Yi-Qian Xu, Hao-Lin Wu, Xing-Yue Fan, Hao-Fei Fan, Rui Wang, Qi-Bing Liu,3?

1. Department of Pharmacology, School of Basic Medicine and Life Sciences, Hainan Medical University, Haikou 571199, China

2. Department of Pharmacology, Ningxia Medical University, Yinchuan 750004, China

3. Key Laboratory of Brain Science Research and Transformation in Tropical Environment of Hainan Province, Haikou 571199, China

Keywords:G protein coupled receptor Signal transduction Physiological effect Pharmacological function Cardio-cerebrovascular disease

ABSTRACT G protein-coupled receptors(GPCRs) are the largest protein superfamily in the body, expressed in various tissues and organs, and are currently one of the most important clinical drug targets. Recently, a class of GPCRs without endogenous ligands(orphan GPCRs) have been discovered. They exhibit different physiological functions in the body and act extensively on the cardiovascular and cerebrovascular systems. Among them, G protein-coupled receptor 124(GPR124) is an orphaned member of the G protein coupled receptor adhesion family that has attracted much attention. It plays a key role in promoting cerebral angiogenesis and maintaining the stability of the blood-brain barrier. It also associated with cardiovascular and cerebrovascular diseases such as cerebral ischemia and atherosclerosis. However, the role of GPR124 in these diseases, the associated signaling pathways, and possible drug intervention targets are still unclear. This article summarizes the physiological effects, pharmacological effects and related signal pathways of GPR124 published in the field of cardiovascular and cerebrovascular diseases published in recent years, in order to provide a reference for the study of the role of GPR124 in the occurrence and development of diseases.

G Protein-Coupled Receptors (GPCRs) are a kind of superfamily of membrane proteins on the cell surface, which can sense different physical and chemical signals outside the cell. As a member of the GPCRs subfamily, adhesion G protein coupled receptors are expressed in different parts of the human body, especially in the immune system, central nervous system and reproductive system,which indicates that they may participate in many physiological processes. G Protein-Coupled Receptors 124(GPR124) is an important member of the family of adhesion G Protein-Coupled Receptors, it is associated with ischemic stroke, arteriosclerosis(AS) , hypertension, multiple sclerosis and cancer. Therefore, indepth study of the physiological and pharmacological functions of GPR124 is expected to provide new therapeutic targets for these diseases. In this article, we will summarize the functional characteristics of GPR124, introduce its main research progress in physiology and pharmacology, and describe the signal transduction and related pathway of GPR124 interaction with cerebrovascular disease.

1. General situation of G protein-coupled receptor

GPCRs is a large family of proteins that carry a variety of physical chemistry signals, including neurotransmitters, hormones, growth factors, and fragrances. The GPCRs superfamily consists of about 800 receptors and is divided into five subfamilies based on amino acid sequence similarity and common physiological characteristics:the largest is rhodopsin family, which has about 284 members in humans, next came the gluttonous family with 33 members, followed by the Glutamate family, the Secrettin family, and the Frizzled family with 22,15, and 11 members, respectively. Structurally,GPCRs is a large family of seven transmembrane proteins, whose core region consists of seven Transmembrane domain, these domains divide receptors into n-terminal, c-terminal, 3 outer rings and 3 inner rings, and are therefore also known as seven transmembrane class of receptors (7TM) proteins[1]. In addition to the 7TM α-helix, the adhesiveness family has a large number of conserved extracellular n-terminal domains and a GPCRs self-induced protein hydrolytic domain (GPS) , which have attracted much attention because of their unique structure. In addition to the 7TM α-helix, the adhesiveness family has a large number of conserved extracellular n-terminal domains and a GPCRs self-induced protein hydrolytic domain(GPS)[2]. GPCRs is one of the three major membrane receptors for transmembrane signal transduction, distributed in almost all tissues and organs, involved in many physiological processes, and closely related to many diseases. At present, more than 36% of the drugs on the market are targeted at GPCRs, which is the most popular drug target[3]. Therefore, to master the regulation mechanism of GPCRs is a key step in the development of new drugs and plays an important role in the prevention and treatment of related diseases.

2. Discovery and biological characteristics of GPR124

GPR124 is also known as tumor endothelial marker 5 because it is found in endothelial cells of human colorectal cancer neovascularization and is the first to be associated with vascular biology[4]. GPR124 is located at 8p11.22 in the human genome and 16q12.4 in the rat, and has 84% homology in amino acid sequence. At the tissue level, GPR124 is expressed differently in humans and mice. The expression of GPR124 was low in human heart, placenta and small intestine, high in skeletal muscle, prostate,testis and colon, and also in liver, kidney, pancreas and spleen.The expression of GPR124 was only detected in brain, heart, lung,skeletal muscle, pancreas and intestine of rats, but not in human brain, thymus, adrenal, cortex, medulla, liver, spleen and stomach of rats[5]. At the cellular level, GPR124 is widely expressed in a variety of tissue endothelial cells, including brain, heart, retina, kidney and pancreas, suggesting that GPR124 may be involved in angiogenesis,arteriosclerosis and other physiological processes[5].

GPR124, as a member of the adhesion family, has the typical structural characteristics of the family members: it contains 1338 amino acids, among which 760 amino acids are found in the extracellular N-terminal region, it consists of several leucinerich regions (leucine-rich repeats, LRR), immunoglobulin (Ig)-like domains, hormone-binding domains (Hor) , and GPS[6]. The intracellular domain of GPR124 contains a conserved PDZ binding motif at the c-terminal[7]. Recent studies have shown that the c-terminal PDZ binding motif is linked to the Elmo-Dock family and the Nucleotide exchange factor, and that the Heterotrimer G protein-dependent pathway is activated by Rho GTPases[8]. The G βγ subunit directly binds to and activates downstream effectors,initiating the GPR124 signal transduction pathway (Fig. 1) [8].

Figure 1 GPR124 signal transduction pathway model

3. GPR124 associated signal transduction molecules

First,we obtained the GPR124 protein-protein interaction network from the protein-protein interaction database(https://string-db.org/).It includes not only known interacting proteins,but also proteins not yet explicitly associated with GPR124. In figure, nodes represent compounds or proteins, and edges represent proteinprotein interactions that encode proteins. The main types of GPR124 associated signaling molecules reported in the literature are as follows (Fig. 2) :

Figure 2 GPR124 protein interaction network diagram Figure 2 depicts the GPR124 protein interaction network. The network consists of 11 nodes and 12 edges. The blue node represents GPR124, and the red node represents the protein interacting with it.

3.1 Wnt/ -catenin

Wnt/β-catenin signals are mediated by FZD receptors and their accessory receptors LRP5/6 through the plasma membrane[9].GPR124 signal transduction complex specifically acts on Wnt7a/7b angiogenesis signal in central nervous system endothelial cells.Studies by Chang et al. have shown that knocking out GPR124 in normal mice at birth or at 2-3 months of age does not reduce the expression of GPR1 and BBB markers, nor did it affect the vascular architecture of the central nervous system and the survival rate of the mice[10]. In order to study the function of GPR124 in embryonic mice, Anderson et al made GPR124(GPR124-/-) deleted in mice,and found that GPR124-/-mice can survive, look normal and have fertility[11].The offspring of GPR124-/-were examined at Day 15.5(E15.5) or the day of birth. All offspring developed abnormalities of cerebral vessels, palate and lungs, and all offspring died in perinatal period. These data suggest that although the elimination of GPR124 after birth does not affect the survival of mice, GPR124 is essential for the normal formation and development of blood vessels in the brain during the embryonic period.

In E12.5 day mouse embryos, the classic Wnt signal and endothelial cells are located in the central nervous system. Wnt7a and Wnt7b are typical Wnt ligands, expressed in the forebrain and neural tubes, and are the starting site of central nervous system angiogenesis. In mouse embryos, knocking out either the neuroepithelial Wnt7a/7b or the endothelial GPR124 caused central nervous system vessels to stop growing, producing abnormal vascular structures that do not feature the blood brain barrier, known as glomeruli[12]. Although GPR124 knock-out in adult mouse endothelial cells does not affect the integrity of the homeostatic blood-brain barrier, under pathological conditions (such as ischemic stroke, Glioblastoma multiforme,etc.) , knock-out of GPR124 will result in many neurovascular Wnt target genes, the expression of AXIN2, APCDD1 and CLDN5 was decreased, which was accompanied by the decrease of classic Wnt/β-catenin signal expression in cerebral vessels[13, 14]. Thus, in a disease state, a lack of GPR124 leads to an interruption of specific Wnt7a/7b signaling, resulting in brain edema, hemorrhage, and blood-brain barrier leakage. This suggests that the role of GPR124 in central nervous system angiogenesis and blood brain barrier is coupled to Wnt7a/7b mediated classical signaling pathways in endothelial cells.

In E10.5 normal mouse embryos, the Reck gene was expressed in a large number of vascular and neural precursor cells, while the Reck deficient mice died at about E10.5, accompanied by tissue integrity impairment, intraperitoneal hemorrhage and early differentiation of neural cells[15]. Silencing Reck gene with tamoxifen in E11 resulted in cerebral hemorrhage and vascular defects in E15.5 mouse embryos,even resulting in fetal death. Therefore, the Reck Gene is essential for mouse embryonic development[16]. The results of Vanhollebeke et al. showed that Reck and GPR124 had a synergistic effect in response to Wnt7a/7b co-activation Wnt/β-catenin signal, and FZ4 co-transfected with LRP5/6 could further enhance WNT/LRP5/LRP6 signal transduction[17]. It was found that the co-activation of GPR124 with Reck and Wnt7a/7b was the highest, but there was almost no response to other Wnts. Wnt7a/7b triggered the classical Wnt pathway significantly when GPR124 coexisted with Reck and Wnt in vitro[17]. Based on these findings, Reck and GPR124 are specific factors that activate Wnt7a/7b, and a corresponding model is proposed according to GPR124 cooperating with Reck to promote Wnt signaling pathway (Figure 3) [7, 9].

Figure 3 Correlation of GPR124 with central nervous system angiogenesis and blood-brain barrier.

3.2 TGF-β

The role of the Transforming growth factor beta growth factor beta(TGF-β) signaling pathway in central nervous system angiogenesis has been investigated, which is mediated by isomer receptors composed of TGF-βr2 and TGF-βr1 or endothelial specific receptor ALK1[18]. TGF-β1 is a multifunctional cytokine that plays an important role in neuron production, survival, migration,neuroglia differentiation and synapse formation. Anderson et al showed that TGF-β induced up-regulation of GPR124 expression in human umbilical vein endothelial cells in vitro, and GPR124 knockout, TGF-β signaling and target gene expression changes in mice showed that GPR124 may be involved in TGF-β signaling pathway[10,19]. Lacking GPR124 expression in the vascular system,embryos exhibit delayed neurovascular invasion, developmental defects, and bleeding. Deletion of the gene in the TGF-β pathway leads to the formation of abnormal blood vessels specific to the forebrain and spinal cord in mouse embryos, similar to the vascular phenotypes seen in GPR124 and Wnt7a/7b deletion mutants. Cleft palate may occur after deletion of TGF-β3 and integrin αv/Β8, and hypoplasia of the lungs may occur after deletion of TGFβ3 or Wnt7a/7b, and cleft palate and hypoplasia of the lungs may also occur after deletion of GPR124, these results further support the hypothesis that GPR124 may interact with the TGF-β signal pathway[10]. In GPR124-deficient mice, the abnormal distribution and deposition of collagen in the basement layer of blood vessels and the abnormal aggregation of smooth muscle cells were observed by immunohistochemistry[19]. Stimulation of TGF-β specifically induces basement membrane formation and smooth muscle cell recruitment or differentiation, further demonstrating that the absence of GPR124 in blood vessels leads to the elevation of TGF-β signal.In summary, the relationship between the GPR124 and TGF-β signaling pathways provides a potential model (figure 4) .

Figure 4 The interaction model between GPR124 and TGF-β signaling pathway in the central nervous system.

3.3 VEGF

Tumor angiogenesis is the key to the growth and metastasis of primary tumors. Vascular endothelial growth factor (VEGF) in tumor cells induces endothelial cell proliferation, migration and invasion by activating vascular endothelial growth factor (VEGF-RRB--mediated angiogenesis signal, thus forming new blood vessels, is a major driver of tumor angiogenesis[20]. In tumor angiogenesis,VEGF mainly signals VEGFR2 and activates downstream factors such as SRC, FAK, Akt, JNK and ERK to regulate the proliferation,migration and invasion of endothelial cells[20]. Cullen et al. found that up-regulation of the GPR124 gene in tumor blood vessels helps to limit vascular leakage caused by VEGF and other tumor-derived permeability factors[4]. Wang et al. studied the role of GPR124 in tumor angiogenesis and found that silent GPR124 decreased VEGFinduced phosphorylation of VEGFR2, Fak and SRC, significantly decreased VEGF and VEGFR2 expression, and thus decreased tumor angiogenesis, these results suggest that GPR124 may modulate VEGF/VEGFR signaling in tumor angiogenesis[20]. VEGF and its co-receptor NFP-1 play an important role in cerebral angiogenesis and blood-brain barrier differentiation. The expression of VEGF in the brains of GPR124 +/+ mice and GPR124 something/something mice during embryonic development showed that VEGF164 was the main expression form, but the expression level of VEGF120 and VEGF188 was lower, but each VEGF variant was over-expressed in the brains of GPR124 something/something mice[4]. Therefore,GPR124 knock-out may lead to up-regulation of VEGF, cause vascular leakage, affect the formation of blood-brain barrier.

4. GPR124 and related diseases

4.1 Cerebrovascular disease

Knock-out of GPR124 causes fetal death due to central nervous system arrest and bleeding, and overexpression of GPR124 leads to the proliferation of central nervous system vascular malformations,this suggests that GPR124 is associated with vascular pathology in a variety of central nervous system disease. Vallon et al. showed that the extracellular Horm domain of GPR124 contains RGD(ARG-GLY-ASP) sequences that bind to extracellular matrix and glycosaminoglycans on the cell surface, integrin v β3 can regulate cell function, stimulate cell migration, induce cell adhesion and promote angiogenesis[21]. Integrin αvβ3, a dimer glycoprotein,plays an important role in cell adhesion and angiogenesis, and is the main binding receptor of RGD sequence. Xiao et al. selected the extracellular domain (GCPF) containing RGD sequence of GPR124 and evaluated whether the domain could improve the cognitive function of vascular dementia by targeting integrin αvβ3[22].In vitro and in vitro vascular experiments have shown that GCPF has the biological activity of promoting angiogenesis, stimulating cell migration and adhesion, and promoting Endothelium growth.Histological analysis showed that subcutaneous injection of GCPF could increase the number of vessels and neurons in hippocampal CA1 region, induce neovascularization and improve spatial learning and memory in vascular dementia rats[22]. GPR124 can target integrin αvβ3, promote angiogenesis in vivo and in vitro, and improve the cognitive function of mice with vascular dementia. Chen et al studied the role of GPR124 in the polarization and migration of pericytes, which showed that GPR124 was overexpressed in pericytes and involved in the formation of filamentous pseudopodia by mediating the rearrangement of cytoskeleton[6]. The formation of filamentous pseudopodia is essential for the polarization and migration of pericytes in ischemic brain injury. GPR124 regulates the rearrangement of the cytoskeleton of polarized cells, thus promoting the polarization and migration of pericytes[6]. These findings suggest that GPR124 may be a potential target for central nervous system vascular injury, such as ischemic stroke and vascular dementia.

4.2 Cardiovascular disease

Arteriosclerosis can cause cerebrovascular disease such as coronary heart disease, stroke and peripheral vascular disease, the main pathological changes are arterial intimal dysfunction, abnormal lipid metabolism and inflammatory response[23]. To investigate the role of GPR124 in the development of arteriosclerosis in adult mice,endothelial-specific gene overexpression and immunofluorescence were used, these results suggest that GPR124 contributes to arteriosclerosis endothelial cell injury by activating nitrosation stress and NLRP3 inflammatory corpuscles[24]. IL-1β is the most common Proinflammatory cytokine in arteriosclerosis plaques,and NLRP3 can activate Caspase-1 and promote IL-1β release.Therefore, inhibiting the inflammatory bodies of NLRP3 can block the activation of NLRP3, reduce the expression of IL-1β, and decrease the number of foam cells induced by ox-LDL, thus prevent or delay the occurrence of arteriosclerosis. Therefore, GPR124 may be a potential drug target for prevention and treatment of AS by inhibiting the activation of NLRP3 and affecting the development of arteriosclerosis.Kaur et al. found that GPR124 up-regulates the dedifferentiation of skeletal muscle smooth muscle cells and negatively regulates the expression of inflammatory factors in skeletal muscle smooth muscle cells, suggesting that GPR124 may be a potential target for regulating blood pressure[25]. Calder ón-Zamora et al. studied the role of GPR124 in the development of hypertension and found that GPR124 is expressed in the aorta, heart,kidney and brain, but in hypertension, GPR124 is up-regulated in the left atrium and left ventricle, indicating over-expression of GPR124, may increase angiogenesis to reduce hypertension-induced myocardial damage. However, due to the lack of known agonists/antagonists and the lack of data on their role in regulating vascular tone or other smooth muscle cell function, the role of GPR124 in blood pressure regulation remains to be further studied[26].

4.3 Tumor

GPR124 is highly expressed in tumor endothelial cells of colorectal,breast, stomach and lung cancers, but not in the corresponding normal tissues. GPR124 is essential for Vascular endothelial growth factor to induce tumor angiogenesis in vivo, and inhibition of GPR124 protein expression can effectively control tumor growth[20].The expression of miRNA-138-5p was negatively correlated with the expression of GPR124 in clinical samples of non-small-cell lung carcinoma. EGFR tyrosine-kinase inhibitor gefitinib is an effective drug for the treatment of EGFR activated non-small-cell lung carcinoma[27]. In gefitinib-sensitive PC9 cells, high-level miRNA-138-5p directly inhibited the expression of GPR124, which made the cells sensitive to gefitinib[27]. In contrast, loss of miRNA-138-5p expression after resistance to gefitinib resulted in high expression of GPR124. In addition, in vitro knock-out of GPR124 overcomes the resistance of non-small-cell lung carcinoma to gefitinib, making GPR124 a potential therapeutic target. Wang et al. showed that β-elemene combined with paclitaxel could stimulate cell apoptosis,decrease the expression of GPR124 and effectively inhibit the growth and metastasis of tumor[28].

5. Conclusion

In conclusion, GPR124 not only participates in the basic pathophysiological processes such as angiogenesis, maintenance of blood-brain barrier function and endothelial inflammatory response, but also serves as a potential drug target for the treatment of cardiovascular and cerebrovascular diseases antheir complications. At present, experiments and studies on GPR124 have been established in animal models, and the mechanism of GPR124 in human tissues and organs has not been elucidated. Many problems need to be solved, including the identification of GPR124 ligands, the detection of their activities and the mechanisms of their important signal transduction pathways. Therefore, further study on the pathophysiological mechanism of GPR124 and its role in cerebrovascular disease and its potential as a drug target have a good application prospect.