G protein-coupled receptors using pharmacoperones

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G protein-coupled receptors (GPCRs) make up the largest family of transmembrane receptors. Once synthesized in the endoplasmic reticulum (ER), it undergoes inspection by the ER quality control system (QCS). Misfolded GPCRs are recognized by the QCS and are then retained in the ER. Though these GPCRs are misrouted, they still can have intrinsic function. Small molecules called "pharmacoperones" can be used to help assist in refolding of these misfolded GPCRs and redirect them to their proper destination. Endogenous chaperones in the ER themselves can be a therapeutic target, since they also are involved in the QCS. Two important ER chaperones, calnexin and calreticulin, make up what is called the calnexin/calreticulin cycle, which is heavily involved in regulating the trafficking of GPCRs. The gonadotropin-releasing hormone receptor (GnRHR) and the vasopressin type 2 receptor (V2R) are two very well known receptors. A vast amount of knowledge has been gained by using these two receptors as model systems. The following paper will discuss the use of pharmacoperones on misfolded GnRHR and V2R to help restore the proper trafficking of these receptors.

Keywords: GPCR Trafficking, Pharmacoperones, GnRHR, V2R, ER QCS, Calnexin, Calreticulin


GPCRs make up the largest family of the trans-membrane receptors and are extensively studied and targeted in drug development. There are many diseases that are associated with faulty GPCRs, including retinitis pigmentosa, nephrogenic diabetes insipidus, and hypogonadotropic hypogonadism. GPCRs are initially co-translationally translocated into the ER where it undergoes proper folding. The folding processes of these GPCRs are quite complex, involving many protein interactions inside the ER. During folding, these receptors are inspected by the ER QCS to see if they have folded correctly. If they pass the inspection, then they continue on through the secretory pathway. If they fail, then they are retained inside the ER. These misfolded receptors can, however, have the opportunity to refold into the correct conformation by interacting with ER chaperones, which are components of the ER QCS that can assist in proper folding. However, if these chaperones still can't correct the misfolded receptor, then the misfolded receptor will be sent towards the degradation pathway. Two important chaperones in the ER that are involved in the trafficking of the GPCRs are calnexin and calreticulin. These two chaperones are involved in what's called the calnexin/calreticulin cycle, which determines if the GPCRs are either sent out to the Golgi for further processing or retained in the ER. The signaling between secretion and retention involves a quite complex system of modifying the polysaccharide that is linked to the GPCR.

Besides the endogenous ER chaperones, these misfolded receptors can also be rescued by small molecules called pharmacoperones. These pharmacoperones are administered externally and diffuse into the cell, where it binds the misfolded receptor and helps to reform it. For this reason, the use of pharmacoperones for therapeutic use is very attractive. The following review will talk more in depth the ER QCS, in particular the calnexin/calreticulin cycle. Also, the structure of two GPCR class I model systems GnRHR and V2R will be discusses and how pharmacoperones might interact with these misfolded receptors to help relocated them to the proper destination.

Calnexin/Calreticulin Cycle:

Once an integral or secreted membrane protein is synthesized, it needs to enter the ER for proper folding. The ER environment is more oxidizing compared to the intracellular environment, therefore disulfide bonds can be formed. In the case of the GPCR class I receptor GnRHR, it is co-translationally translocated into the ER with the help of the Sec61 translocon. The nascent GPCR is linked with a polysaccharide, Glc3Man9GlcNAc2, which undergoes processing during the secretory pathway. This processing involves the trimming and addition of sugar residues and how it is processed determines where the glycoprotein goes. Initially, when the glycoprotein enters the ER, glucosidase I (GI) and glucosidase II (GII) each cleave off one glucose residue from the polysaccharide attached to the GPCR. This monoglycosylated receptor now binds to the chaperones calnexin or calreticulin, which are key components of the ER QCS, for inspection. If the receptor is folded correctly, then the remaining glucose residue is cleaved for GII and it is released from the chaperone. This correctly folded receptor is then further processed so the enzyme glucosyltransferase (GT) can't recognize it to reglycosylate it and then continues on to the Golgi. If the receptor is not folded correctly, for instance it has an exposed hydrophobic domain, immature glycan or unpaired cysteines, then it will be reglycosylated by GT and will be retained in the ER. This misfolded protein is now monoglycosylated again, which means it can again bind to one of the chaperones for another attempt at folding correctly. This calnexin/calreticulin cycle continues until either the receptor finds the correct conformation, however, there is a point where it will be targeted for degradation if it can't find it in a certain amount of time. The precise mechanism of how the misfolded protein is recognized and sent for degradation is uncertain (Parodi 2008). Since this calnexin/calreticulin cycle can regulate the amount of plasma membrane expression of the GPCRs, it is a potential target for therapy.

If there is an abnormally high amount of misfolded proteins in the ER, it can overwhelm the QCS. BiP/Grp78 is a chaperone in the ER, which is involved in the protective unfolded protein response (UPR), can sense ER stress. If the stress is too overwhelming, then multiple signaling pathways are activated by the dissociation of this chaperone from certain kinases and a transcription factor . The activation of UPR induced genes leads to the overall decrease in protein translation. This buys some time for the ER QCS to correct the folding or send the misfolded receptors for degradation. If the UPR still doesn't help relieve the ER stress, then it leads to apoptosis (Forman, 2003). There is not a lot of focus on the role of ER stress response to misfolded GPCRs. Things that happen in the ER are a bit more complicated than once thought and work is still being done to elucidate the ins and outs of the ER.

GnRHR and V2R Model Systems for GPCR class I:

A lot is known about GnRHR, which makes it a great candidate for a model system. GnRHRs are located in the anterior pituitary and they bind the agonist gonadotropin releasing hormone (GnRH). Once the agonist binds in the transmembrane region, it activates the coupled Gq/11 protein, which then activates phospholipase C (PLC). PLC hydrolysis of phosphatidylinositol (PIP2) leads to formation of 2nd messenger inositol triphosphate (IP3) and diacyl glycerol (DAG). IP3 increases intracellular calcium and results the release of the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH are both involved with reproduction and defective GnRHRs can result in hypogonadotropic hypogonadism or in other words reproductive failure (Naor, 2009).

V2Rs are located in the renal collecting duct of the kidney and is involved in fluid balance. When the agonist arginine-vasopressin, also known as antidiuretic hormone) binds in the transmembrane region, it stimulates the activation of the receptor-coupled Gs protein. This Gs protein then activates adenyl cyclase which in turn produces cAMP and promotes the activation of protein kinase A (PKA). PKA then phosphorylates many downstream targets, which results in the incorporation of the water channel, aquaporin-2, to get inserted in the plasma membrane. Mutations in V2R can result in decreased water retention, which leads to nephrogenic diabetes insipidus. Symptoms include dehydration and elevated sodium levels (Rieg, 2009).

Interestingly, it has been found that misfolded GnRHRs that are retained in the ER can be rescued by small molecules called pharmacoperones. A misfolded GnRHR is retained in the ER, however, this doesn't mean that it has lost its function. With the help of a pharmacoperone, its folding can be corrected and the receptor can be re-routed to its proper destination. Screening for an ideal pharmacoperone will be difficult and laborious. Even choosing the appropriate animal model isn't trivial.

Drug Development and Animal Models:

Primate GnRHR plasma membrane expression levels greatly differ from rodent plasma membrane expression. One major difference between the two is the addition of an extra lysine residue at position 191 in primates. This critical residue destabilizes the formation of the cys14-cys200 disulfide bond, which the formation of this bond is essential for passing the ER QCS (Conn, 2006). This bond is efficiently formed in rodents, which leads to the efficient plasma membrane expression. It is thought that this additional lys191 residue acts as a regulatory mechanism. The presence of this residue results in plasma membrane expression of only half of the newly synthesized receptors. The other 50% is retained in the ER and acts as a 'reserve pool' that can be called upon when needed (Conn, 2007). This gives much better precision in stimulating ovulation at the right moment. It's not surprising that greater regulation is seen in primates, since greater time and energy is invested in creating one offspring, while rodents have large litters. To confirm this, the lys191 was deleted from the human GnRHR. This resulted in the "almost complete plasma membrane expression of the wildtype receptor," (Conn, 2006). Another unique feature that primate GnRHRs have is the virtually non-existent intracellular carboxyl-tail. Intracellular-carboxyl tails add stability to the receptor and reduce internalization (Conn, 2007). Ligand affinity also differs between primate and rodent GnRHRs. The ligand affinity is decreased in primates, compared to rodents. This gives the primate GnRHRs an advantage of being able to ignore background noise (Conn, 2007). Also, modulation of the levels of each gonadotropin depends on frequency of agonist binding and not the amount. High frequency leads to a higher level of LH, while lower frequency leads to a higher level of FSH.

Another disulfide bond that is critical for both primates and rodents is the cys114-cys196. A mutation at either one of these cysteine residues results in an misfolded receptor that could not be rescued. An important salt bridge between glu90-lys121 is another important interaction that can be rescued by the use of a pharmacoperone. A surrogate salt bridge between asp98-lys121 restores the receptor shape and is routed to the plasma membrane (Conn, 2009). Other mutants that affected the plasma membrane expression include residues in the transmembrane region. For instance, the serine to arginine mutations at position 168 and 217 creates a disruption that affects the orientation of the second extracellular loop. The cys14-cys200 bridge if sensitive to these fluctuations in arrangement, since they need to be close enough to each other the form the disulfide bond. Like proline, serine is often associated in tight turns, since it is relatively small. The change from a small, slightly polar residue to a charged amino acid most likely results in a conformation that is too thermodynamically unstable even pharmacoperones to rescue. This indicates that the region involving transmembrane helix 4 and 5, is of great significance, since it will alter the orientation of the second extracellular loop that is involved with the disulfide bridges. In general, most of the mutations that lead to misrouting are seen in the transmembrane region. This is expected since buried hydrophobic residues are usually conserved. "Out of 21 mutants that led to hypogonadotropic hypogonadism, 14 of them involved the change of a single charged residue. The rest involved the shape, with 4 involving a loss or gain of a cysteine, one involving a proline and 2 having large truncations or deletions," (Conn2007). This indicates that the correct conformation of the GPCR is quite sensitive to mutations.

Pharmacoperone Administration:

In the case of GnRHR, suitable pharmacoperones were found by high-throughput screening. The mechanism of how pharmacoperones work is not so trivial. It is thought that the peptidomimetic antagonists that bind at or near the natural ligand binding site, which then pulls together the receptor so it can pass the ER QCS. If this is the case, then it would imply that the formation of the proper active site has something to do with the overall structure of the receptor as a whole. To see if indeed these pharmacoperones indeed rescue misfolded receptors in the ER, a protein inhibitor cycloheximide to halt protein synthesis. The pharmacoperone was then added, to see if it increased GnRHR plasma membrane expression. Indeed, these pharmacoperones increased the level of plasma membrane expression, indicating that it is rescuing already synthesized receptors. This means that the timing of the administration is not an issue. Interestingly, if one class of chemicals could rescue the misfolded receptor, it could be rescued by other unrelated classes as well. This is concurrent with the idea that these antagonists bind in relatively the same locating, around the natural ligand binding site. The classes of chemicals include indoles, quinolones and erythromycin-derived macrolides. It has been reported that mutant receptors can associate with their wild-type counterpart, which results in what is called a double negative (DN) effect. Pharmacoperones can relieve this DN effect, which would result in the rescue of both the misfolded receptor and the associated wild-type receptor, resulting in greater expression (Conn, 2007).

Issues to Address for Pharmacoperone Administration in vivo:

Practically all known pharmacoperones for GnRHRs are antagonists of the native ligand. This creates competition between the two molecules, which is undesirable for the use in a clinical setting. Also, once the antagonist binds and rescues the misfolded receptor it needs to be removed so that the natural agonist can bind properly. Finding a pharmacoperone that doesn't compete with the natural ligand would be ideal, however, it may be the case that the pharmacoperone has to occupy or partially occupy the ligand binding site in order for it to rescue the misfolded receptor. Another complication that needs some attention is the fact that for GRHRs, pituitary signaling depends on the pattern of exposure to the agonist. This means that the timing of pharmacoperone administration is of importance in vivo (Conn, 2007). Also, like all drugs, good efficacy and specificity is paramount for the use of pharmacoperones in a clinical setting.


The knowledge gained by studying these two GPCR class I model systems, GnRHR and V2R, have provides great insight in GPCR trafficking. Things are a lot more complex than once thought. For instance, the calnexin/calreticulin cycle is only one component of the ER QCS, yet this cycles involved a huge array of reactions and all of them are not known. Understanding this cycle can give us a better understanding of how GPCR trafficking works. GPCR structural analysis using mutagenesis has provided valuable insight on the essential features that are required for a properly folded receptor. Knowing the important features can help in designing the proper pharmacoperone that can help rescue the conformation and restore the receptor to its proper location. This type of therapy has enormous potential, however, more needs to be known in order for the use of pharmacoperones is wide spread in a clinical setting. Shortsightedness is something that has been seen in the past, for instance in gene therapy and in making vaccines. Another setback like the one seen with the Merck HIV vaccine would definitely have an negative impact, not only for pharmacoperones, but for science as a whole.


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