Elsevier

Cellular Signalling

Volume 75, November 2020, 109748
Cellular Signalling

Thrombin-activated PAR1 membrane expression is regulated by Rab11a-RCP complex dissociation

https://doi.org/10.1016/j.cellsig.2020.109748Get rights and content

Highlights

  • Thrombin induces calpain-dependent Rab11a/RCP complex disassembly.

  • Rab11a/RCP complex regulates thrombin-activated PAR1 internalization.

  • Rab11a/RCP complex disassembly promotes PAR1 targeting to lysosomes.

  • Rab11a/RCP complex induces an increase in migration and proliferation of RPE cells.

Abstract

PAR1 activation by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration, characteristic of fibroproliferative eye diseases. Due to the cleavage of PAR1 N-terminal domain, carried by thrombin, the arrest of PAR1 signaling is achieved by transport into lysosomes and degradation. Recent findings suggest that the GTPase Rab11a in conjunction with its effector RCP may direct PAR1 to lysosomes. Hereby we demonstrate that thrombin-induced PAR1 internalization and lysosomal targeting requires the disassembly of the Rab11a/RCP complex, and that this process depends on thrombin-induced intracellular calcium increase and calpain activation. These findings unveil a novel mechanism that regulates thrombin activated PAR1 internalization and degradation.

Introduction

The serine protease thrombin is better known by its essential role in the coagulation cascade. However, it also triggers a variety of responses not only in blood cells such as platelets, but also in endothelial cells, and other tissues. It achieves such function through the activation of Protease-Activated Receptors (PARs), a family of G-protein-coupled-receptors (GPCRs) composed by PAR1, PAR2, PAR3 and PAR4 [1]. The four receptors of this family are activated by the cleavage of the extracellular N-terminal domain, which unmasks a new N-terminal sequence that functions as an intra-molecular ligand [2]. PAR1, the prototype of this receptor family is activated by the proteolytic cleavage at the Arg41-Ser42 by thrombin which exposes a new N-terminal (42-SFLLRN-47) that acts as a tethered ligand.

PARs play a very important role in hemostasis, thrombosis and inflammatory responses to tissue injury by regulating cellular responses such as cytokine secretion, integrin activation, metabolic regulation, and changes in cell shape and motility; of particular importance is the induction of cell proliferation in pathological contexts [2].

The retinal pigment epithelium (RPE), a monolayer of differentiated quiescent cells located between the neural retina and the choroid is involved in the trans-epithelial transport of nutrients, the storage and metabolism of vitamin A derivatives, the renewal of photoreceptor outer segments, and the ionic homeostasis of the sub-retinal space [3]. As the predominant component of the outer blood-retina barrier (BRB), the RPE plays an essential role in the maintenance of the functional and structural integrity of the neural retina, required for visual function. Under pathological conditions RPE cells may be exposed to serum-contained growth factors and pro-inflammatory agents, including thrombin generated by the activation of the coagulation cascade. Thrombin concentration can exhibit fast variations, reaching around 250 nM within minutes, in rhegmatogenous retinal detachment [4]. Exposure of RPE cells to thrombin results in the proliferation, dedifferentiation, and migration of RPE cells to the vitreous, and the subsequent assembly of contractile membranes on both retinal surfaces, which promotes retinal detachment. Based on these findings, exposure of RPE cells to thrombin has been associated with the development of Proliferative Vitreoretinopathy (PVR) [5]. Our previous work has demonstrated that thrombin promotes RPE cell proliferation [6], cytoskeletal remodeling [7] and cell migration [8]. However, molecular targets for the prevention of PVR have not been defined.

Given the nature of the catalytic and irreversible activation of PARs, different from classical GPCRs, their activity is tightly regulated [9]. The inactivation of thrombin-induced PAR1 signaling requires C-terminal phosphorylation and β-arrestin binding, which in turn uncouples G proteins from the receptor, desensitizing the receptor. Additionally, β-arrestin interaction with components of the endocytic machinery facilitates the recruitment of PAR1 to clathrin-coated pits and its internalization from the plasma membrane [10]. However, activated PAR1 sorting into early endosomes has been shown to be coincident with p38 phosphorylation, suggesting that p38 signaling may be initiated or sustained by PAR1 at endosomes [11]. Therefore, sorting into lysosomes for degradation is a critical issue for the termination of thrombin-induced PAR1 signaling.

Among the proteins known to regulate the internalization and recycling of GPCRs, the family of Rab GTPases controls cellular processes such as endocytosis, trafficking, endosome fusion and exocytosis. Among these proteins, Rab11a and Rab11b are required for PAR1 trafficking and the recycling of non-activated PAR1, whereas activated PAR1 is directed to lysosomes for degradation by Rab11a [11].

GTP-Rab activates different effector molecules [12,13] such as Rab11-family interacting proteins (Rab11-FIPs) which bind to a conserved 20 amino acid C-terminal domain called RBD (Rab11-binding domain). FIPs have been subdivided into two classes: Class I FIPs are characterized by an N-terminal C2 domain involved in phospholipid binding; whereas Class II FIPs bare an additional calcium-binding EF-hand motif [14]. Within class I FIPS, Rab coupling protein (RCP) is a ~ 85 KDa protein effector for Rab11a. RCP localizes predominantly to the Endocytic Recycling Compartment (ERC) and regulates transport from this compartment to the plasma membrane. The C2 domain of RCP mediate targeting to the plasma membrane by binding to Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and phosphatidic acid [15]. In addition to N-terminal C2 domain and C-terminal RBD domain, RCPs have 3 functional PEST (Pro-Glu-Ser-Thr) motifs (182/249/336), which are believed to be a target for calcium-dependent calpain proteolysis [16]. Calpains are a family of calcium-activated cysteine proteases that catalyze the proteolysis of a number of cellular proteins in eukaryotes [17], and have been shown to regulate the internalization of membrane proteins through clathrin-coated vesicles [18].

The assembly/disassembly of the Rab11a/RCP complex is involved in the regulation of protein trafficking at the plasma membrane, and stimulates cell migration [19]. Interestingly, the inhibition of calpain promotes RCP and Rab11a accumulation at the plasma membrane [15,16,20], suggesting the requirement of calpain-driven proteolysis for the internalization of Rab/RCP.

We have previously shown that thrombin stimulation of PAR1 induces an increase in intracellular calcium concentration and the subsequent activation of calpain. Moreover, we showed that thrombin-induced calpain activation regulates PAR1 internalization through α-spectrin degradation, since calpain activation and α-spectrin degradation are not induced by the activation of PARs 2,3 or 4. These data suggest that the mechanism that regulates PAR1 internalization differs from other PARs [21]. In the present study we further analyzed the regulation of PAR1 internalization in RPE cells and demonstrated that this process depends on the disassembly of the Rab11a/RCP complex through a novel mechanism not described previously. These findings contribute to the elucidation of the processes underlying the development of proliferative eye pathologies such as PVR.

Section snippets

Reagents

All reagents used were cell culture grade. Thrombin (605157) and Calpain inhibitor III (208722) were purchased from Calbiochem/EMD Millipore (Billerica, MA, USA). PAR1 (SFLLRNPNDKYEPF-NH2), PAR2 (SLIGRL-NH2), PAR3 (SFNGGP-NH2) and PAR4 (GYPGKF-NH2) agonist peptides were from Bachem (Torrance, CA, USA). DMEM/F12 and Opti-MEM, Fetal bovine serum (FBS), Lipofectamine RNAi-MAX Transfection Reagent-Delivery of siRNA (13778–075), Lipofectamine LTX with Plus Reagent (15338–100), and Hoechst 33258

Thrombin promotes RCP degradation

RCP and Rab11a are involved in the trafficking of membrane proteins. RCP includes 3 functional PEST (Pro-Glu-Ser-Thr) motifs (182/249/336) which are calpain substrates [16]. We have shown that thrombin induces intracellular Ca2+ increase and the activation of calpain in ARPE19 cells [21]. Using western-blot anti-RCP, we here show that thrombin promotes the time-dependent degradation of RCP (Fig. 1 A). Results demonstrate that stimulation of ARPE19 cells by 2 U/mL thrombin for 12 h. decreases

Discussion

Thrombin effects are mediated by the activation of Protease-Activated Receptors (PARs), a family of G-protein-coupled receptors (GPCRs) activated by the proteolytic cleavage of the N-terminal sequence by thrombin. Given the catalytic nature of this process, the activation of PARs is irreversible, and differs from that of ligand-induced classical GPCR activation [9]. Consequently, the elucidation of the mechanisms involved in the termination of thrombin-induced PAR1 signaling and recycling is

Funding

This work was partially supported by Grants from Consejo Nacional de Ciencia y Tecnologia (CONACyT) (Grant 254333) and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT/UNAM) (Grant IN205317) to Ana María López-Colomé. Alejandro Alvarez-Arce is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received fellowship 573317 from CONACyT.

Acknowledgments

Authors acknowledge Rytis Prekeris, Department of Cell and Developmental Biology, School of Medicine, University of Colorado Anschutz Medical Campus, for the donation of CFP-Rab11a and RPC-YFP plasmids; Dr. Ruth Rincón Heredia and Dr. Abraham Rosas Arellano for technical assistance in FRET and confocal microscopy; Dr. Karla Méndez-Maldonado for support in plasmid amplification and purification.

References (42)

  • J.A. Trejo et al.

    The cytoplasmic tails of protease-activated receptor-1 and substance P receptor specify sorting to lysosomes versus recycling

    J. Biol. Chem.

    (1999)
  • J.A. Trejo et al.

    Protease-activated receptor-1 down-regulation. A mutant HeLa cell line suggests novel requirements for PAR1 phosphorylation and recruitment to clathrin-coated pits

    J. Biol. Chem.

    (2000)
  • B. Chen et al.

    Adaptor protein complex-2 (AP-2) and epsin-1 mediate protease-activated receptor-1 internalization via phosphorylation- and ubiquitination-dependent sorting signals

    J. Biol. Chem.

    (2011)
  • B. Chen et al.

    Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins

    J. Biol. Chem.

    (2014)
  • P. Lall et al.

    Structure-function analyses of the interactions between Rab11 and Rab14 small GTPases with their shared effector Rab coupling protein (RCP)

    J. Biol. Chem.

    (2015)
  • N.L. Grimsey et al.

    Cannabinoid receptor 2 undergoes Rab5-mediated internalization and recycles via a Rab11-dependent pathway

    Biochim Biophys Acta Mol Cell Res

    (2011)
  • S.E. Jacobsen et al.

    The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway

    J. Biol. Chem.

    (2017)
  • K. Mihara et al.

    Thrombin-Mediated Direct Activation of Proteinase-Activated Receptor-2 : Another Target for Thrombin Signaling

    (2016)
  • O. Strauss

    The retinal pigment epithelium in visual function

    Physiol. Rev.

    (2005)
  • L.J.A.G. Ricker et al.

    High subretinal fluid procoagulant activity in rhegmatogenous retinal detachment

    Investig. Ophthalmol. Vis. Sci.

    (2010)
  • J. Bastiaans et al.

    The role of thrombin in proliferative vitreoretinopathy

    Invest. Ophthalmol. Vis. Sci.

    (2014)
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