G-Protein-Coupled Receptor Trafficking: Understanding the

The primary function of cell surface receptors is to recognize specific chemical signals from other substances and produce a biological response. Poin...
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PERSPECTIVE G-Protein-Coupled Receptor Trafficking: Understanding the Chemical Basis of Health and Disease Alfredo Ulloa-Aguirre†,‡, Jo Ann Janovick†, Alfredo Leaños Miranda‡, and P. Michael Conn†,‡,§,* † Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, Oregon 97006, ‡Research Unit in Reproductive Medicine, Instituto Mexicano del Seguro Social, México D. F., Mexico, §Departments of Physiology and Pharmacology, and Cell and Developmental Biology, Oregon Health & Science University, Portland, Oregon 97239

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-protein-coupled receptors (GPCRs) are a large and functionally diverse superfamily of plasma membrane (PM) receptors that consist of single-polypeptide chains that traverse the lipid bilayer seven times, forming characteristic transmembrane (TM) helices. After binding ligand, GPCRs interact with G proteins and transduce signals from outside the cell to the intracellular environment (1). This Perspective summarizes the emerging role of GPCR trafficking as a posttranslational control mechanism, using the gonadotropin-releasing hormone (GnRH) receptor (GnRHR) as a model for evolved inefficiency in trafficking and PM expression of a receptor. The GnRHR Is a Key Regulator in Reproduction. The GnRHR is a GPCR located in the anterior pituitary (Figure 1) that mediates responses to its ligand GnRH, a decapeptide produced by hypothalamic neurons (2, 3). The GnRHR integrates the neural system of the hypothalamus with the anterior pituitary endocrine system by producing peripheral signaling that is mediated by the pituitary gonadotropins, glycoprotein hormones that enter the peripheral circulation and regulate gonadal function (Figure 1). The GnRHR is a therapeutic target for the regulation of fertility and the treatment of several reproductive disorders (2). Diversity in Reproductive Patterns. Although the GnRHR recognizes the same endogenous ligand in most mammals and many non-mammalian vertebrates, it also serves different regulatory patterns of reproduction. Among animals with particular specializations are dogs (which show pseudopregnancy), mice and rats (shortcycling animals), opossums (non-placental mammals that give birth from an external pouch), and guinea pigs

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A B S T R A C T The primary function of cell surface receptors is to recognize specific chemical signals from other substances and produce a biological response. Point mutations in cell surface receptors may result in production of misfolded proteins that are translated but do not reach their proper functional destination in the cell. Also, for some G-protein-coupled receptors, large amounts of wild-type receptor may be destroyed without arriving at the plasma membrane (PM). For the human gonadotropin-releasing hormone receptor, this “inefficiency” has resulted from strong and convergent evolutionary pressure, producing receptor molecules that are sensitive to single changes in chemical charge and are delicately balanced between expression at the PM or retention/degradation in the endoplasmic reticulum. This Perspective focuses on the evolved mechanisms that control PM expression of this receptor at this post-translational level.

*Corresponding author, [email protected].

Received for review August 18, 2006 and accepted October 12, 2006. Published online November 17, 2006 10.1021/cb600360h CCC: $33.50 © 2006 by American Chemical Society

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Figure 1. Functional relations of the hypothalamic–pituitary axis. GnRH is synthesized and secreted by specialized neurons located mainly in the arcuate nucleus of the medial basal hypothalamus and the preoptic area of the anterior hypothalamus. In contrast to other hypothalamic neurons that project to the posterior lobe of the pituitary (e.g., the supra-optic hypophyseal tract), GnRHproducing neurons project to many sites within the brain and also to the median eminence, terminating in an extensive plexus of boutons on the primary portal vessel, which delivers GnRH to its target cell, the gonadotrope of the adenohypophysis. The secretion and interaction of GnRH with its cognate receptor occur in a pulsatile and intermittent manner; such an episodic signaling allows the occurrence of distinct rates and patterns of synthesis and pulsatile secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), whose coordinated release allows for an extremely precise control of gonadal function. These trophic hormones are responsible for stimulating the synthesis and secretion of gonadal hormones and for effecting the process of gametogenesis. The characteristics of the pulsatile release of GnRH, LH, and FSH appear to be regulated by several hypothalamic neurotransmitters (e.g., adrenergic and opioidergic regulation), as well as by the gonadal hormone environment.

(rodents with a long luteal phase). In primates, the tight regulation of reproduction is imposed by the need for efficiency in a process of long gestations, monoovulatory cycles, and single-offspring births. In a broader evolutionary view of reproduction, it is interesting to compare the length of time, metabolic cost, and number of offspring in humans to fish or reptiles. In the latter, small numbers of eggs survive to hatch and a still smaller number actually survive to reproduce themselves. The metabolic investment in each egg is quite modest. Clearly, the management of the human reproductive process must be more closely regulated to protect the greater investment of metabolic energy and time commitment by making the process “work” effectively a greater proportion of the time. It is therefore interesting to consider how nature has accommodated such changes without modifying the basic components of the system. The mammalian GnRHR type I (hereafter referred to only as GnRHR) is among the smallest members of the GPCR superfamily and bears unique structural features, including the lack of a carboxyl-terminal intracellular extension (3, 4) (Figure 2). Fish, reptiles, birds, and the primate type II GnRHR (3) do possess this carboxyl tail, whose presence is associated with differential physiological receptor regulation; when added to the mammalian GnRHR, it dramatically increases PM expression levels of this receptor (4). Unique Chemical Features of GnRHR from Different Species Influence Cellular Trafficking. To enable agonist binding, the GnRHR needs to be at the PM. In the case of the human (h) GnRHR [and, potentially, other GPCRs (5–7)], nature has chemically modified the receptor so that the translation product is delicately balanced between routing to the PM or retention/degradation in the endoplasmic reticulum (ER) (8). This receptor is 632

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extremely sensitive to mutations; a single change in charge can affect the balance (9). A particular feature of primate GnRHRs is the presence of a lysine residue at position 191, which is located in the extracellular loop 2 (EL2) (Figure 3) and restricts the GnRHR PM expression (10). Non-primate mammals utilize a less-effective Glu191 in this position (or Gly191 in the opossum, all 328 amino acids), whereas rats and mice do not have this insertion at all [327 amino acids (3)] and a higher proportion of translation product of both rodent receptors is expressed at the PM (Figure 3). Protein (Mis)folding and Intracellular Trafficking. The GnRHR is scrutinized by the quality-control system of the cell. This system employs recognition mechanisms to examine newly synthesized proteins and ensure that correctly folded proteins are placed into function (8, 11). Among the components of the “conformationscreening” mechanisms are protein chaperones of the ER; these assist in folding of nascent proteins, allowing them to achieve their “proper” conformation and preventing conformationally defective proteins from accumulating within the cell and causing disease (8, 9, 11). Because the quality-control system monitors the structural form of the protein rather than the biological function, misfolded/misrouted mutant proteins can be functionally competent proteins. Therapeutic approaches that redirect misrouted proteins to their correct destination within the cell may restore them to proper functions (12). That a misfolded protein may still behave as a functionally competent protein upon rescue will depend on how the structural defect disrupts the structure of the protein. In the case of the hGnRHR, insertion or removal of prolines or the insertion or removal of cysteines (that leads to loss or gain of cysteine bridges) may result in an uncorrectable misfolded/misrouted protein (9, 13, 14). Conversely, agents that stabilize and/or reshape the defective structure caused by mutations may still allow www.acschemicalbiology.org

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Figure 2. Sequence of the hGnRHR and location of the inactivating (loss-of-function) mutations identified to date. The GnRHR consists of a single polypeptide chain that traverses the cell surface membrane seven times, forming characteristic TM helices interconnected by alternating extracellular (EC) and intracellular (IC) loops. The circle corresponding to the lysine residue at position 191 in the EL2 is enlarged and rose-colored. Mutants with an asterisk (N10K, T32I, E90K, Q106R, C200Y, S217R, R262Q, L266R, and Y284C) were reconstructed in the corresponding mouse and rat receptor sequences and tested for function (see text).

the receptor to “escape” from proteasomal degradation (8, 12, 14–16). In this case, the rescued misfolded competent receptor may be expressed at the PM and affect function (9). Pharmacological Chaperones (Pharmacoperones) and Misfolded GnRHR Mutant Rescue. Early studies demonstrated that the function of a mutant hGnRHR (E90K), which causes severe hypogonadotropic hypogonadism (HH) in humans (17) (Figure 2), was completely restored when the primate-specific amino acid Lys191 was deleted (10). HH is a disease characterized by decreased release of gonadotropins, leading to impaired gonadal function (9). Lys191 is absent in wildtype (WT) mouse GnRHR (mGnRHR) and rat GnRHR (rGnRHR), which route with higher efficiency to the PM (8, 18) than the human counterpart. Studies (15, 16) employing a number of pharmacoperones (lowmolecular-weight molecules that enter cells and are templates for correcting misfolded mutants and restorwww.acschemicalbiology.org

ing function) revealed that E90K and most other naturally occurring mutant receptors, thought to bear defects in domains involved in receptor function, were actually misfolded proteins whose routing to the PM was compromised (9, 14–16). Two particular hGnRHR mutants involving changes in charge, S168R and S217R, were recalcitrant to rescue by different classes of pharmacoperones (9). These findings initially suggested that these sites were unrescuable because they may involve domains important for receptor function. However, further studies demonstrated that this was not the case and that residues at positions 168 and 217 in the hGnRHR were important in regulating the position of the EL2 and the intimacy of residues Cys14 and Cys200 necessary to form a disulfide bond and a properly folded receptor in the hGnRHR (Figure 4) (20). Because of charge considerations, the unfavorable exchange of serine and arginine at the 4 and 5 TM helices likely moved the EL2 into a VOL.1 NO.10 • 631–638 • 2006

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Figure 3. Evolutionary changes of the GnRHR. The GnRHR is larger in birds, fish, and reptiles; in these species, the receptor bears an intracellular carboxyl-terminal extension (dark-brown circles within the light-brown square). In mammals, the GnRHR is shorter and the carboxyl tail is absent; the blue circle represents the amino acid residue in position 191, which is frequently glutamic acid or glycine but is replaced by lysine in primates. In rat and mice GnRHR, this amino acid is absent. In most mammals, an association between cysteine residues 14 and 200 (red-filled circles) must form for proper routing of the receptor from the ER to the PM; in primates this association is formalized by a covalent bond (red line). Glutamic acid or lysine at position 191 destabilizes the association between these two cysteines. The disulfide bridge between the first and second extracellular loops (formed by cysteine residues 114 and 195 or 196 in most mammals; lower red line) is a structural feature present in almost all GPCRs known and is associated with the fundamental stability of the structure containing seven TM helices.

position from which the formation of a cysteine bridge is unlikely and the mutant with the unformed cysteine bridge cannot pass the quality-control system even in the presence of pharmacoperones. Studies with pharmacoperones also showed that PM expression of the WT hGnRHR but not the WT rGnRHR and WT mGnRHR increased substantially upon exposure to these agents (18, 21, 22). This indicates that a large portion of the hGnRHR is normally inefficiently trafficked to the PM, retained by the quality-control system, and likely degraded. The observation that the hGnRHR was so susceptible to alterations of single charges in the receptor structure supported the view that the human receptor is precariously balanced between retention in the ER and routing to its final destination; this is not seen in rats or mice, animals that routed the GnRHRs to the PM with higher efficiency. Because deletion of Lys191 from the hGnRHR led to almost complete PM expression of the WT and E90K mutant receptors, we concluded that the presence of this residue was associated with routing regulation. For reasons that were buried in the physically diffuse amino acid differences between the rodent and human sequences, the simple addition of Lys191 to WT rodent GnRHRs did not decrease routing (20, 22). As discussed below, removing Lys191 from the hGnRHR obviates the need for the Cys14–Cys200 bond (20). 634

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Mutations Leading to Misfolded hGnRHRs Have Less Impact on Trafficking in Rat and Mouse WT GnRHRs. Reconstruction of nine naturally occurring mutations leading to misfolded GnRHRs in humans (Figure 2) in the corresponding mouse and rat receptor sequences revealed that only the E90K, S217R (G216R and S216R in the mouse and rat GnRHR, respectively), L266R, and Y284C (L265R and Y283C in both rodent GnRHR sequences) substitutions significantly impact rat and mouse GnRHR function (20, 22). Notably, pharmacoperone treatment restored function of all but the G216R and S216R mutants (which corresponded to the pharmacoperone-insensitive S217R mutant in the hGnRHR), an indication that the effect of these mutations was to cause severe protein misfolding and loss of the ability to translocate the protein to the PM (22). In KEYWORDS Disulfide bridge: A covalent linkage formed between two sulfhydryl groups on cysteines. Evolution: A change in the properties of populations of organisms that transcend the lifetime of a single individual. Gonadotropin-releasing hormone receptor: The receptor for the hypothalamic peptide hormone gonadotropinreleasing hormone that stimulates the release of gonadotropins. G-protein-coupled receptors: A large and functionally diverse superfamily of plasma membrane receptors that consist of single-polypeptide chains that traverse the lipid bilayer seven times, forming characteristic transmembrane helices.

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PERSPECTIVE Figure 4. Structures of hGnRHR a) Predicted structure of the hGnRHR based on homology modeling with the structure of bovine rhodopsin (19). The coiled structures represent the antiparallel ␣-helices of transmembrane domains 1–7 connected by the extra- and intracellular loops of the receptor (curved cords). Intermolecular associations between Cys14 (at the NH2-terminal segment) and Cys200 (at the EL2, colored in red) and between Cys114 (at the COOH-terminal end of the EL1) and Cys196 (at the EL2) are shown (colored sticks). Top right: magnification of the upperthird portion of the receptor showing the Cys14 –Cys200 (red sticks) and Cys114 –Cys196 (blue sticks) disulfide bridges. b) A model of the hGnRHR showing the seven TM helices displayed as rods. HGnRHR mutants at Ser168 and Ser217 (red circles) cannot be rescued by pharmacoperone treatment. The approximate location of amino acid residues that differ between the human and rat GnRHR and that represent thermodynamically unfavored substitutions are represented by small lavendar circles (human/rat in enlarged roseate circles) (amino acids 7, 168, and 203; 202 in rat and mouse); amino acid 189 is included for its physical proximity to the Cys14 –Cys200 bridge. Amino acids 7, 168, and 189 frequently coevolved with the appearance of the “extra” amino acid in position 191 (lysine in primates, glutamic acid or glycine in all non-rat/mouse mammalian species sequenced to date). The locations of some other residues that impact on the net levels of receptor PM expression and allow the effect of Lys191 are shown in yellow. Positions of these residues are simply pictorial. Residues located in the NH2 extracellular segment and in the EL2 as well as sequences flanking this loop (i.e., within TM helices 4 and 5) and those that abut on that area (ELs 1 and 3) presumably control the destabilizing role of Lys191 (blue circle) on the formation of the Cys14 –Cys200 bridge as disclosed by mutagenesis experiments (21). This bridge is magnified in the right figure; the covalent bond between the two sulfur atoms is shown by gold sticks. The non-optimized models shown in this figure were generated with the molecular visualization program PyMol (DeLano Scientific, San Francisco, CA) and kindly provided by Angel Piñeiro and Eduardo Jardón-Valadez from the Faculty of Chemistry of the National University of Mexico, Mexico City, Mexico.

addition, the mGnRHR appeared to be less forgiving than the rGnRHR to substitutions, because the mouse mutations more greatly reduced PM expression of the mutant receptors compared with the rat mutant counterparts (22). These observations indicated that these rodent GnRHRs appear to be more tolerant of mutation than is the hGnRHR and that the small number of semiand non-conservative differences between the mouse and rat GnRHRs (at positions 11, 24, 160, and 216) might have a physiological influence on receptor function. In fact, creation of “rat-like” mGnRHR misfolded mutants indicated that a modest amino acid change, that is, substitution of glycine in position 216 in the mGnRHR with serine (present in the rGnRHR), resulted in markedly increased PM expression of the mGnRHRE90K sequence; modification of the corresponding sequence in the rGnRHR, to make it more “mouse-like”, was associated with a 2-fold decrease of expression (22). Further substitutions exchanging proline with glycine in position 11 and isoleucine with threonine in position 24 or 160 resulted in a substantial decrease in mGnRHR-E90K function, which was more prominent www.acschemicalbiology.org

whenever Gly216 was present. Ser216 in the rGnRHR sequence increased the efficiency of routing to the PM of rat mutants that are otherwise misrouted as disclosed by pharmacoperone treatment. Mutant GnRHRs Exert Dominant-Negative Effects Due to an Influence on Trafficking. Receptor oligomerization and interactions with accessory proteins are welldocumented features of GPCR function (23, 24). In some cases, intracellular association of receptors as homo- or heterodimers led to either cell surface targeting (a dominant-positive effect) or the intracellular retention of the complex (a dominant-negative effect) (6, 9, 24). In the case of the hGnRHR, misfolded hGnRHR mutants may form intracellular complexes at the ER, affecting proper delivery and trafficking of the receptor to the cell surface (8, 21, 25). Cotransfection experiments revealed that this dominant-negative effect of mutants on their WT counterpart resulted from a physical interaction between these molecules (21). Despite the high homology between rat, mouse, and human GnRHRs, pharmacoperone-sensitive rat and mouse misfolded receptors exhibited different VOL.1 NO.10 • 631–638 • 2006

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dominant-negative actions on their corresponding receptors: (i) The WT rGnRHR escapes the dominantnegative effects of mutant rGnRHRs (e.g., rGnRHR-E90K) but not those from human and mouse mutants, thus indicating that this receptor retains the ability to oligomerize. (ii) Mutant mGnRHRs are more effective in dominant-negative actions on both rodent receptors. (iii) Mutant hGnRHRs are more effective, as dominantnegative regulators, on the human WT receptor than on rat and mouse WT receptors, which lack Lys191 (22). The effect of Ser216 in the rGnRHR appeared to be important for the loss of dominant-negative effects of mutant rGnRHRs on the WT counterpart; experiments showed that the Gly216 (mouse) ¡ Ser216 (rat) substitution in the mGnRHR mutants sequence resulted in loss of dominant-negative effect of mouse mutants on the rat receptor species (22). In the hGnRHR, substitution of Ser217 with glycine led to a modest reduction in receptor function and concomitantly to loss of the dominantnegative effect of the mutant on the WT receptor, whereas removal of Lys191 from the S217G mutant allowed functional recovery. These findings implicate the absence of Lys191 and presence of Ser216/217 as determinants for PM expression and loss of the dominant-negative effect of mutant GnRHRs. In this scenario, the counteracting effect of Ser217 on the dominant-negative action of mutant hGnRHRs may be mitigated by the insertion of Lys191, because deletion of this residue from the hGnRHR sequence led to loss of the dominant-negative effect of misfolded mutants on WT receptor expression (18, 22). Cys14–Cys200 Bridge Formation and Tolerance to Mutations. The amino acid sequence of the GnRHR predicts two disulfide bridges at extracellular regions (Cys14–Cys195/196 and Cys14–Cys199/200; Figure 4). The first bridge is a structural feature present in KEYWORDS almost all known GPCRs and Intracellular trafficking: The passage of newly is associated with the stabilsynthesized proteins from the ER to their final destination within the cell. ity of the heptahelical strucMutant receptors: Protein molecules with an ture (1); mutation of the cysaltered amino acid sequence due to changes teine residue at either end of in the nucleotide sequence of their corresponding gene. this bridge in the GnRHR Pharmacological chaperones: Low-molecularresulted in a complete loss weight molecules that enter cells and are of activity, which could not templates for correcting misfolded mutants. Pharmacoperone: A pharmacological chaperone. be rescued by pharmacoperProtein folding: The process whereby a protein ones (20). The functional sigmolecule assumes its 3D shape. nificance of the Cys14– 636

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Cys199/200 bridge differed depending on the receptor species: (i) It was not essential for optimal function of the rGnRHR because replacement of the cysteine residues at either end of the bridge does not affect agoniststimulated intracellular signaling. (ii) In the mGnRHR, breakage of this bridge results in ⬃50% decrease in receptor function. (iii) For the hGnRHR, formation of this bridge was an absolute requirement for correct routing and PM expression of the receptor because bridgebreaking mutants exhibit either no activity or marginal activity (14, 22). Further, exposure of these mutants to pharmacoperones normalizes receptor function, an indication that the absence of the bridge resulted in a misfolded protein. What are the chemical determinants that lead to the requirement of the Cys14–Cys200 bridge in folding of the hGnRHR? Removal of Lys191 from misfolded hGnRHR mutants (including Cys14 and Cys200 mutants) led to functional rescue of the altered receptors, an indication that the association between Cys14 and Cys200 may be potentially disrupted or diminished by the presence of Lys191 (10, 20). In contrast with the majority of mutations resulting in HH in humans (Figure 2), the effect of Lys191 is not wholly an effect of charge because replacement by alanine, glutamic acid, or glutamine also produced inefficient PM expression of human sequence compared with the hGnRHR-desLys191 variant. The observation that removal of Lys191 obviates the need for this bridge in the hGnRHR and that inserting Lys191 alone into the rat or mouse sequence (⬎88% homologous with the human sequence) did not impact the requirement for this bridge between the NH2terminal and the EL2 led to the search for additional components of the requisite motif. This problem was approached by locating the thermodynamically unfavored changes among GnRHR sequences for various animal species and identifying those amino acid residues that frequently coevolved with the appearance of the “extra” amino acid in position 191 (lysine in primates, glutamic acid or glycine in all non-rat/mouse mammalian species sequenced to date; Figure 3) or that were proximal to it and to the Cys14–Cys200 bridge (Figure 4). When the molecule as a whole is considered, the modifications associated with orientation of EL2 (positions 7, 168, 189, and 202/ 203) all involve the gain or loss of proline or serine residues. Proline formed a five-membered nitrogencontaining ring, a feature found in very tight turns in www.acschemicalbiology.org

PERSPECTIVE protein structures. Serine, with a slightly polar nature, small size, and propensity of the side-chain hydroxyl oxygen to H-bond with the protein backbone, also was found in association with tight turns of the protein structure. The rest of the motifs were identified by making guesses based on the physical relation between amino acids in the 3D state of the receptor molecule. With this information, human receptors that were more “rat-like” were constructed and tested; these expressed at the higher levels associated with rat receptor and lacked the requirement for the Cys14–Cys200 bridge, which is another structural feature of the rGnRHR (20). The spatial alignment was, therefore, quite important, because these two residues must be sufficiently close to each other to allow bridge formation. When the bridge formed, the hGnRHR was recognized by the qualitycontrol system as correctly folded. On the contrary, when it did not form, it was viewed as defective and was retained in the ER. Thus, the presence of Lys191 limited the number of hGnRHR molecules that were exported from the ER to the PM. Inefficient WT hGnRHR Trafficking: A PostTranslational Regulatory Mechanism? It seems that the cell is exploiting the insertion of Lys191 in the hGnRHR for controlling routing in normally functioning cells. Why waste half the amount of newly synthesized receptor molecules? Several possibilities exist. First, having a pool of receptors that can be recruited during very demanding conditions (e.g., during the mid-follicular phase of the menstrual cycle) may be very important; the increased synthesis of the receptor at these times would allow the receptor to escape from proteosomal

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degradation. Second, by regulating the amount of receptors expressed, the pituitary may more precisely modulate gonadotropin release and maturation of a single oocyte during each ovulatory cycle. In this scenario, the complexity of reproduction appears to have evolved as the investment in creating a single offspring increased. A look at GnRHR in animals whose sequences appeared odd revealed that several species display marked differences in their reproductive patterns when compared with their evolutionarily close relatives. Among rodents (animals with large litters), only the guinea pig is known to have added an amino acid (glutamic acid) at position 191 of the GnRHR; this particular rodent, a hystricomorph that diverged very early in rodent evolution, exhibits a long luteal phase, which is a primate characteristic. Most non-rodent mammals also contain glutamic acid at this position; this suggests that loss of an amino acid in the homologous position may represent a specialization associated with very short reproductive cycles like those present in rats and mice. In the opossum (a non-placental mammal that places fetuses in a marsupium), the presence of the uncharged glycine at position 191 may reflect the divergence of this group and the specialization needed for this unique form of reproduction. The large number of biological systems that appear to rely on assessment of fidelity of protein structure (26) and folding suggests that regulation at this level may prove to be more common than presently appreciated. Acknowledgment: This work is supported by CONACYT grant 45991-M; NIH grants HD-19899, RR-00163, HD-18185, TW/HD00668.

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