Cui ZJ. To activate a G protein-coupled receptor permanently with cell surface photodynamic action in the gastrointestinal tract. World J Gastroenterol 2025; 31(12): 102423 [DOI: 10.3748/wjg.v31.i12.102423]
Corresponding Author of This Article
Zong-Jie Cui, PhD, Professor, Department of Biology, College of Life Sciences, Beijing Normal University, No. 19 Xinjiekouwai Street, Haidian District, Beijing 100875, China. zjcui@bnu.edu.cn
Research Domain of This Article
Pharmacology & Pharmacy
Article-Type of This Article
Opinion Review
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Zong-Jie Cui, Department of Biology, College of Life Sciences, Beijing Normal University, Beijing 100875, China
Zong-Jie Cui, The Ministry of Education Laboratory for Cell Proliferation and Regulation, College of Life Sciences, Beijing Normal University, Beijing 100875, China
Author contributions: Cui ZJ conceived the idea of this review, analyzed the relevant literature, wrote the initial drafts of this paper, wrote and approved the final submitted version of this review.
Supported by the National Natural Science Foundation of China, No. 32271278 and No. 31971170.
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Zong-Jie Cui, PhD, Professor, Department of Biology, College of Life Sciences, Beijing Normal University, No. 19 Xinjiekouwai Street, Haidian District, Beijing 100875, China. zjcui@bnu.edu.cn
Received: October 18, 2024 Revised: January 14, 2025 Accepted: January 17, 2025 Published online: March 28, 2025 Processing time: 160 Days and 5.8 Hours
Abstract
Different from reversible agonist-stimulated receptor activation, singlet oxygen oxidation activates permanently G protein-coupled receptor (GPCR) cholecystokinin 1 (CCK1R) in type II photodynamic action, with soluble photosensitizer dyes (sulphonated aluminum phthalocyanine, λmax 675 nm) or genetically encoded protein photosensitizers (KillerRed λmax 585 nm; mini singlet oxygen generator λmax 450 nm), together with a pulse of light (37 mW/cm2, 1-2 minutes). Three lines of evidence shed light on the mechanism of GPCR activated by singlet oxygen (GPCR-ABSO): (1) CCK1R is quantitatively converted from dimer to monomer; (2) Transmembrane domain 3, a pharmacophore for permanent photodynamic CCK1R activation, can be transplanted to non-susceptible M3 acetylcholine receptor; and (3) Larger size of disordered region in intracellular loop 3 correlates with higher sensitivity to photodynamic CCK1R activation. GPCR-ABSO will add to the arsenal of engineered designer GPCR such as receptors activated solely by synthetic ligands and designer receptors exclusively activated by designer drugs, but show some clear advantages: Enhanced selectivity (double selectivity of localized photosensitizer and light illumination), long-lasting activation with no need for repeated drug administration, antagonist-binding site remains intact when needed, ease to apply to multiple GPCR. This type of permanent photodynamic activation may be applied to functional proteins other than GPCR.
Core Tip: G protein-coupled receptors (GPCR) are a large group of functional proteins, whose ligands account for a large proportion of clinical drugs. Important as GPCR may be, their ligand-independent pharmacology is poorly investigated. This minireview shows that the A-class GPCR cholecystokinin 1 receptor, predominantly expressed in pancreatic acini, could be activated permanently by singlet oxygen in photodynamic action. Three lines of evidence regarding mechanisms of photodynamic activation is presented. Clues suggest that permanent photodynamic activation of other GPCR and other functional proteins are possible, permanent photodynamic activation may be a general principle, both in gastrointestinal tract and elsewhere.
Citation: Cui ZJ. To activate a G protein-coupled receptor permanently with cell surface photodynamic action in the gastrointestinal tract. World J Gastroenterol 2025; 31(12): 102423
The G protein-coupled receptors (GPCR) are a major class of functional proteins, in this Opinion Review we present published data to show that the A class GPCR cholecystokinin 1 receptor (CCK1R) is permanently activated by the excited state molecular oxygen, the singlet oxygen, in type II photodynamic action, together with description of clarified molecular mechanisms. We present data available in the literature to show that other functional proteins might also be photodynamically activated, and propose that functional proteins, cellular functions and organism behaviors could all be modulated by a pulsed dose of photodynamic action, and suggest that this be termed photodynamic biology (PDB) to distinguish it from photodynamic therapy that is extensively studied for the killing of cancer cells and pathogenic microbes.
GPCR ACTIVATED BY SINGLET OXYGEN
In conventional receptor pharmacology, the resting receptor is activated after binding an orthosteric agonist or is inhibited by a reverse agonist; agonist binding is inhibited competitively by a neutral antagonist. Allosteric ligands bind to target GPCR, to either sensitize or desensitize the receptor to agonist stimulation, but may not by themselves change the activity level of the resting receptor in the absence of an agonist; such allosteric ligands are therefore either positive or negative allosteric modulators[1]. When positive allosteric modulator directly activates the target receptor, it becomes an orthosteric agonist[2-4]. An orthosteric pharmacophore could be combined with an allosteric pharmacophore by a flexible linker, to obtain the innovative dualsteric or bitopic ligands, which are able to bind simultaneously both the orthosteric and allosteric sites on the same receptor[5]. Orthosteric, allosteric or dualsteric ligands bind to specific receptor with varied affinities (dissociation constant Kd usually from pM to μM)[5,6], but could all be washed out by the circulating extracellular fluid.
The agonist-stimulated mode of receptor activation has been utilized in designer G protein-specific GPCR, to regulate the physiological functions of multiple GPCR receptors in vivo. These designer GPCR include the early version of receptor activated solely by synthetic ligands[7] and the more recently popularized designer receptor exclusively activated by designer drugs[8-10]. Both versions of these designer GPCR are activated exclusively by synthetic drugs/ligands/agonists, but significantly less activated or not activated at all by their endogenous natural agonists[11,12].
In sharp contrast to conventional ligand-based pharmacological intervention of GPCR, we have found that some GPCR are activated permanently, by delta singlet oxygen (1O2), the lowest lying excited singlet state of molecular oxygen generated in type II photodynamic action. Such ligand-independent receptor activation involves the trans-fixation or photodynamic welding of the GPCR in the activated conformation or state, therefore are named GPCR activated by singlet oxygen (GPCR-ABSO)[13].
It may be noted here that this general principle of permanent photodynamic activation (instead of inactivation, of multiple categories of functional proteins, studied by others at mega doses of singlet oxygen)[14-17] by a measured low dose of 1O2 may be applied to some member of most categories of functional proteins as classified by the International Union for Pharmacology[18] (see below).
Table 1 below summarizes the characteristics of GPCR-ABSO and their differences from receptor activated solely by synthetic ligands and designer receptor exclusively activated by designer drugs. The advantages of the recently discovered GPCR-ABSO are obvious, including one-off activation, double selectivity, and the option of inhibition of permanently activated receptor with antagonist, for example (Table 1).
Table 1 Comparison of the properties of novel types of G protein coupled receptors.
The most convenient way to produce 1O2 in biological settings in the aqueous extracellular fluid or at the plasma membrane is photodynamic action, involving a photosensitizer, light at the appropriate wavelength for efficient absorption by the photosensitizer, and well-oxygenated microenvironment in the proximity of the photosensitizer. In type II photodynamic action, only 1O2 is generated[19,20]. Unlike other reactive oxygen species, 1O2 has a lifetime on the scale of μs and a diffusion distance in the cellular milieu of 10 nm to 150 nm[21]. The excitation energy of 94 kJ/mole (0.9772 eV, emitting a near infrared phosphorescence photon of 1268.7 nm) of 1O2 ensures some reactive specificity[21,22].
1O2 can be generated also during the physiological process of respiratory burst in phagocytes (such as neutrophils, macrophages and others)[23-25]. Of the mixed reactive oxygen species which is also named biological plasma (or cold atmospheric plasma, as well as plasma-activated medium)[26] generated in a respiratory burst, hydrogen peroxide, nitric oxide, hypochlorous acid are stable molecules per se, whereas peroxynitrite anion, O2-., 1O2, hydroxyl radical have a lifetime of 10 ms, 1 ms, 1 μs, and ns respectively (Figure 1). The short lifetime and limited diffusion distance and restricted reactivity of 1O2 are all reflected in its specific activation of CCK1R - a receptor widely implicated in multiple physiological functions in vivo, such as satiety sensing and exocrine pancreatic secretion[27].
Figure 1 Reactive oxygen species and their average lifetime.
The nicotinamide adenine dinucleotide phosphate oxidase of phagocytes catalyzes the generation and release of superoxide (O2-.) after transfer of an electron to molecular oxygen (O2), O2-. is transformed into hydrogen peroxide (H2O2) by superoxide dismutase. H2O2 reacts with chloride anion, a major anion in the extracellular fluid, catalyzed by myeloperoxidase, to produce hypochlorous acid (HOCl), a bactericidal chemical. Nitric oxide (NO) produced by NO synthase reacts with O2-. to generate peroxynitrite anion. HOCl reacts with H2O2 to generate delta singlet oxygen (1O2). Ferrous iron reacts with H2O2 in Fenton reaction to produce ferric ion and hydroxyl radical (OH.). Note that H2O2, NO, HOCl are relatively stable, but peroxynitrite anion, O2-., 1O2, OH., has decreasing lifetimes of 10 ms, ms, μs, and ns respectively. 1O2 shows specificity in oxidative reactions (than OH-, for example) and has a diffusion distance from 10 nm to 150 nm, depending on the site of generation and methods of measurement and detection. NE: Neutrophil elastase; NADPH: Nicotinamide adenine dinucleotide phosphate; NAD: Nicotinamide adenine dinucleotide; NOS: Nitric oxide synthase; NO: Nitric oxide; H2O2: Hydrogen peroxide; HOCl: Hypochlorous acid; ONOO: Peroxynitrite anion; O2-.: Superoxide; 1O2: Delta singlet oxygen; OH.: Hydroxyl radical; DPI: Diphenyleneiodonium chloride; MPO: Myeloperoxidase; NOX: Nicotinamide adenine dinucleotide phosphate oxidase; Cl-: Chloride anion; SOD: Superoxide dismutase; H+: Proton; Fe2+: Ferrous iron; Fe3+: Ferric ion.
PERMANENT PHOTODYNAMIC ACTIVATION OF CCK1R
In the freshly isolated rat pancreatic acini, photodynamic action at the plasma membrane with water-soluble photosensitizer sulphonated aluminum phthalocyanine (SALPC) (1 μmol/L, λmax 675 nm at 53000 Lux / 72 mW/cm2) was found to trigger persistent cytosolic calcium oscillations, which were completely blocked with CCK1R antagonist FK480; after this blockade by FK480, the muscarinic agonist bethanechol could still elicit a robust calcium response, indicating the very healthy status of the pancreatic acinar cells after photodynamic CCK1R activation with SALPC[28] (Figure 2A). Therefore, it is very clear that the calcium-mobilizing (i.e., coupled to Gq protein) CCK1R is specifically activated by SALPC photodynamic action, but photodynamic action does not affect the muscarinic acetylcholine M3 receptor which is also coupled to Gq and calcium signaling[28] (data on M3R are not shown in Figure 2).
In experiments as those shown in Figure 2, the relative plasma membrane localization and specificity of the water-soluble photosensitizer SALPC is enhanced by very brief incubation of the freshly isolated rat pancreatic acini (SALPC: 1 μmol/L for only 10 minutes, see Figure 2 above). It is established that the water-soluble photosensitizer SALPC (also named aluminum phthalocyanine tetrasulphonate-AlPcS4) remains on the surface of the cells after brief incubation. The Krammer group have noted particularly that “Twenty minutes after aluminum phthalocyanine tetrasulphonate 12 μmol/L incubation, the dye is not able to enter the cell”[29]. So even at a SALPC concentration 10-times of that used in Figure 2 for permanent photodynamic activation of CCK1R and over a longer incubation time period of 20 minutes, SALPC does not cross the plasma membrane to enter the cell interiors at any detectable amount. It may be noted here that for the purpose of inducing different modes of cell death programs to kill cancer or other undesired non-malignant cells, photosensitizers are often applied to cancer cells for many hours (without washout) before being irradiated with light. Therefore, photodynamic physiology/pharmacology/biology (PDB) should be clearly distinguished from photodynamic therapy.
To rule out any possible link between photodynamic activation of CCK1R and the special microenvironment of rat pancreatic acinar cell plasma membrane, the human cholecystokinin type 1 receptor (CCK1R) was ectopically expressed in HEK293 cells (Figure 2B). It was found that the human CCK1R (hCCK1R) could be readily activated by photodynamic action with SALPC as photosensitizer as well, to similarly trigger persistent calcium oscillations[13] (Figure 2C). Note that in the same hCCK1R-HEK293 cell, stimulation of the agonist CCK at a physiological concentration of 20 pM resulted in regular calcium oscillations which were readily washed out, but photodynamic activation (photosensitizer SALPC 2 μmol/L plus subsequent light irradiation at > 580 nm, 36.7 mW/cm2) later on in the same cell triggered persistent calcium oscillations which were not washed out by the perifusion buffer. This pattern of CCK1R activation was named permanent activation, in contrast to the agonist-induced reversible activation[13].
Such permanent photodynamic activation of rat CCK1R that was found in isolated rat pancreatic acini or permanent photodynamic activation of hCCK1R ectopically expressed in a cell line (such as HEK293) as shown in Figure 2 has also been observed in the freshly isolated mouse and Peking duck pancreatic acini, the photodynamically activated CCK1R in all cases has been readily and reversibly inhibited by specific CCK1R antagonists[28,30-32]. This clearly indicates that after permanent photodynamic activation of CCK1R, the antagonist-binding site in the activated receptor is completely intact and functional. These experiments will probably rule out also any likelihood of direct activation by photodynamic action of downstream targets, since, for example, receptor blockade by antagonist would not have affected Gq protein activation or signaling directly triggered by other means at the levels of Gq protein, or phospholipase C.
Photodynamic activation of CCK1R is within the physiological ranges, because photodynamic CCK1R activation-elicited cytosolic calcium oscillations (such as those shown in Figure 2) are similar in wave forms and frequencies to calcium oscillations induced by CCK octapeptide in the physiological range of concentrations (10 to 100 pM) in the isolated exocrine pancreatic acini[30,33-35]. Only repetitive calcium spikes arising from the baseline are observed in this range of CCK concentrations when applied to the freshly isolated and perifused pancreatic acini[33-35]. The appearance of persistent cytosolic calcium oscillations is taken as a hallmark for the permanent photodynamic activation of CCK1R.
Other than the free-standing water-soluble chemical photosensitizers SALPC[13,28,30,32] and gadolinium porphyrin-like macrocycle B[31], CCK1R could be photodynamically activated with genetically encoded protein photosensitizers (GEPP) specifically expressed at the plasma membrane in native CCK1R-expressing AR4-2J cells[36], or after tagging (with or without a short linker sequence in between) GEPP to either the N- or C-terminal of CCK1R in cell lines (HEK293, CHO-K1) without natively-expressing CCK1R[13,21]. All GEPP examined have been found to be able to catalyze photodynamic activation of CCK1R, although those GEPP demonstrate varied levels of 1O2 quantum yield (ϕ1O2)[36] (Table 2).
Table 2 Genetically encoded protein photosensitizers[89-91].
In comparison with chemical photosensitizers whose relatively specific localization at subcellular sites such as at the plasma membrane (or endoplasmic reticulum, mitochondria, lysosomes and other subcellular organelles) is determined by their physicochemical properties, GEPP could be expressed highly specifically at defined subcellular sites after building a fusion construct with a leader peptide sequence, or after tagging to the N-terminus, C-terminus or after inserting in the middle of the sequence of target proteins of interest. GEPP mediated photodynamic activation of intracellular CCK1R has also been established in AR4-2J cells[36]. Photodynamic activation or modulation of intracellular GPCR with GEPP is advantageous over conventional ligand-based receptor pharmacology in that the plasma membrane or intracellular membranes would then pose no diffusion barriers in photodynamic pharmacology with GEPP. Because with GEPP photodynamic receptor activation, no ligands need to be added to the extracellular medium[36]. With photodynamic CCK1R activation, however, one must consider the limited range of the optical window of light penetration of biological tissues (600 nm-1400 nm)[37], when doing in vivo experiments, if external light but not internal bioluminescence light is used to drive the photodynamic activation process.
It may be noted here that in the A-class GPCR, the opsins from lower animals and mammalian rhodopsin are activated by light irradiation directly (a physiological vision process), by way of 11-cis-retinal isomerization to all-trans-retinal (reversible by enzyme-catalyzed reaction steps in the pigmented epithelial cells and Müller cells), the effects do not last after light off[38]. Interestingly, chlorophyll derivative photosensitizer chlorin e6 (Ce6) (λmax 660 nm) has been found to accumulate in the rod photoreceptors in the retina of the deep-sea dragonfish Malacosteus niger (the black loosejaw), and of some invertebrates, to extend their normally blue/green-tuned retina to far-red vision[39]. Molecular dynamics simulation and quantum chemistry calculation studies have shown that the far-red absorbing Ce6 could bind to the extracellular loops of rhodopsin, to generate singlet oxygen and initiate the 11-cis-retinal to all-trans-retinal isomerization, starting the visual phototransduction cascade, and night vision[40]. Whether the enhanced vision by Ce6 relates to direct photodynamic activation of the apo-rhodopsin protein itself, just like photodynamic activation of CCK1R protein, will probably need further experimentation.
It has been possible to shed some light on the mechanism of permanent photodynamic CCK1R activation at the physiological level, as detailed below.
MECHANISM FOR PERMANENT PHOTODYNAMIC CCK1R ACTIVATION
Quantitative dimer-to-monomer conversion of CCK1R protein
When the membrane proteins prepared from the freshly isolated rat pancreatic acini were subject to reduced electrophoresis and Western blot, the CCK1R protein was found to be present very clearly as both dimers and monomers (Figure 3A)[41]. Most remarkably, after photodynamic action, with SALPC as the photosensitizer, at the photodynamic intensity/dose (SALPC: 1 μmol/L, red light at 36.7 mW/cm2) that is identical or similar to that to photodynamically activate CCK1R therefore would trigger persistent cytosolic calcium oscillations in live cells[28,30,32], the resting receptor protein dimer was converted nearly quantitatively to the monomer[41] (Figure 3A). This CCK1R protein monomerization process after photodynamic action, is highly similar to agonist-stimulated rat CCK1R receptor protein monomerization, as revealed by the bioluminescence resonance energy transfer (BRET) technique in rat CCK1R gene-transfected COS cell line[42]. This would further suggest that the photodynamic activation of CCK1R that has been demonstrated[13,21,28,30,32,43] is likely highly physiological. This monomerization process of the A-class CCK1R activation is exactly the opposite to B-class GPCR, where the resting monomers dimerize after agonist-stimulated activation.
It has been demonstrated also that the TM3 in the CCK1R structure plays a critical role in photodynamic CCK1R activation[43].
TM3 domain as a transplantable pharmacophore
Of the dozen or so class-A calcium-mobilizing receptors that have been examined in the author’s laboratory over many years, it has been found that the A-class GPCR could be divided into 4 categories according to their susceptibility to photodynamic modulation: Those that are sensitized after photodynamic action for agonist activation (equivalent to the ligand-dependent positive allosteric modulation), those that are desensitized (equivalent to the ligand-dependent negative allosteric modulation), not affected (no effect or neutral antagonism possible), and those that are directly activated (equivalent to the ligand-dependent orthosteric agonism). In this sense the effect of 1O2 could be referred loosely to as being dualsteric/bitopic as in ligand-dependent conventional receptor pharmacology[1,3], although 1O2 is clearly not a ligand of any type in GPCR receptors containing no heme molecules (unlike hemoglobin monomer which has an oxygen-binding heme molecule).
Whereas CCK1R is directly activated specifically by 1O2 in type II photodynamic action, the type 3 muscarinic acetylcholine receptor (M3R) is not affected at all by photodynamic action with either SALPC or mini singlet oxygen generator (miniSOG) as the photosensitizer[28,43]. This might imply that photodynamic activation does show some specificity at such low photodynamic intensity/dose, doses for PDB studies in comparison with mega doses for photodynamic killing of undesirable cells or pathogens in photodynamic therapy.
Both structural[44-47] and point mutation studies[48,49] point to the importance of TM3 in CCK1R activation. Whereas CCK1R is and M3R is not susceptible to photodynamic activation, when the TM3 region of CCK1R is transplanted to M3R to replace the TM3 of M3R, the resultant chimeric receptor M3R-TM3CCK1R now becomes activatable by photodynamic action, after tagging the protein photosensitizer miniSOG to the N-terminus of the chimeric receptor[43] (Figure 3B). The calculated extent of miniSOG photodynamic activation of M3R, chimeric M3R-TM3CCK1R, and CCK1R are 0%, 65%, 100% respectively[43] (Figure 3B). Comparison of TM3 residue composition of CCK1R with the list of 1O2-susceptible residues (Met/M, Cys/C, Tyr/Y, Trp/W, His/H)[22,50,51] reveals that the triplet motif of Y119/3.30F120/3.31M121/3.32 might play a vital role in photodynamic activation of CCK1R[43].
In a study to compare the varied sensitivity of CCK1R to photodynamic activation in pancreatic acini isolated from both mammalian and avian species, sensitivity to photodynamic activation of CCK1R was found correlated to the size of the secondary-structure-free region of intracellular loop 3 (ICL3).
The length of the disordered region in ICL3 correlates positively with sensitivity for photodynamic activation of CCK1R
Comparative studies with pancreatic acini isolated freshly from the rat, mouse and Peking duck revealed that the sensitivity for photodynamic activation of CCK1R in these species was, in descending order: Mouse > rat > Peking duck, the same order of sensitivity for agonist stimulated receptor activation[32]. Comparison of the modeled three-dimensional structure of rat, mouse, and Peking duck CCK1R with hCCK1R (PDB: 7F8X) found them to align/overlap almost completely, with the conspicuous exception in ICL3[32]. Even the ICL3 show good overlap in their secondary structures, but secondary-structure-free part immediately after TM5 diverge significantly (Figure 3C). This region is named as the structure-free region of ICL3. The size or length of this region (mouse A250-K286, 37 residues; rat A265-L296, 32 residues; Peking duck S253-D270, 18 residues) correlates positively with sensitivity to photodynamic activation of CCK1R receptor from these different species[32] (Figure 3C). This observation is corroborated by data from other groups to suggest that ICL3 is important for receptor coupling to downstream G proteins[52], the size of ICL3 is used to selectively couple to different types of G protein subunit including αq that is coupled to CCK1R[53,54]. It is interesting to note that the C-terminal of ICL3 might be stretched and pulled in an outward movement (towards the extracellular fluid) by TM6 (by a distance of 14 Å) during agonist-stimulated receptor activation, as observed with nanobody-bound β2 adrenergic receptor[55]. It might be noted also that the disordered region in the C-terminal of GPCR has been proposed to be important for interaction with the plasma membrane[56].
OUTLOOK–GPCR-ABSO AND THE EMERGING FIELD OF PHOTODYNAMIC BIOLOGY
Other than CCK1R, the pharmacologically and structurally similar CCK2R is photodynamically activated also, after tagging with protein photosensitizer miniSOG[57]. Both receptors (CCK1R and CCK2R) are known to be involved in vital physiological functions in vivo: In exocrine pancreatic secretion[27], central satiety sensation[58], sinoatrial node pace making[59], memory formation and storage[60], and many other important physiological functions. Their photodynamic activation implies that all these and other relevant physiological functions could be modulated in vivo. The fact that such property of permanent photodynamic activation could be transplanted from CCK1R to other members of the A-class of GPCR family such as M3R (M3R per se is not susceptible to photodynamic activation)[28,43] suggests that photodynamic activation of GPCR could be used to study the roles and functions of vast numbers of important GPCR in the A-class, and possibly in other classes also. Even if direct photodynamic activation of GPCR is not ascertained for each GPCR, photodynamic sensitization/desensitization could be expected for ligand-independent positive or negative allosteric modulations (Table 1). The changed fate or degradation, the activated-state sojourn time in receptor protein dynamics, and characterization of the transfixed structure of photo-oxidatively activated GPCR receptor proteins after photodynamic activation in comparison with agonist-stimulated activation, will provide fertile grounds for future investigations.
Permanent photodynamic activation of GPCR would be most helpful to investigate GPCR physiology and pharmacology in vivo to manipulate intact animal behaviors. The endogenous sources of 1O2 in photodynamic activation of GPCR could be 1O2 generated in type II photodynamic action after light irradiation of an endogenous photosensitzer[61-63], of exogenous photosensitizer after microinjection to the target site of interest, or of GEPP protein photosensitizers target expressed in specific regions, tissues, organs or cell types, or tagged to the GPCR proteins of interest[21]. The well-developed bio-photonics and trans-genetics technologies used for optogenetics[64], for photodynamic therapy to kill cancer cells, and readily available clinically approved photosensitizers[37,65] will facilitate future in vivo investigations, and probably the clinical translations of GPCR-ABSO.
Most interestingly, other transmembrane proteins such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2, an enzyme important in intermediate metabolism, in neutrophil physiology and inflammation, could be activated by ultraviolet A-driven photodynamic action involving apoprotein-bound endogenous photosensitizers[66]. Other than NADPH oxidase 2, NADPH oxidase 5 and dual oxidase 2 are now known to be photodynamically activated permanently[67]. An array of important transmembrane and cytosolic proteins, possessing the intrinsic property of being oxidatively activated, could all potentially be photodynamically activated locally by focused light irradiation. These proteins of interest will include calcium (Ca2+)- and voltage-gated potassium channel BK/Slo (after oxidation of calcium bowl domain residues Met536, Met712, Met739)[68], the multifunctional Ca2+/calmodulin-dependent protein kinase II (after oxidation of regulatory domain residues of Met281, Met282)[69-71], or riboflavin (an efficient photosensitizer) transporter of SLC52A1[18]. Therefore, some member of a large majority of categories of functional proteins (out of GPCR, ionic channels, transporters, enzymes, catalytic receptors, nuclear hormone receptors, and others) as classified by the International Union for Pharmacology[18] could be subject to photodynamic activation.
Future works on the photodynamic activation and modulation of functional proteins will be facilitated greatly by the emerging protein tagging technologies, which typically include (1) SNAP-Tag (the tagging of an engineered O6-alkylguanine alkyltransferase - AGT); (2) CLIP-Tag (AGT-based tag which reacts specifically with O2-benzylcytosine derivatives); (3) Halo-Tag (haloalkane dehalogenase-tag); and (4) Trimethoprim (TMP)-Tag, and others. Halo-Tag[72], for example, could be genetically fused to functional proteins of interest such as CCK1R. The Halo-Tag (more specifically, the side chain of residue D106) will then serve as an anchor for covalent attachment (via the chloro-containing end) of the specific small linear molecular ligand chloroalkane, the opposite end of the chloroalkanes could be pre-conjugated to a great many of red-absorbing chemical photosensitizers[65] (see also https://www.internationalphotodynamic.com/interactive-photosensitizer-spectraviewer). If endogenously generated bioluminescence from high intensity bright luciferases such as NanoLuc[73] is harvested to drive the photodynamic activation process such as shown for NanoLuc-miniSOG BRET-driven photodynamic CCK1R activation in the rat pancreatic acinar cells AR4-2J[74] and for photodynamic eradication of deep-seated tumors in mice bearing NanoLuc-miniSOG expressing tumors (with tumor-specific lentiviral vector used)[75], even photosensitizers with maximum absorption peaks outside of the optical window (600 nm-1400 nm)[37] of light penetration of biological tissues may be used. Photodynamic activation might also be applied to modulate the function of CCK1R- or other GPCR-expressing cancer cells and multiple types of immune cells (such as mast cells, lymphocytes, neutrophils).
Interestingly, the permanent photodynamic activation of CCK1K summarized in this article might as well be related to the “irreversible photoactivation of a pancreatic secretagogue receptor” initially noted by the Jamieson group at Yale some 45 years ago when performing receptor ultraviolet (> 320 nm) photoaffinity labeling with 2-nitro-5-azidobenzoyl-Gly-CCK-8 in the isolated guinea pig pancreatic acini[76] but has not been followed up in detail before.
Study limitations
This review focuses on permanent photodynamic activation of an A-class GPCR, the CCK1R, but related works are available in the literature that are collateral for what is presented here: (1) Works have been carried out previously on oxidative modulation of cell surface adrenergic receptors[77], and the constitutive activity of viral GPCR[78-85]. The constitutive activation of the viral GPCR might shed some further light on the detailed mechanisms of permanent photodynamic CCK1R activation; (2) Although we are confident that what we have found so far and presented in this review suggests that other than photodynamic therapy, a related and probably much wider field of PDB needs more attention, this area apparently will require more involvements of other laboratories world-wide for validation and discussion; and (3) Although currently approved photosensitizers could be repurposed for clinical photodynamic pharmacology/therapeutics (other than to kill the undesired cancer and unmaligned cells), it is expected that the use of GEPP may require additional works on the use of viral vectors and trans-genetics[86-88].
CONCLUSION
CCK1R is a GPCR activated by singlet oxygen (GPCR-ABSO) and will be a useful toolkit for GPCR studies, along with other engineered GPCR such as designer receptors exclusively activated by designer drugs. Some members of the major categories of functional proteins as classified by International Union of Pharmacology could potentially be activated by singlet oxygen therefore by type II photodynamic action. Technologies such as advanced genetically encoded protein photosensitizers, specific protein tagging technology, animal trans-genetics and tissue-specific viral vector delivery technologies are now available, facilitating the emerging field of photodynamic biology of GPCR and other functional proteins.
Footnotes
Provenance and peer review: Unsolicited article; Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Gastroenterology and hepatology
Country of origin: China
Peer-review report’s classification
Scientific Quality: Grade A, Grade B
Novelty: Grade A, Grade A
Creativity or Innovation: Grade A, Grade A
Scientific Significance: Grade A, Grade B
P-Reviewer: Cui YN; Gao B S-Editor: Fan M L-Editor: A P-Editor: Zheng XM
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