G Protein-Coupled Receptors for PGE2: Many Effects from One Lipid
By Tom Brock, Ph.D.
PGE2 is undoubtedly the bad boy of lipids. While cholesterol may be ‘bad cholesterol’ to some people, no one is immune to the pain, fever, and inflammatory effects of PGE2. And that’s just a short list of PGE2’s effects, but it’s enough to fuel a market for veterinary NSAIDs of $17.5 million, prescription NSAIDs at $30 billion, and over-the-counter NSAIDs in excess of $90 billion per year. Add to that the diverse and important effects of PGE2 in cancer, reproduction and parturition, bone formation, cardiovascular tone and blood pressure, gastrointestinal and kidney maintenance, lipolysis, sleep, and neuroprotection and you have an amazing resumé for a single molecule. How does PGE2 do all of these things? The secret involves combining four different receptors with their immediate intracellular signaling pathways and cell-specific responses. This article provides a brief overview of cellular responses to PGE2.
G Protein-Coupled Receptors
GPCRs are plasma membrane-integrated receptors that are coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins). All GPCRs have a common structure consisting of an amino-terminal random loop leading to seven membrane-spanning hydrophobic α-helices connected by hydrophilic chains of variable length. Agonist binding produces a change in receptor conformation, exposing intracellular sites involved in the interaction with the G protein. The G protein, consisting of α, β, and γ subunits, interacts with the receptor, leading to the release of guanosine diphosphate (GDP) from the α subunit and its replacement with guanosine triphosphate (GTP). The binding of GTP activates Gα so that it dissociates from the Gβγ dimer and triggers a Gα-specific pathway. In some cases, the Gβγ complex can also stimulate a downstream pathway.
The four GPCRs that respond to PGE2 are called E-prostanoid receptors, of which there are four subtypes (EP1-4). All prostanoid receptors belong to a subclass of GPCRs typified by the rhodopsin receptors. In many GPCRs, the agonist interacts with an extended extracellular amino-terminal binding site. However, in rhodopsin receptors, the amino-terminal loop is truncated and the target for receptor activation is buried deep in the membrane between the membrane-spanning helices. As a result, the key amino acid residues that are conserved across photoreceptors are located within the hydrophobic residues of the α-helices. Interestingly, sequence alignment of the four EP receptors from diverse organisms reveals several perfectly conserved residues within the α-helices. In fact, the binding site for the ligand, PGE2, is localized between helices within the hydrophobic environment of the lipid bilayer. This raises the intriguing possibility that PGE2 may approach the receptor from either side of the membrane. That is, PGE2 produced within the cell may never need to physically leave the cell to find the binding pocket of EP receptors on the cell surface.
EP1 Effects Through PLCβ
The EP1 receptor is particularly abundant in kidney, less abundant in lung, spleen, and skeletal muscle, and is also found in testes, brain, heart, and eye. In the kidney, it has been described on preglomerular arterioles, connecting segments, cortical and medullary collecting ducts, and media of arteries and arterioles. In part, this distribution parallels the distribution of smooth muscle that is associated with vascular elements throughout the kidney. As a general rule, EP1 is also abundant on smooth muscle associated with vessels in other organs, including the gastrointestinal tract, lung, spleen, and eye. It has also been found on other cell types, including certain fibroblast and endothelial cell populations.
EP1 is a GPCR that signals primarily through Gαq, which produces a transient rise in intracellular calcium (Figure 1). In this pathway, the interaction of PGE2 with EP1 converts GDP/Gαq to GTP/Gαq, evoking the release of GTP/Gαq from the αβγ trimer and activation of PLCβ. PLCβ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). IP3 binds to IP3 receptors on the endoplasmic reticulum (ER), which act as calcium (Ca2+) channels, allowing the release of Ca2+ from intracellular stores within the ER to the cytoplasm. DAG, Ca2+ and other second messengers activate cell-specific Ca2+ channels at the cell surface, producing a further increase in cytoplasmic calcium. This transient increase in intracellular Ca2+ alters the activity of many proteins, including several isoforms of PKC. Certain types of PKC have binding sites for DAG and Ca2+, and these co-factors stimulate PKC to phosphorylate and activate several substrates. In this way, PGE2 evokes Ca2+- and PKC-mediated effects in cells expressing EP1. There are also reports that EP1 can act through Gαi, which is described below.
The activation of EP1 produces an increase in atrial contractility with renal vasoconstriction in the kidney and contraction of pulmonary venous smooth muscle with airway constriction in the lung. It seems likely that PGE2 increases vascular tone through EP1-mediated Ca2+ influx in other sites as well. EP1 has also been linked with hyperalgesia and allodynia in rats and mice, as well as acid-induced visceral pain hypersensitivity in humans. On the other hand, EP1 may also be involved in mechanical and thermal analgesia. EP1 has also been implicated in resetting the peripheral circadian clock, gastric cytoprotection, colon cancer, hyperthermia, sleep inhibition, and dopamine-related behavioral changes.
cAMP at the Heart of EP Signaling
The remaining three EP receptors, EP2-4, vie for control of cAMP production. EP2 and EP4 are coupled to Gαs, which directs the synthesis of cAMP, while EP3 is Gαi-linked and acts to inhibit cAMP production (Figure 2). The principle enzyme, adenylate cyclase (AC), converts ATP to cAMP. Importantly, the inhibition of cAMP production by Gαi is relevant for both reducing basal cAMP production, which can be appreciable in unstimulated cells, and blocking the increased cAMP generation in cells stimulated with ligands that activate Gαs-coupled receptors, like EP2,4.
The prototypical pathway activated by cAMP involves PKA. In resting cells, PKA exists as a tetramer of two regulatory subunits holding two catalytic subunits in an inactive state. The association of cAMP with the regulatory components causes dissociation of the tetramer, allowing the free and active catalytic subunits of the kinase to phosphorylate target proteins. A second pathway that is cAMP-dependent involves the membrane-associated exchange proteins activated by cAMP (Epac), which includes Epac-1 and Epac-2. These are two of several guanine nucleotide exchange factors (GEFs) that modify the Ras GTPase homologs Rap1 and Rap2. Like the Gα subunits associated with GPCRs, the Rap proteins are activated when bound GDP is replaced with GTP by a GEF, like Epac. Hydrolysis of GTP to GDP in situ inactivates Rap. The cAMP-Epac-Rap pathway is involved in regulating a variety of different cell-specific processes, ranging from cell motility to gene expression.
As noted above, the Gβγ dimer, which is released from the heterotrimer following receptor activation, can also stimulate signaling pathways. There are multiple isoforms of Gγ, and some of those that associate with Gαi can activate PLCβ. This triggers the usual signaling leading to increased intracellular Ca2+ and PKC activation, reminiscent of signaling through Gαq. Thus, the activation of EP3 can be monitored by measuring either cAMP or Ca2+.
Many Effects from One Pathway
While it is convenient to illustrate three receptors together in one diagram, in real life they may act together in one cell type or be dispersed on different cells and tissues. For example, both EP2 and EP4 occur on vascular smooth muscle, on the corpus cavernosum of the penis, overlap considerably throughout the brain and kidney, and are common to many types of leukocytes. However, EP2 appears to be common on the spinal cord and articular cartilage, whereas EP4 is more common on osteoblasts, the gastrointestinal tract, and lung. Within the eye, EP2 is present on the sclera, corneal epithelium, and choriocapillaries, while EP4 is on corneal endothelium and keratocytes, as well as conjunctival and iridal stroma cells. EP3, on the other hand, is abundant on kidney tubules, gastrointestinal tract neurons, and medial nuclei of the thalamus. There is an interesting co-expression of EP3 and EP4 throughout the eye. Also, EP3 is found in the uterus, particularly on the myometrium.
Both EP2 and EP4 promote renal vasodilation, whereas EP2 may be more broadly involved in vascular smooth muscle relaxation. EP4 is clearly involved in bone formation, while the role of EP2 may be more relevant to fracture healing. Activation of either receptor impairs various immune cell functions (chemotaxis, phagocytosis, and TNF-α and IL-8 expression), although the effects differ between types of leukocytes and appear to be more EP2-dependent. Evidence also indicates that EP2 mediates spinal inflammatory hyperalgesia, neuroprotection, and inhibition of sleep. EP4, but not EP2, regulates gastric acid secretion, duodenal bicarbonate secretion ,and renin secretion, suggesting that EP4 plays a more prominent role in the maintenance of kidney and gastrointestinal homeostasis.
Mice lacking EP3 exhibit increased PGE2-mediated renal vasodilation, reduced PGE2-induced airway responsiveness, and lack of PGE2-mediated thermal hyperalgesia, compared to wild-type mice. These findings indicate that EP3 is involved in smooth muscle contraction, the constriction of vessels, venules and airway, and the sensation of pain. EP3, like EP4, also appears to promote duodenal bicarbonate secretion, which is relevant to intestinal protection.
Due to space limitations, only certain actions and effects of PGE2 could be presented. Recent reviews on the roles of EP receptors in stress responses,1 neurologic disease,2 kidney,3 and bone,4 as well as on GPCRs in general,5 are recommended for further information.
1. Furuyashiki, T. and Narumiya, S. Curr. Opin. Pharmacol. 9, 31-38 (2009).
2. Cimino, P.J., Keene, C.D., Breyer, R.M., et al. Curr. Med. Chem. 15, 1863-1869 (2008).
3. Hao, C.M. and Breyer, M.D. Annu. Rev. Physiol. 70, 357-377 (2008).
4. Hikiji, H., Takato, T., Shimizu, T., et al.. Prog. Lipid Res. 47, 107-126 (2008).
5. Oldham, W.M. and Hamm, H.E. Nat. Rev. Mol. Cell Biol. 9, 60-71 (2008).
Figure 1. Signaling through EP1 leads to an increase in intracellular calcium and activation of PKC
Figure 2. Three EP receptors act through adenylate cyclase and cAMP. EP3 also activates PLCβ to increase Ca2+
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