May M. Paing

Caveolae are required for protease-selective signaling by protease-activated receptor–1

Protease-activated receptor-1 (PAR1) is a G-protein–coupled receptor uniquely activated by proteolysis. Thrombin, a coagulant protease, induces inflammatory responses and endothelial barrier permeability through the activation of PAR1. Activated protein C (APC), an anti-coagulant protease, also activates PAR1. However, unlike thrombin, APC elicits anti-inflammatory responses and protects against endothelial barrier dysfunction induced by thrombin. We found that thrombin and APC signaling were lost in PAR1-deficient endothelial cells, indicating that PAR1 is the major effector of protease signaling. To delineate the mechanism responsible for protease-selective signaling by PAR1, we examined the effect of APC and thrombin on the activation of RhoA and Rac1, small GTPases that differentially regulate endothelial barrier permeability. Thrombin caused robust RhoA signaling but not Rac1 activation, whereas APC stimulated a marked increase in Rac1 activation but not RhoA signaling, consistent with the opposing functions of these proteases on endothelial barrier integrity. Strikingly, APC signaling and endothelial barrier protection effects were abolished in cells lacking caveolin-1, whereas thrombin signaling remained intact. These findings suggest that compartmentalization of PAR1 in caveolae is critical for APC selective signaling to Rac1 activation and endothelial barrier protection. We further report that APC induces PAR1 phosphorylation and desensitizes endothelial cells to thrombin signaling but promotes limited receptor cleavage and negligible internalization and degradation even after prolonged APC exposure. Thus, APC selective signaling and endothelial barrier protective effects are mediated through compartmentalization of PAR1 in caveolae and a novel mechanism of PAR1 signal regulation.

ALIX binds a YPX3L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/ MVB sorting

A novel MVB/lysosomal sorting pathway for signaling receptors bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be broadly applicable to GPCRs containing YPXnL motifs.

Clathrin Adaptor AP2 Regulates Thrombin Receptor Constitutive Internalization and Endothelial Cell Resensitization

ABSTRACT Protease-activated receptor 1 (PAR1), a G protein-coupled receptor for the coagulant protease thrombin, is irreversibly activated by proteolysis. Unactivated PAR1 cycles constitutively between the plasma membrane and intracellular stores, thereby providing a protected receptor pool that replenishes the cell surface after thrombin exposure and leads to rapid resensitization to thrombin signaling independent of de novo receptor synthesis. Here, we show that AP2, a clathrin adaptor, binds directly to a tyrosine-based motif in the cytoplasmic tail of PAR1 and is essential for constitutive receptor internalization and cellular recovery of thrombin signaling. Expression of a PAR1 tyrosine mutant or depletion of AP2 by RNA interference leads to significant inhibition of PAR1 constitutive internalization, loss of intracellular uncleaved PAR1, and failure of endothelial cells and other cell types to regain thrombin responsiveness. Our findings establish a novel role for AP2 in direct regulation of PAR1 trafficking, a process critically important to the temporal and spatial aspects of thrombin signaling.

A Tyrosine-based Sorting Signal Regulates Intracellular Trafficking of Protease-activated Receptor-1 MULTIPLE REGULATORY MECHANISMS FOR AGONIST-INDUCED G PROTEIN-COUPLED RECEPTOR INTERNALIZATION

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly proteolytically activated, internalized, and then sorted to lysosomes and degraded. Internalization and lysosomal sorting of activated PAR1 is critical for termination of receptor signaling. We previously demonstrated that activated PAR1 is rapidly phosphorylated and internalized via a clathrin- and dynamin-dependent pathway that is independent of arrestins. Toward understanding the mechanisms responsible for activated PAR1 internalization through clathrin-coated pits we examined the function of a highly conserved tyrosine-based motif, YXXL, localized in the cytoplasmic carboxyl tail of the receptor. A mutant PAR1 in which tyrosine 383 and leucine 386 were replaced with alanines (Y383A/L386A) was significantly impaired in agonist-triggered internalization and degradation compared with wild-type receptor. In contrast, constitutive internalization, and recycling of unactivated PAR1 Y383A/L386A mutant was not affected, suggesting that tonic cycling of the mutant receptor remained intact. Strikingly, a PAR1 C387Z truncation mutant in which the YXXL motif was exposed at the C terminus constitutively internalized and degraded in an agonist-independent manner, whereas C387Z truncation mutant in which the critical tyrosine and leucine were mutated to alanine (C387Z-Y383A/L386A) failed to internalize. Inhibition of PAR1 C387Z mutant constitutive internalization with dominant-negative K44A dynamin blocked agonist-independent degradation of the mutant receptor. Together these findings strongly suggest that internalization of activated PAR1 is controlled by multiple regulatory mechanisms involving phosphorylation and a highly conserved tyrosine-based motif, YXXL. This study is the first to describe a function for a tyrosine-based motif, YXXϕ, in GPCR internalization and reveal novel complexities in the regulation of GPCR trafficking.

Structural and Functional Basis for Inhibition of Erythrocyte Invasion by Antibodies that Target Plasmodium falciparum EBA-175.

Disrupting erythrocyte invasion by Plasmodium falciparum is an attractive approach to combat malaria. P. falciparum EBA-175 (PfEBA-175) engages the host receptor Glycophorin A (GpA) during invasion and is a leading vaccine candidate. Antibodies that recognize PfEBA-175 can prevent parasite growth, although not all antibodies are inhibitory. Here, using x-ray crystallography, small-angle x-ray scattering and functional studies, we report the structural basis and mechanism for inhibition by two PfEBA-175 antibodies. Structures of each antibody in complex with the PfEBA-175 receptor binding domain reveal that the most potent inhibitory antibody, R217, engages critical GpA binding residues and the proposed dimer interface of PfEBA-175. A second weakly inhibitory antibody, R218, binds to an asparagine-rich surface loop. We show that the epitopes identified by structural studies are critical for antibody binding. Together, the structural and mapping studies reveal distinct mechanisms of action, with R217 directly preventing receptor binding while R218 allows for receptor binding. Using a direct receptor binding assay we show R217 directly blocks GpA engagement while R218 does not. Our studies elaborate on the complex interaction between PfEBA-175 and GpA and highlight new approaches to targeting the molecular mechanism of P. falciparum invasion of erythrocytes. The results suggest studies aiming to improve the efficacy of blood-stage vaccines, either by selecting single or combining multiple parasite antigens, should assess the antibody response to defined inhibitory epitopes as well as the response to the whole protein antigen. Finally, this work demonstrates the importance of identifying inhibitory-epitopes and avoiding decoy-epitopes in antibody-based therapies, vaccines and diagnostics.

AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway

A GPCR ubiquitin-independent MVB/lysosomal sorting pathway is regulated by the adaptor protein complex-3 (AP-3) and ALIX, a noncanonical ESCRT component. AP-3 binds to a PAR1 C-tail–localized, tyrosine-based motif and mediates PAR1 lysosomal degradation. AP-3 also facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.

Multimeric assembly of host-pathogen adhesion complexes involved in apicomplexan invasion

Apicomplexan parasites are the causative agents of diseases that include malaria, toxoplasmosis, and coccidiosis. These obligate intracellular parasites have evolved to use a conserved mechanism for host-cell invasion. The apicomplexan phylum is defined by the presence of micronemes and rhoptries, which are distinct organelles located at the apical end of the parasite. These organelles secrete molecules necessary for host-cell invasion [1]. Apicomplexan parasites can invade disparate cell types, including hepatocytes, erythrocytes, lymphocytes, macrophages, and cells lining the digestive tract. Unlike viruses and intracellular bacteria, apicomplexans actively invade host cells without relying on host uptake pathways. As such, host-cell sensing and subsequent invasion are driven entirely by the parasite in a dynamic and rapid process. Intracellular residence protects the parasite from immune attack and enables parasite replication prior to host-cell lysis and subsequent invasion of neighboring host cells. The repertoire of ligand-receptor complexes utilized by parasites for entry into host cells is diverse. Some interactions occur through cell-specific receptors resulting in high-affinity interactions, while others occur through multiple lower-affinity interactions via surface moieties found on several cell types. Receptor-specific and general cell binding may explain host-cell tropism of different pathogens, although additional factors are important. There is growing evidence that multimeric assembly of parasite ligands and host surface molecules strengthens the host-pathogen interactions necessary for invasion. We discuss recent work that has advanced our knowledge of the assembly of adhesive complexes from two critical apicomplexan pathogens and highlight areas of research that require further investigation.

Critical Glycosylated Residues in Exon Three of Erythrocyte Glycophorin A Engage Plasmodium falciparum EBA-175 and Define Receptor Specificity

ABSTRACT Erythrocyte invasion is an essential step in the pathogenesis of malaria. The erythrocyte binding-like (EBL) family of Plasmodium falciparum proteins recognizes glycophorins (Gp) on erythrocytes and plays a critical role in attachment during invasion. However, the molecular basis for specific receptor recognition by each parasite ligand has remained elusive, as is the case with the ligand/receptor pair P. falciparum EBA-175 (PfEBA-175)/GpA. This is due largely to difficulties in producing properly glycosylated and functional receptors. Here, we developed an expression system to produce recombinant glycosylated and functional GpA, as well as mutations and truncations. We identified the essential binding region and determinants for PfEBA-175 engagement, demonstrated that these determinants are required for the inhibition of parasite growth, and identified the glycans important in mediating the PfEBA-175–GpA interaction. The results suggest that PfEBA-175 engages multiple glycans of GpA encoded by exon 3 and that the presentation of glycans is likely required for high-avidity binding. The absence of exon 3 in GpB and GpE due to a splice site mutation confers specific recognition of GpA by PfEBA-175. We speculate that GpB and GpE may have arisen due to selective pressure to lose the PfEBA-175 binding site in GpA. The expression system described here has wider application for examining other EBL members important in parasite invasion, as well as additional pathogens that recognize glycophorins. The ability to define critical binding determinants in receptor-ligand interactions, as well as a system to genetically manipulate glycosylated receptors, opens new avenues for the design of interventions that disrupt parasite invasion. IMPORTANCE Plasmodium falciparum uses distinct ligands that bind host cell receptors for invasion of red blood cells (RBCs) during malaria infection. A key entry pathway involves P. falciparum EBA-175 (PfEBA-175) recognizing glycophorin A (GpA) on RBCs. Despite knowledge of this protein-protein interaction, the complete mechanism for specific receptor engagement is not known. PfEBA-175 recognizes GpA but is unable to engage the related RBC receptor GpB or GpE. Understanding the necessary elements that enable PfEBA-175 to specifically recognize GpA is critical in developing specific and potent inhibitors of PfEBA-175 that disrupt host cell invasion and aid in malaria control. Here, we describe a novel system to produce and manipulate the host receptor GpA. Using this system, we probed the elements in GpA necessary for engagement and thus for host cell invasion. These studies have important implications for understanding how ligands and receptors interact and for the future development of malaria interventions. Plasmodium falciparum uses distinct ligands that bind host cell receptors for invasion of red blood cells (RBCs) during malaria infection. A key entry pathway involves P. falciparum EBA-175 (PfEBA-175) recognizing glycophorin A (GpA) on RBCs. Despite knowledge of this protein-protein interaction, the complete mechanism for specific receptor engagement is not known. PfEBA-175 recognizes GpA but is unable to engage the related RBC receptor GpB or GpE. Understanding the necessary elements that enable PfEBA-175 to specifically recognize GpA is critical in developing specific and potent inhibitors of PfEBA-175 that disrupt host cell invasion and aid in malaria control. Here, we describe a novel system to produce and manipulate the host receptor GpA. Using this system, we probed the elements in GpA necessary for engagement and thus for host cell invasion. These studies have important implications for understanding how ligands and receptors interact and for the future development of malaria interventions.