The Biased Activities of Prostanoids and Their Receptors: Review and Beyond

Abstract

Since the discovery of β-arrestin, a new concept/viewpoint has arisen in G-protein coupled receptor (GPCR)-mediated signaling. The Lock and Key concept of GPCR was previously recognized as basically a single- or mono-originated pathway activated from a single receptor. However, the new concept/viewpoint allows for many- or more-than-one-originated pathways activated from a single receptor; namely, biased activities. It is well-recognized that prostanoids exhibit preferences for their corresponding cognate receptors, while promiscuous cross-reactivities have also been reported among endogenous prostanoids and their receptor family. However, of particular interest, such cross-reactivities have led to reports of their physiologically significant roles. Thus, this review discusses and considers that the endogenous prostanoids are not showing random cross-reactivities but what are showing important physiological and pathological activities as biased ligands. Moreover, why and how the biased activities are evoked by endogenous structurally similar prostanoid ligands are discussed. Furthermore, when the biased activities of endogenous prostanoids first arose is also discussed and considered. These biased activities of endogenous prostanoids are also discussed from the perspective that they may provide many benefits and/or disadvantages for all living things, any-where on this planet, who/which are utilizing, had utilized, and will utilize the prostanoids and their receptor system, as a marked driving force for evolution.

1. INTRODUCTION

1.1. What Is Biased Activity?

The “Lock and Key” concept has long been considered useful to explain a ligand and receptor interaction for G-protein coupled receptors (GPCR). However, since the discovery of β-arrestin, a new concept/viewpoint has arisen in GPCR signaling. The old Lock and Key concept was basically a single- or mono-originated pathway activated from a single receptor, but the new concept/viewpoint allows for many- or more-than-one-originated pathways activated from a single receptor. Indeed, the receptor is able to be differently activated by each biased ligand dependently/specifically: the ligand-biased signaling pathway(s). These ligands are now called biased ligands. Each biased ligand can activate its selective/preferential pathway without altering the activity of other pathways. Some biased ligands may inhibit their specific signaling pathway(s) without changing other signaling pathways through the exact same receptor. For example, noradrenalin/norepinephrine can activate Gαs-protein-mediated cAMP signaling as well as β-arrestin/G protein-coupled receptor kinase (GRK)-mediated receptor internalization to maximal levels as a full agonist of adrenalin β2 receptors.¹⁾ On the other hand, dopamine can also activate Gαs-protein-mediated cAMP signaling to maximal levels via an adrenalin β2 receptor, while the activity of β-arrestin/GRK-mediated receptor internalization is very weak so that dopamine may act on this pathway as a partial agonist.¹⁾ Thus, dopamine is a biased ligand toward Gαs-protein-mediated signaling. In others, carvedilol used to be considered as a β2-adrenergic receptor antagonist; however, it was reported to activate the β-arrestin-mediated pathway as a biased β2-adrenergic receptor agonist. Thus, carvedilol does not activate the regular Gαs-protein-mediated pathway, but the β-arrestin-mediated pathway as a biased ligand.²⁾

Here, some important findings from the last couple of years of research of ligand-biased activities of prostanoids are introduced, with a focus on 3 important recent topics regarding prostanoid receptors and endogenous ligand-mediated signaling. Moreover, 2 possible reasons why biased activities have emerged were discussed, and their significance and perspectives were considered from the viewpoint of evolution.

2. PROSTANOIDS RECEPTOR AND ENDOGENOUS LIGAND-MEDIATED BIASED ACTIVITIES: 3 TOPICS

2.1. Topic 1, Prostaglandin (PG)E₁, PGE₂, and PGE₃ Act as Biased Ligands of E-Type Prostanoid (EP)4 Receptors

Among the prostanoids, PGE₂ is a major and the most abundant lipid mediator to regulate many physiological and pathological activities through the activation of EP receptors.^3,4) EP receptors have four main subtypes: EP1 to EP4. Among them, EP4 receptors have been reported to couple with Gαs-protein and can activate adenylyl cyclase to form cAMP. EP4 receptors have also been reported to couple with Gαi-protein, and its pathway involves the activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinases (ERKs).^5,6) Because two different G-proteins couple with EP4 receptors, each G-protein-dependent biased signaling pathway will be differently activated depending on the ligand binding to the receptor.

The main cognate ligand of EP receptors is PGE₂, which is well-known to be synthesized from arachidonic acid by the action of cyclooxygenase (COX).^7,8) However, if dihomo-γ-linolenic acid or eicosapentaenoic acid is metabolized to COX, PGE₁ or PGE₃ is synthesized, respectively.⁹⁾ The difference among PGE₁, PGE₂, and PGE₃ is the number of doublebonds in their structures; PGE₁ has one, PGE₂ has two, and PGE₃ has three doublebonds. PGE₂ is well-known as a pro-cancer prostanoid.^7,8) Indeed, PGE₂ and COX-2 are biomarkers of colorectal cancer.¹⁰⁾ Another major hallmark of colorectal cancer is an increase in β-catenin/T-cell factor (TCF) transcriptional activity.¹¹⁾ Interestingly, the β-catenin/TCF-mediated pathway is also primarily regulated by the Gαi-protein-mediated pathway of EP4 receptors activated by PGE₂.^12–14)

On the other hand, PGE₁ and PGE₃ function as anti-cancer prostanoids,^15,16) albeit with a similar structure to PGE₂. PGE₁ and PGE₃ were previously reported to be able to induce cAMP formation effectively as full agonists of EP4 receptors, but they only partially activate β-catenin/TCF-mediated signaling.⁹⁾ Since β-catenin/TCF-signaling is a hallmark of colorectal cancer, these results indicate that PGE₁ and PGE₃ may act as negative biased agonists of EP4 receptors to mediate anti-cancer effects by selectively not fully activating β-catenin/TCF-mediated signaling.⁹⁾

The negative biased activities of PGE₁ and PGE₃ for β-catenin/TCF-mediated signaling may be due to the numbers and patterns of bonding formation between each PGE and EP4 receptors. EP4 receptors form hydrogen bonds with three key functional groups of all PGEs: at positions 1, 9 of carbonyl functional groups and at position 15 of the hydroxyl functional group.⁹⁾ However, each PGE forms hydrogen bonds with the receptor slightly differently, particularly at positions 9 and 15.

Arginine (R) 291 in the transmembrane (TM) 6 domain of EP4 receptors forms one hydrogen bond with PGE₂ at position 9 of the carboxyl group in the cyclopentane ring. Serine (S) 307 of the TM7 domain of EP4 receptors also forms two hydrogen bonds with PGE₂ at position 15 of the hydroxyl group in the side chain.⁹⁾ EP4 receptors form two hydrogen bonds with R291 in TM6 to carbonyl functional groups at position 9 of the cyclopentane rings of PGE₁ and PGE₃, but only a single hydrogen bond with S307 in the TM7 domain to hydroxyl functional groups at position 15 in their side chains.⁹⁾

Thus, these results suggest one possible reason why PGEs function as either pro- or anti-cancer prostanoids by biased activity for EP4 receptors. The anti-cancer and/or pro-cancer effects are both mediated by the same EP4 receptors, but the outputs depend on the biased functions of each endogenous PGE; PGE₁ and PGE₃ may not be able to transform the receptor conformation fully to activate signaling for β-catenin/TCF-mediated signaling like PGE₂, due to the numbers and patterns of bonding formation with EP4 receptors; hence, they showed negative biased activities to signaling (Fig. 1A).

Fig. 1. The Biased Activities Evoked by Endogenous Prostanoids and Their Receptors

(A) PGE₁, PGE₂, and PGE₃ exert opposing effects on cancer development through EP4 receptors as biased agonists. (B) PGE₂ and its metabolite 15-keto-PGE₂ exert opposing effects on inflammation by switching from EP4 receptors to EP2 receptors as biased/switched agonists. (C) PGD₂ and PGE₂ exert opposing effects on inflammation through the DP and/or EP2 receptors as biased agonists.

2.2. Topic 2, 15-Keto-PGE₂, a Metabolite of PGE₂, Acts as a Biased and Switched Agonist of EP Receptors

After PGE₂ has evoked its physiological and pathological responses, i.e., pro-inflammatory effects, it is metabolized to 15-keto-PGE₂ by the action of 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Therefore, 15-keto-PGE₂ is widely used and has long been considered as an inactive metabolite of PGE₂.^17,18) Although the efficacies are lower than PGE₂, 15-keto-PGE₂ has been reported to be able to bind to EP2 and EP4 receptors and accumulate cAMP to some extent.¹⁹⁾ Thus, 15-keto-PGE₂ may not be just an inactive metabolite of PGE₂, but might also have an additional role as a biased/partial agonist of EP2 and EP4 receptors.²⁰⁾

Although the EC₅₀ value of 15-keto-PGE₂ to EP2 receptors is approximately 200 times larger than PGE₂, the E_max values of both prostanoids are similar, indicating that 15-keto-PGE₂ is able to activate EP2 receptors as a full agonist. However, the EC₅₀ value of 15-keto-PGE₂ to EP4 receptors is more than 2000 times larger than that of PGE₂, and also the E_max value of 15-keto-PGE₂ is almost half that of PGE₂.²⁰⁾ These results indicate that 15-keto-PGE₂ may work as a partial agonist of EP4 receptors in terms of cAMP formation.

Previously PGE₂ stimulation of EP4 receptors, but not EP2 receptors, was reported to be able to induce the phosphorylation of ERKs via the Gαi-protein-mediated pathway.^5,6) The EC₅₀ value of 15-keto-PGE₂ to EP4 receptors is approximately 200 times larger than that of PGE₂, and the E_max value of 15-keto-PGE₂ is almost 70% that of PGE₂, indicating that 15-keto-PGE₂ acts as a partial agonist when compared with full-agonist PGE₂ on the phosphorylation of ERKs to EP4 receptors. However, in the case of the EP2 receptor-stimulated ERKs pathway, 15-keto-PGE₂ has few effects, if any, on the phosphorylation of ERKs via activation of EP2 receptors, similar to PGE₂-stimulated ones.²⁰⁾

On the other hand, EP2 receptors as well as EP4 receptors when stimulated with PGE₂ induced β-catenin/TCF-mediated transcriptional activities with similar potencies and efficacies.^12,20) Interestingly, the EP2 receptor-activated β-catenin/TCF-mediated transcriptional activity is mainly via Gαs-protein activation, whereas the EP4 receptor-activated β-catenin/TCF-mediated activity is predominantly via Gαi-protein activation.¹²⁾ 15-Keto-PGE₂ can also stimulate β-catenin/TCF-mediated pathways via both EP2 and EP4 receptors in a concentration-dependent manner. However, on signaling through the EP2 receptors, the maximal activation was about 80%, close to that of the PGE₂-stimulated E_max value, and the EC₅₀ value of 15-keto-PGE₂ to EP2 receptors is approximately 200 times larger than PGE₂.²⁰⁾ On signaling through EP4 receptors, the E_max value of 15-keto-PGE₂ is approximately 50% and the EC₅₀ value of 15-keto-PGE₂ to EP4 receptors is approximately 300 times larger than PGE₂.²⁰⁾ Therefore, in terms of β-catenin/TCF-mediated transcriptional activities, 15-keto-PGE₂ functioned as a partial agonist of both EP2 and EP4 receptors but 15-keto-PGE₂ showed lower efficacy as well as potency for EP4 receptors than that for EP2 receptors.

Based on the results obtained from the receptor-ligand binding assay, on comparing IC₅₀ values between EP2 receptors and EP4 receptors to PGE₂, the value is about 10 times lower in EP4 receptors.²⁰⁾ Of particular interest, the differences in IC₅₀ values between PGE₂ and 15-keto-PGE₂ are around 100 times to EP2 receptors but around 10000 times to EP4 receptors. Therefore, PGE₂ may tend to bind to EP4 receptors rather than EP2 receptors, whereas 15-keto-PGE₂ may more easily bind to EP2 receptors than EP4 receptors. The lower potencies and efficacies of 15-keto-PGE₂ for EP4 receptors in terms of cAMP formation, phosphorylation of ERKs, and β-catenin/TCF-mediated transcriptional activity could be due to the lower binding affinity of this prostanoid for EP4 receptors, at least in part.²⁰⁾

Taking all the results together, 15-keto-PGE₂ could have an additional role as a biased/partial agonist to take over the actions of PGE₂ to gradually terminate reactions as soft-landing ways through EP2 and EP4 receptors.²⁰⁾ Therefore, 15-keto-PGE₂ may not be a simple inactive metabolite of PGE₂, but actually a biased agonist that also acts as a “switched agonist” for cellular signaling to the EP2 receptor-mediated pathway from the EP4 receptor-mediated pathway, for restoring/terminating inflammatory reactions evoked by PGE₂ and/or maintaining homeostasis, such as in the colorectal tissues/cells functions together with PGE₂²⁰⁾ (Fig. 1B).

2.3. Topic 3, PGD₂ and PGE₂, as the Biased Ligands

Interestingly, not only PGE family prostanoids and/or metabolites of PGE₂ but also other endogenous prostanoids may act as biased ligands to their non-cognitive prostanoid receptors.^21,22) For example, both PGD₂ and PGE₂ have been shown to activate β-catenin/TCF-mediated signaling and cAMP-mediated signaling via D-type prostanoid (DP) receptors and EP2 receptors.²¹⁾

Regarding human DP and EP2 receptors, they are the most homologically closely related prostanoid receptors.^4,23–25) Indeed, the genes of DP and EP2 receptors are considered to be generated by tandem duplication since they are located on the same chromosome, 14q22 in humans,^26–29) so that DP and EP2 receptors consist of similar amounts of amino acids in humans: 359 and 358 amino acids, respectively.³⁰⁾

As for the ligands, the structures of PGD₂ and PGE₂ are similar to each other since they are positional isomers in which the hydroxyl group and carbonyl group at positions 9 and 11, respectively, in their cyclopentane rings are oppositely bonded.^21,31)

Hence, the most homologically related DP and EP2 receptors can be activated by structurally similar ligands, PGD₂ and PGE₂. Therefore, aside from their different affinities, both ligands have the ability to activate both receptors to similar maximal levels, i.e., they show similar efficacies, as full agonists, in terms of both cAMP-mediated signaling and β-catenin/TCF-mediated signaling.^21,31)

When focusing on the ligand-receptor interaction, tyrosine (Y) 199 on TM5 as well as R284 on TM6 in DP receptors, and Y196 on TM5 as well as glutamate (E) 288 on TM6 in EP2 receptors, have the potential to form hydrogen bonding interactions with the ligands, PGD₂ and PGE₂. Thus, in regards to DP receptors, the 11 position of carbonyl in the cyclopentane ring of PGD₂ can bind to and form a hydrogen bond with Y199 on TM5 of DP receptors. Similarly, the first position of the carboxyl functional group of PGD₂ can form a hydrogen bond with R284 on TM6 of the receptors. While in the case of PGE₂, the first position of the carboxyl functional group of PGE₂ also forms a hydrogen bond with R284 in the DP receptor, but the cyclopentane ring of PGE₂ does not form a hydrogen bond with DP receptors.^21,31)

On the other hand, in the case of the EP2 receptor, PGD₂ neither formed a hydrogen bond with Y196 on TM5 nor E288 on TM6 of EP2 receptors. Whereas the 15 position of the hydroxyl functional group of PGE₂ formed a hydrogen bond with E288 in the EP2 receptor. Moreover, the 10 position of PGE₂ formed a non-classical CH–π hydrogen bond with the phenol ring structure of Y196 in the EP2 receptor. Thus, although PGD₂ and PGE₂ act as full agonists of both DP and EP2 receptors, the numbers and styles of hydrogen bonding between each receptor and ligand are different, which may define the potencies of each receptor and ligand. However, more importantly, different ligand-binding patterns may induce different conformations of each receptor; each ligand has the potential to act as a biased agonist to regulate the physiological functions of PGD₂ or PGE₂, and/or DP or EP2 receptors. Indeed, molecular dynamics simulation analysis showed that the distance between PGD₂ and EP2 receptors was shortened after ligand binding, indicating that PGD₂ binding to EP2 receptors could markedly change the conformation of the receptor, whereas the apparent distance change was not detected in PGE₂-activated EP2 receptors or PGD₂ or PGE₂-activated DP receptors.^21,31)

Meanwhile, PGD₂-activated EP2 receptors were found to exert a greater effect on phosphodiesterase 4 (PDE4) activity than PGE₂-activated EP2 receptors.³²⁾ Thus, the conformational changes induced by PGD₂ binding to EP2 receptors may recruit PDE4 to the receptors. Therefore, although PGD₂ has the potential to accumulate EP2 receptor-mediated cAMP as a full agonist, because of the greater activity of PDE4 induced by PGD₂, accessible levels of cAMP may be decreased due to rapid degradation by the recruitment of PDE4. As discussed previously, these results strongly indicate that EP2 receptors may have an ability to distinguish between PGD₂ and PGE₂, but DP receptors may not, at least in terms of cAMP-mediating pathways, through the differential functional coupling of PDE4 probably with β-arrestin.^31,32)

With respect to physiological functions, PGD₂ and PGE₂ have been shown to act sometimes opposing to each other.^30,31) For example, PGD₂ has been shown to lower the body temperature, whereas PGE₂ elevates it.³³⁾ PGD₂ has also been reported to increase food intake, whereas PGE₂ suppresses it.³³⁾ While PGD₂ is known to be crucially involved in the deterioration of asthma, PGE₂ may have potentially beneficial effects on this disease.³⁴⁾ PGD₂ has been reported to be important for inducing sleep, whereas the onset and maintenance of wakefulness is considered to be regulated by PGE₂.^35,36) Furthermore, since PGE₂ is regarded as a biomarker of colorectal cancer, this prostanoid is believed to be involved in cancer development; however, PGD₂ is believed to protect against and/or ameliorate cancer development.³¹⁾

Generally, the cAMP-mediated signaling pathway inhibits cellular growth.³⁷⁾ On the other hand, the β-catenin/TCF-signaling pathway is involved in proliferation, differentiation, and tumorigenesis.^38,39) As previously shown and discussed, when DP receptors are stimulated by a physiological concentration of PGD₂, approximately 10 nM,²¹⁾ proliferation regulated by the β-catenin/TCF-mediated signaling pathway may coordinate with the growth-inhibitory cAMP signaling pathway, resulting in the maintenance of cellular homeostasis, which may protect against and/or ameliorate cancer-related damage.^40,41) However, if DP receptors are stimulated with approximately 10 nM PGE₂, they may only activate β-catenin/TCF-mediated proliferation/developmental signaling, without activating the cellular growth-inhibitory cAMP signaling pathway, resulting in β-catenin/TCF-mediated proliferation signaling, which may be continuously activated. Therefore, the physiological concentration of PGE₂ can ultimately lead to tumorigenesis, as a biased ligand of DP receptors.

In the case of EP2 receptors, the physiological concentration of PGE₂ can activate both β-catenin/TCF-mediated proliferation signaling and growth-inhibitory cAMP signaling pathways, thereby contributing to the maintenance of cellular homeostasis. However, 10 nM PGD₂ did not activate EP2 receptors at all, which may explain why PGD₂ is not generally considered a factor in cancer development, unlike a well-known biomarker of cancer development, PGE₂. Although this is just one explanation for a particular example, the above biased activities may provide a more detailed understanding of why structurally similar PGD₂ and PGE₂ show opposing effects on cancer development. Taken together, it is widely accepted that PGD₂ exerts anti-inflammatory effects, whereas PGE₂ shows pro-inflammatory effects. These opposing effects of PGD₂ and PGE₂ may be attributed to the biased activities evoked by their activated receptors, i.e., DP and EP2 receptors (Fig. 1C).

3. WHY BIASED ACTIVITIES APPEARED; FROM THE VIEWPOINT OF EVOLUTION: 2 HYPOTHESES

3.1. Hypothesis 1, Same Ancestor but Diverging

The partiality, i.e., affinity, efficacy and/or potency, of ligands for receptors has been considered to drive evolution for newly duplicated receptors. The evolutional transition occurred from common receptors for different ligands to a specific receptor for each ligand, in other words, from a receptor-directed mechanism to ligand-directed mechanism.⁴²⁾

The coding DNA sequences of human DP receptors as well as human EP2 receptors were previously extracted from 1092 persons worldwide, revealing that DP receptors show more mutations/variations than EP2 receptors among people across the globe.³⁰⁾ Thus, in human DP receptors, 20 mutations/variations were detected including 16 amino acid replacement mutations/variations with 4 silent mutations/variations. Whereas in human EP2 receptors, 12 mutations/variations were detected including 4 amino acid replacement mutations/variations with 8 silent mutations/variations.³⁰⁾ Generally, silent mutations should accumulate at a constant rate over time since they are not affected by natural selection. As described earlier, the genes of DP and EP2 receptors are considered to be generated by tandem duplication.²⁹⁾ As DP receptors showed 4 times more amino acid mutations/variations than EP2 receptors, DP receptors may still be in a rapid evolutionary stage, and/or EP2 receptors are constrained by amino acid replacement.³⁰⁾ Therefore, DP receptors may acquire novel functions, either neo-functionalization and/or sub-functionalization, as a duplicated copy of EP2 receptors.³⁰⁾

Of particular interest, not only DP and EP2 receptors, but also PGD₂ and PGE₂ may have been synthesized/produced by enzymes that diverged from the same ancestors. Indeed, microsomal PGE synthase-2 and hematopoietic PGD synthase have similar catalytic mechanisms because they may have originated from a common ancestor, cytoplasmic glutathione S-transferase.⁴³⁾ On considering a previous study showing that PGE₂-sensitive receptors were the ancestral receptors,⁴⁴⁾ possibly ancestral EP2 or EP2-like receptors, and the result described above that under the physiological condition, PGD₂ is not able to activate EP2 receptors, PGD₂ may have originally emerged without activity on the ancestral EP2 or EP2-like receptors in order to avoid cross-reactivity, as discussed previously.³¹⁾

Moreover, the biased activity of PGE₂ may be due to the incomplete selectivity of DP receptors in terms of the evolutional time frame, which may explain why DP receptors have more mutations/variations than EP2 receptors.^30,31) Thus, one possibility of biased agonism may come from the receptor in a rapid evolutional stage and its incomplete ligand selectivity.

3.2. Hypothesis 2, Different Ancestor and Overtaking

Using DP receptor-knockout mice, DP receptor signaling was shown to be involved in the suppression of tumor-associated angiogenesis and plasma leakage, i.e., tumor-associated vascular hyper-permeability.⁴⁰⁾ Also in an intact mouse study, stimulation of DP receptors led to inhibition of airway inflammation to suppress asthma.⁴⁵⁾ Thus, DP receptors themselves may have anti-inflammatory functions. However, PGD₂ sometimes showed pro-inflammatory effects, which may be mediated though the activation of chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) receptors, aka DP2 receptors.^4,46,47) This is partly supported by the clinical trial of a DP receptor antagonist, laropiprant, that showed no efficacy in patients with allergic rhinitis and asthma.⁴⁸⁾

As above, DP and CRTH2 receptors are very closely related to allergic inflammation such as atopy and asthma by stimulation with PGD₂ although they show opposing effects. Thus, one possible explanation is that the pro-inflammatory effects of PGD₂ are mediated by activation of CRTH2 receptors, whereas the anti-inflammatory effects of PGD₂ are via activation of DP receptors. This opposite/diverged function of PGD₂ is probably due to the lack of structural identity between DP and CRTH2 receptors.⁴⁾ Among the prostanoid receptors, only the CRTH2 receptors do not share gene homology; they are distant from the other prostaoid receptors in the phylogenic tree.⁴⁹⁾ The original roles of CRTH2 receptors may be to reinforce the adaptive immune system in vertebrates as chemo-attractant receptors since they are selectively expressed in inflammation-related cells to induce chemotactic responses and cytokine inductions.^50–52) So far, CRTH2 receptors have been found only in vertebrates, so that PGD₂ might become a CRTH2 receptor ligand incidentally during the evolutional time frame. CRTH2 receptors might have been stimulated by PGD₂ as a biased agonist when cross-reaction occurred, although the original cognate ligand itself and whether it still exists remain unknown.

Taken together, the anti-inflammatory DP receptor system may have been intercepted when CRTH2 receptors emerged by PGD₂ as a biased agonist of CRTH2 receptors to evoke opposite/diverged effects of DP receptors.

4. CONCLUSIONS AND PERSPECTIVES

4.1. Either Way, Evolutional Events? — Why, How, What, When, Where, and Who

One possibility is that these biased ligands, e.g., PGE₁, PGE₃, and/or 15-keto-PGE₂, may share the same receptors with PGE₂ at this moment, and will gain cognate receptors plausibly by duplication of EP receptors in the future. Thus, these ligands and receptors are in the stage of a receptor-directed mechanism but before the stage of a ligand-directed mechanism, as described earlier.⁴²⁾ Similar biased activities were also shown in PGE₂ and FP receptors²²⁾ and PGF_2α and EP2 or DP receptors.²¹⁾ Therefore, the biased activities evoked by endogenous structurally similar prostanoid ligands may be attributed to the transitional stage of the evolutional process. These activities are somehow preserved and/or not regressed until they gain new cognate receptor pairs (why).

How the biased activities of endogenous prostanoids are plausibly attributed to the receptors forming distinct ligand-dependent conformations (how). So far, non-cognate endogenous prostanoids have been recognized as non-cognate or slow-reacting alternate ligands. However, what endogenous prostanoid biased ligands serving/operating are important physiological and pathological activities (what).^20,21,31,32) Moreover, endogenous prostanoid biased activities may start when the ancestral prostanoid receptors, possibly ancestral EP2 or EP2-like receptors which have gained their new ligand, plausibly PGD₂, and will continue as long as evolution occurs (when). These biased activities may provide many benefits and/or disadvantages for all living things, anywhere on this planet (where), who/which are utilizing, had utilized, and will utilize the prostanoid system (who).

Given the discussion above, the biased prostanoid activities could be temporal effects arising during the process of evolution. Considering the almost everlasting timeframe, complete one-to-one correspondence between a ligand and receptor may be rare, and the many-to-many system, such as chemokines and their receptors, would probably be common in nature, at least in, to some degree, evolved creatures. Therefore, each biased activity may represent temporal effects during the evolutional stage; however, at the same time, each biased activity itself would be a marked driving force for evolution (Fig. 2).

Fig. 2. The Endogenous Prostanoid Biased Activities May Have Started When the Ancestral EP2 or EP2-Like Receptors Gained Their New Ligand during the Evolutional Time Frame

Each biased activity may be a temporal effect during the evolutional stage; however, at the same time, each biased activity itself would be a marked driving force for evolution.

Finally, aside from the evolutional viewpoint, since there is a vast amount of structurally similar endogenous prostanoids including their metabolites at present, it will provide a marked advantage for understanding physiological roles of these biased mechanisms between ligands and their receptor family. Thus, if the biased mechanisms can be properly utilized, those prostanoids would have the potential to act as beneficial endogenous biased ligands and/or leading compounds for developing and providing novel therapies in the future.

Acknowledgments

I would like to acknowledge Dr. Toshihiko Murayama and past and current laboratory members, Dr. Hiroki Takahashi, Dr. Yutaka Tamura, and Dr. Akiko Suganami in Chiba University, as well as Dr. John W. Regan and past and current laboratory members in The University of Arizona, Dr. Keijo Fukushima, and my past and current laboratory members in Tokushima University for their contributions to this work.

This research was supported in part by MEXT KAKENHI Grant 20K07084 (JAPAN).

Conflict of Interest

The author declares no conflict of interest.

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