Cellular Regulation of Macropinocytosis.

Interest in macropinocytosis has risen in recent years owing to its function in tumorigenesis, immune reaction, and viral infection. Cancer cells utilize macropinocytosis to acquire nutrients to support their uncontrolled proliferation and energy consumption. Macropinocytosis, a highly dynamic endocytic and vesicular process, is regulated by a series of cellular signaling pathways. The activation of small GTPases in conjunction with phosphoinositide signaling pivotally regulates the process of macropinocytosis. In this review, we summarize important findings about the regulation of macropinocytosis and provide information to increase our understanding of the regulatory mechanism underlying it.

PRL1 and PRL3 promote macropinocytosis via its lipid phosphatase activity.

PRL1 and PRL3, members of the protein tyrosine phosphatase family, have been associated with cancer metastasis and poor prognosis. Despite extensive research on their protein phosphatase activity, their potential role as lipid phosphatases remains elusive. Methods: We conducted comprehensive investigations to elucidate the lipid phosphatase activity of PRL1 and PRL3 using a combination of cellular assays, biochemical analyses, and protein interactome profiling. Functional studies were performed to delineate the impact of PRL1/3 on macropinocytosis and its implications in cancer biology. Results: Our study has identified PRL1 and PRL3 as lipid phosphatases that interact with phosphoinositide (PIP) lipids, converting PI(3,4)P2 and PI(3,5)P2 into PI(3)P on the cellular membranes. These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis, facilitating nutrient extraction, cell migration, and invasion, thereby contributing to tumor development. These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis. Additionally, we found a correlation between PRL1/3 expression and glioma development, suggesting their involvement in glioma progression. Conclusions: Combining with the knowledge that PRLs have been identified to be involved in mTOR, EGFR and autophagy, here we concluded the physiological role of PRL1/3 in orchestrating the nutrient sensing, absorbing and recycling via regulating macropinocytosis through its lipid phosphatase activity. This mechanism could be exploited by tumor cells facing a nutrient-depleted microenvironment, highlighting the potential therapeutic significance of targeting PRL1/3-mediated macropinocytosis in cancer treatment.

Procedures to Measure Dictyostelium Phagocytosis and Macropinocytosis.

Uptaking particulate objects and bulk liquid by eucaryotic cells is critical for their growth, survival, and defense. Dictyostelium is a model organism spearheaded to uncover mechanisms behind various types of uptaking activities. Here, we describe assays measuring phagocytosis and macropinocytosis using Dictyostelium discoideum.

Dissecting the roles of dynamin and clathrin in platelet pinocytosis.

Platelets endocytose many molecules from their environment. However, this process of pinocytosis in platelets is poorly understood. Key endocytic regulators such as dynamin, clathrin, CDC42 and Arf6 are expressed in platelets but their roles in pinocytosis is not known. Stimulated platelets form two subpopulations of pro-aggregatory and procoagulant platelets. The effect of stimulation on pinocytosis is also poorly understood. In this study, washed human platelets were treated with a range of endocytosis inhibitors and stimulated using different activators. The rate of pinocytosis was assessed using pHrodo green, a pH-sensitive 10 kDa dextran. In unstimulated platelets, pHrodo fluorescence increased over time and accumulated as intracellular puncta indicating constituently active pinocytosis. Stimulated platelets (both pro-aggregatory and procoagulant) had an elevated pinocytosis rate compared to unstimulated platelets. Dynamin inhibition blocked pinocytosis in unstimulated, pro-aggregatory and procoagulant platelets indicating that most platelet pinocytosis is dynamin dependent. Although pinocytosis was clathrin-independent in unstimulated and procoagulant populations, clathrin partially contributed to pinocytosis in pro-aggregatory platelets.

The multifunction Coxiella effector Vice stimulates macropinocytosis and interferes with the ESCRT machinery.

Intracellular bacterial pathogens divert multiple cellular pathways to establish their niche and persist inside their host. Coxiella burnetii, the causative agent of Q fever, secretes bacterial effector proteins via its Type 4 secretion system to generate a Coxiella-containing vacuole (CCV). Manipulation of lipid and protein trafficking by these effectors is essential for bacterial replication and virulence. Here, we have characterized the lipid composition of CCVs and found that the effector Vice interacts with phosphoinositides and membranes enriched in phosphatidylserine and lysobisphosphatidic acid. Remarkably, eukaryotic cells ectopically expressing Vice present compartments that resemble early CCVs in both morphology and composition. We found that the biogenesis of these compartments relies on the double function of Vice. The effector protein initially localizes at the plasma membrane of eukaryotic cells where it triggers the internalization of large vacuoles by macropinocytosis. Then, Vice stabilizes these compartments by perturbing the ESCRT machinery. Collectively, our results reveal that Vice is an essential C. burnetii effector protein capable of hijacking two major cellular pathways to shape the bacterial replicative niche.

Leep2A and Leep2B function as a RasGAP complex to regulate macropinosome formation.

Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation.

Liposome nanoparticle conjugation and cell penetrating peptide sequences (CPPs) enhance the cellular delivery of the tau aggregation inhibitor RI-AG03.

Given the pathological role of Tau aggregation in Alzheimer's disease (AD), our laboratory previously developed the novel Tau aggregation inhibitor peptide, RI-AG03. As Tau aggregates accumulate intracellularly, it is essential that the peptide can traverse the cell membrane. Here we examine the cellular uptake and intracellular trafficking of RI-AG03, in both a free and liposome-conjugated form. We also characterize the impact of adding the cell-penetrating peptide (CPP) sequences, polyarginine (polyR) or transactivator of transcription (TAT), to RI-AG03. Our data show that liposome conjugation of CPP containing RI-AG03 peptides, with either the polyR or TAT sequence, increased cellular liposome association three-fold. Inhibition of macropinocytosis modestly reduced the uptake of unconjugated and RI-AG03-polyR-linked liposomes, while having no effect on RI-AG03-TAT-conjugated liposome uptake. Further supporting macropinocytosis-mediated internalization, a 'fair' co-localisation of the free and liposome-conjugated RI-AG03-polyR peptide with macropinosomes and lysosomes was observed. Interestingly, we also demonstrate that RI-AG03-polyR detaches from liposomes following cellular uptake, thereby largely evading organellar entrapment. Collectively, our data indicate that direct membrane penetration and macropinocytosis are key routes for the internalization of liposomes conjugated with CPP containing RI-AG03. Our study also demonstrates that peptide-liposomes are suitable nanocarriers for the cellular delivery of RI-AG03, furthering their potential use in targeting Tau pathology in AD.

Impact of Oncogenic Changes in p53 and KRAS on Macropinocytosis and Ferroptosis in Colon Cancer Cells and Anticancer Efficacy of Niclosamide with Differential Effects on These Two Processes.

Mutations in p53 and KRAS are seen in most cases of colon cancer. The impact of these mutations on signaling pathways related to cancer growth has been studied in depth, but relatively less is known on their effects on amino acid transporters in cancer cells. This represents a significant knowledge gap because amino acid nutrition in cancer cells profoundly influences macropinocytosis and ferroptosis, two processes with opposing effects on tumor growth. Here, we used isogenic colon cancer cell lines to investigate the effects of p53 deletion and KRAS activation on two amino acid transporters relevant to macropinocytosis (SLC38A5) and ferroptosis (SLC7A11). Our studies show that the predominant effect of p53 deletion is to induce SLC7A11 with the resultant potentiation of antioxidant machinery and protection of cancer cells from ferroptosis, whereas KRAS activation induces not only SLC7A11 but also SLC38A5, thus offering protection from ferroptosis as well as improving amino acid nutrition in cancer cells via accelerated macropinocytosis. Niclosamide, an FDA-approved anti-helminthic, blocks the functions of SLC7A11 and SLC38A5, thus inducing ferroptosis and suppressing macropinocytosis, with the resultant effective reversal of tumor-promoting actions of oncogenic changes in p53 and KRAS. These findings underscore the potential of this drug in colon cancer treatment.

CYRI-B-mediated macropinocytosis drives metastasis via lysophosphatidic acid receptor uptake.

Pancreatic ductal adenocarcinoma carries a dismal prognosis, with high rates of metastasis and few treatment options. Hyperactivation of KRAS in almost all tumours drives RAC1 activation, conferring enhanced migratory and proliferative capacity as well as macropinocytosis. Macropinocytosis is well understood as a nutrient scavenging mechanism, but little is known about its functions in trafficking of signalling receptors. We find that CYRI-B is highly expressed in pancreatic tumours in a mouse model of KRAS and p53-driven pancreatic cancer. Deletion of Cyrib (the gene encoding CYRI-B protein) accelerates tumourigenesis, leading to enhanced ERK and JNK-induced proliferation in precancerous lesions, indicating a potential role as a buffer of RAC1 hyperactivation in early stages. However, as disease progresses, loss of CYRI-B inhibits metastasis. CYRI-B depleted tumour cells show reduced chemotactic responses to lysophosphatidic acid, a major driver of tumour spread, due to impaired macropinocytic uptake of the lysophosphatidic acid receptor 1. Overall, we implicate CYRI-B as a mediator of growth and signalling in pancreatic cancer, providing new insights into pathways controlling metastasis.

Pancreatic cancer is an aggressive disease with limited treatment options. It is also associated with high rates of metastasis ­ meaning it spreads to other areas of the body. Environmental pressures, such as a lack of the nutrients metastatic cancer cells need to grow and divide, can change how the cells behave. Understanding the changes that allow cancer cells to respond to these pressures could reveal new treatment options for pancreatic cancer. When nutrients are scarce, metastatic cancer cells can gather molecules and nutrients by capturing large amounts of the fluid that surrounds them using a mechanism called macropinocytosis. They can also migrate to areas of the body with higher nutrient levels, through a process called chemotaxis. This involves cells moving towards areas with higher levels of certain molecules. For example, cancer cells migrate towards high levels of a lipid called lysophosphatidic acid, which promotes their growth and survival. A newly discovered protein known as CYRI-B has recently been shown to regulate how cells migrate and take up nutrients. It also interacts with proteins known to be involved in pancreatic cancer progression. Therefore, Nikolaou et al. set out to investigate whether CYRI-B also plays a role in metastatic pancreatic cancer. Experiments in a mouse model of pancreatic cancer showed that CYRI-B levels were high in pancreatic tumour cells. And when the gene for CYRI-B was removed from the tumour cells, they did not metastasise. Further analysis revealed that CYRI-B controls uptake and processing of nutrients and other signalling molecules through macropinocytosis. In particular, it ensures uptake of the receptor for lysophosphatidic acid, allowing the metastatic cancer cells to migrate. The findings of Nikolaou et al. reveal that CYRI-B is involved in metastasis of cancer cells in a mouse model of pancreatic cancer. This new insight into how metastasis is controlled could help to identify future targets for treatments that aim to prevent pancreatic cancer cells spreading to distant sites.

Na,K-ATPase activity promotes macropinocytosis in colon cancer via Wnt signaling.

Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the ß-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.

TET2-STAT3-CXCL5 nexus promotes neutrophil lipid transfer to fuel lung adeno-to-squamous transition.

Phenotypic plasticity is a rising cancer hallmark, and lung adeno-to-squamous transition (AST) triggered by LKB1 inactivation is significantly associated with drug resistance. Mechanistic insights into AST are urgently needed to identify therapeutic vulnerability in LKB1-deficient lung cancer. Here, we find that ten-eleven translocation (TET)-mediated DNA demethylation is elevated during AST in KrasLSL-G12D/+; Lkb1L/L (KL) mice, and knockout of individual Tet genes reveals that Tet2 is required for squamous transition. TET2 promotes neutrophil infiltration through STAT3-mediated CXCL5 expression. Targeting the STAT3-CXCL5 nexus effectively inhibits squamous transition through reducing neutrophil infiltration. Interestingly, tumor-infiltrating neutrophils are laden with triglycerides and can transfer the lipid to tumor cells to promote cell proliferation and squamous transition. Pharmacological inhibition of macropinocytosis dramatically inhibits neutrophil-to-cancer cell lipid transfer and blocks squamous transition. These data uncover an epigenetic mechanism orchestrating phenotypic plasticity through regulating immune microenvironment and metabolic communication, and identify therapeutic strategies to inhibit AST.

Pathways for macrophage uptake of cell-free circular RNAs.

Circular RNAs (circRNAs) are stable RNAs present in cell-free RNA, which may comprise cellular debris and pathogen genomes. Here, we investigate the phenomenon and mechanism of cellular uptake and intracellular fate of exogenous circRNAs. Human myeloid cells and B cells selectively internalize extracellular circRNAs. Macrophage uptake of circRNA is rapid, energy dependent, and saturable. CircRNA uptake can lead to translation of encoded sequences and antigen presentation. The route of internalization influences immune activation after circRNA uptake, with distinct gene expression programs depending on the route of RNA delivery. Genome-scale CRISPR screens and chemical inhibitor studies nominate macrophage scavenger receptor MSR1, Toll-like receptors, and mTOR signaling as key regulators of receptor-mediated phagocytosis of circRNAs, a dominant pathway to internalize circRNAs in parallel to macropinocytosis. These results suggest that cell-free circRNA serves as an "eat me" signal and danger-associated molecular pattern, indicating orderly pathways of recognition and disposal.

Presynaptic structural and functional plasticity are coupled by convergent Rap1 signaling.

Dynamic presynaptic actin remodeling drives structural and functional plasticity at synapses, but the underlying mechanisms remain largely unknown. Previous work has shown that actin regulation via Rac1 guanine exchange factor (GEF) Vav signaling restrains synaptic growth via bone morphogenetic protein (BMP)-induced receptor macropinocytosis and mediates synaptic potentiation via mobilization of reserve pool vesicles in presynaptic boutons. Here, we find that Gef26/PDZ-GEF and small GTPase Rap1 signaling couples the BMP-induced activation of Abelson kinase to this Vav-mediated macropinocytosis. Moreover, we find that adenylate cyclase Rutabaga (Rut) signaling via exchange protein activated by cAMP (Epac) drives the mobilization of reserve pool vesicles during post-tetanic potentiation (PTP). We discover that Rap1 couples activation of Rut-cAMP-Epac signaling to Vav-mediated synaptic potentiation. These findings indicate that Rap1 acts as an essential, convergent node for Abelson kinase and cAMP signaling to mediate BMP-induced structural plasticity and activity-induced functional plasticity via Vav-dependent regulation of the presynaptic actin cytoskeleton.

ATP2B4 is an essential gene for epidermal growth factor-induced macropinocytosis in A431 cells.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.

Macropinocytosis at the crossroad between nutrient scavenging and metabolism in cancer.

Macropinocytosis (MP), the actin-dependent bulk uptake of extracellular fluids, plays a central role in nutrient scavenging, allowing cancer cells to sustain their growth in the hypoxic and nutrient-deprived microenvironment often found in solid tumours. The lack of soluble nutrients and several oncogenic signalling pathways, with RAS being the most studied, push MP-dependent internalisation of extracellular proteins, which are then digested in the lysosomes, replenishing the intracellular nutrient pools. This review will highlight recent advances in understanding how MP is regulated in hypoxic cancers, how it impinges on chemoresistance, and how different MP cargos facilitate tumour growth. Finally, I will highlight the crosstalk between MP and extracellular matrix receptors.

Extracellular Microvesicles Modified with Arginine-Rich Peptides for Active Macropinocytosis Induction and Delivery of Therapeutic Molecules.

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), transfer bioactive molecules from donor to recipient cells in various pathophysiological settings, thereby mediating intercellular communication. Despite their significant roles in extracellular signaling, the cellular uptake mechanisms of different EV subpopulations remain unknown. In particular, plasma membrane-derived MVs are larger vesicles (100 nm to 1 µm in diameter) and may serve as efficient molecular delivery systems due to their large capacity; however, because of size limitations, receptor-mediated endocytosis is considered an inefficient means for cellular MV uptake. This study demonstrated that macropinocytosis (lamellipodia formation and plasma membrane ruffling, causing the engulfment of large fluid volumes outside cells) can enhance cellular MV uptake. We developed experimental techniques to induce macropinocytosis-mediated MV uptake by modifying MV membranes with arginine-rich cell-penetrating peptides for the intracellular delivery of therapeutic molecules.

CX-4945 (Silmitasertib) Induces Cell Death by Impairing Lysosomal Utilization in KRAS Mutant Cholangiocarcinoma Cell Lines.

BACKGROUND/AIM: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. MATERIALS AND METHODS: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. RESULTS: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. CONCLUSION: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.

Alveolar-capillary endocytosis and trafficking in acute lung injury and acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is associated with high morbidity and mortality but lacks specific therapeutic options. Diverse endocytic processes play a key role in all phases of acute lung injury (ALI), including the initial insult, development of respiratory failure due to alveolar flooding, as a consequence of altered alveolar-capillary barrier function, as well as in the resolution or deleterious remodeling after injury. In particular, clathrin-, caveolae-, endophilin- and glycosylphosphatidyl inositol-anchored protein-mediated endocytosis, as well as, macropinocytosis and phagocytosis have been implicated in the setting of acute lung damage. This manuscript reviews our current understanding of these endocytic pathways and subsequent intracellular trafficking in various phases of ALI, and also aims to identify potential therapeutic targets for patients with ARDS.

Proton pump inhibitors enhance macropinocytosis-mediated extracellular vesicle endocytosis by inducing membrane v-ATPase assembly.

Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical drugs modulating EV levels are still lacking. Here, we show that proton pump inhibitors (PPIs) reduce EVs by enhancing macropinocytosis-mediated EV uptake. PPIs accelerate intestinal cell endocytosis of autocrine immunosuppressive EVs through macropinocytosis, thereby aggravating inflammatory bowel disease. PPI-induced macropinocytosis facilitates the clearance of immunosuppressive EVs from tumour cells, improving antitumor immunity. PPI-induced macropinocytosis also increases doxorubicin and antisense oligonucleotides of microRNA-155 delivery efficiency by EVs, leading to enhanced therapeutic effects of drug-loaded EVs on tumours and acute liver failure. Mechanistically, PPIs reduce cytosolic pH, promote ATP6V1A (v-ATPase subunit) disassembly from the vacuolar membrane and enhance the assembly of plasma membrane v-ATPases, thereby inducing macropinocytosis. Altogether, our results reveal a mechanism for macropinocytic regulation and PPIs as potential modulators of EV levels, thus regulating their functions.

The shear rate promotes pinocytosis of extracellular dextran in platelets.

BACKGROUND: Several conventional studies focused on platelet pinocytosis for possible utilization as drug delivery systems. Although platelet pinocytosis is important in such utilization, the impact of the shear rate on pinocytosis is unclear. OBJECTIVE: Our objective was to investigate the relationship between shear rate and platelet pinocytosis in vitro. In addition, this study addressed the change in platelet aggregation reactivity with adenosine diphosphate (ADP) stimulation after pinocytosis. METHOD: Porcine platelet-rich plasma was mixed with fluorescein isothiocyanate (FITC)-conjugated dextran and incubated for 15 min under shear conditions of 0, 500, and 1500 s-1. After incubation, confocal microscopic scanning and three-dimensional rendering were performed to confirm the internalization of FITC-dextran into platelets. The amount of FITC-dextran accumulated via platelet pinocytosis was compared using flow cytometry at each shear rate. In addition, light transmission aggregometry by ADP stimulation was applied to platelets after pinocytosis. RESULTS: The amount of intracellular FITC-dextran increased with higher shear rates. Platelets with increased amounts of intracellular FITC-dextran did not show changes in the aggregation reactivity to ADP. CONCLUSIONS: A higher shear rate promotes platelet pinocytosis, but enhanced pinocytosis does not affect aggregation sensitivity, which is stimulated by ADP.