperones that assist in folding (or can retain incorrectly folded proteins) and are transferred to the plasma membrane where they exert their physiological functions. We summarize trafficking of the gonadotropin-releasing hormone receptor (GnRHR) to the plasma membrane. The trafficking of GnRHR is among the best characterized due in part to its small size and the consequent ease of making mutant proteins. Human mutations that cause disease through the misrouting of GPCRs including GnRHR are also reviewed. Special emphasis is placed on therapeutic opportunities presented by pharmacological chaperone drugs, or pharmacoperones, that allow misrouted mutants to be routed correctly and restored to function.

Figure 1

Functional relations of the hypothalamic–pituitary axis. Gonadotropin-releasing hormone (GnRH) is synthesized and secreted by specialized neurons located mainly in the arcuate nucleus (AN) of the medial basal hypothalamus and the preoptic area of the anterior hypothalamus. GnRH producing neurons project to the median eminence (ME) where they terminate in an extensive plexus of boutons on the primary portal vessel, which delivers GnRH to its target cell, the gonadotrope of the adenohypophysis (AH). The secretion and interaction of GnRH with its cognate receptor occurs in a pulsatile and intermittent manner; such episodic signaling allows the occurrence of distinct rates and patterns of synthesis and pulsatile release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotrophic hormones are responsible for stimulating the synthesis and secretion of gonadal hormones and for affecting the process of gametogenesis. The characteristics of the pulsatile release of GnRH, LH, and FSH appear to be positively or negatively regulated by several hypothalamic neurotransmitters (e.g., adrenergic and opioidergic regulation), as well as by the gonadal hormone environment.

Figure 2

(a) Sequence of the human gonadotropin-releasing hormone receptor and location of the inactivating mutations identified to date. Circles represent amino acids; those colored dark-red or grey are residues in which the indicated mutation leads to complete and partial HH, respectively; those colored green form a motif of four non-contiguous residues that are required in the human GnRHR for Lys¹⁹¹ to destabilize the formation of the Cys¹⁴-Cys²⁰⁰ bridge [29]. The circle corresponding to the Lys residue at position 191 in the second extracellular loop is enlarged and colored blue. The red lines indicate the position of the Cys¹⁴-Cys²⁰⁰ and Cys¹¹⁴-Cys¹⁹⁶ disulfide bridges; the light purple dashed lines indicate the Glu⁹⁰-Lys¹²¹ (residues in orange circles) salt bridge and the black dashed lines show the association of Asp⁹⁸ and Lys¹²¹ enabled by pharmacoperones. The light orange shadow corresponds to the portion of the receptor where the TMs 2 and 3 are located and that are stabilized by the conserved Glu⁹⁰-Lys¹²¹ salt bridge or the surrogate Asp⁹⁸-Ph-Lys¹²¹ bridge resulting from pharmacoperone action; the light green shadow corresponds to the “zone of death” in TMs 4 and 5, where mutations are completely recalcitrant (Ser¹⁶⁸Arg and Ser²¹⁷Arg) or marginally responsive (Ala¹⁷¹Thr) to pharmacoperones [23]. (b) Superposition of the WT hGnRHR conformation (green structure) and the hGnRHR lacking Lys¹⁹¹ (orange structure), showing the positions of the Cys¹⁴–Cys²⁰⁰ and Cys¹¹⁴–Cys¹⁹⁶ disulfide bridges (highlighted in both structures). (Reproduced from [31] with permission from the Society for Endocrinology). (c) Close-up showing specific interactions between Glu⁹⁰ (in TM2), Asn³¹⁵ (TM7), Lys¹²¹ (TM3), and Ser¹²⁴ (TM3), forming a microdomain that is important for GnRHR stability [25, 32, 53]. Also shown is Asp⁹⁸ (in TM2), which forms the surrogate Asp⁹⁸-Lys¹²¹ salt bridge (dashed red line) upon pharmacoperone action (shown as “Ph” in the white box) [32]. (Reproduced from [53] with permission from the American Chemical Society).

Figure 3

Prediction of functional response of the (a) WT hGnRHR and (b–d) heterozygous hGnRHR mutants to pharmacological treatment in vivo based on in vitro co-expression studies [39]. (a) In the case of the WT hGnRHR, administration of pharmacoperones would presumably lead to improved function, whereas, for the different naturally occurring heterozygous combinations (b–d) leading to complete or partial HH, the in vitro response would predict complete or partial clinical recovery or nearly complete failure to pharmacological rescue. The extent of clinical responses following pharmacological rescue depends on potential interactions between mutant receptors, including the dominant negative effects imposed by one of the defective heterozygous receptors. For example, in the case of HH patients with hGnRHR genotypes bearing the Ala¹⁷¹Thr, Ala¹²⁹Asp, Ser²¹⁷Arg, or L³¹⁴X^(stop) alleles, the dominant effect exerted by these defective receptors may lead to a less than expected clinical response to pharmacoperones. For others, the potential interactions between mutant receptors would not negatively affect or might even favor the outcome to pharmacoperone treatment [39]. In these latter cases, a full clinical response may be an achievable goal. The oval forms represent hGnRHR molecules with a conformation compatible with endoplasmic reticulum (ER) export; the free forms represent conformationally defective receptors whose intracellular traffic to the cell surface plasma membrane is impaired. These misfolded receptors are retained in the ER and eventually degraded through the polyubiquitination/proteasome pathway.