The heptahelical G protein-coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine-and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs.

The family of G protein-coupled receptors (GPCRs) constitutes the largest class of signalling receptors in the human genome, controlling vast physiological responses and are the target of many drugs. After activation, GPCRs are rapidly desensitized by phosphorylation and b-arrestin binding. Most classic GPCRs are internalized through a clathrin, dynamin and b-arrestindependent pathway and then recycled back to the cell surface or sorted to lysosomes for degradation. Given the vast number and diversity of GPCRs, different mechanisms are likely to exist to precisely regulate the magnitude, duration and spatial aspects of receptor signalling. The G protein-coupled protease-activated receptors (PARs) provide elegant examples of GPCRs that are regulated by distinct desensitization and endocytic sorting mechanisms, processes that are critically important for the spatial and temporal fidelity of PAR signalling. PARs are irreversibly activated through proteolytic cleavage and transmit cellular responses to extracellular proteases. Activated PAR1 internalizes through a clathrin-and dynamin-dependent pathway independent of b-arrestins. Interestingly, PAR1 is basally ubiquitinated and deubiquitinated after activation and traffics from endosomes to lysosomes independent of ubiquitination. In contrast, b-arrestins mediate activated PAR2 internalization and function as scaffolds that promote signalling from endocytic vesicles. Moreover, activated PAR2 is modified with ubiquitin, which facilitates lysosomal degradation. Activated PARs also adopt distinct active conformations that signal to diverse effectors and are likely regulated by different mechanisms. Thus, the identification of the molecular machinery important for PAR signal regulation will enable the development of new strategies to manipulate receptor signalling and will provide novel targets for the development of drugs.

Coagulation is a host defense system that limits the spread of pathogens. Coagulation proteases, such as thrombin, also activate cells by cleaving PARs. In this study, we analyzed the role of PAR-1 in coxsackievirus B3-induced (CVB3-induced) myocarditis and influenza A infection. CVB3-infected Par1 -/-mice expressed reduced levels of IFN-β and CXCL10 during the early phase of infection compared with Par1 +/+ mice that resulted in higher viral loads and cardiac injury at day 8 after infection. Inhibition of either tissue factor or thrombin in WT mice also significantly increased CVB3 levels in the heart and cardiac injury compared with controls. BM transplantation experiments demonstrated that PAR-1 in nonhematopoietic cells protected mice from CVB3 infection. Transgenic mice overexpressing PAR-1 in cardiomyocytes had reduced CVB3-induced myocarditis. We found that cooperative signaling between PAR-1 and TLR3 in mouse cardiac fibroblasts enhanced activation of p38 and induction of IFN-β and CXCL10 expression. Par1 -/-mice also had decreased CXCL10 expression and increased viral levels in the lung after influenza A infection compared with Par1 +/+ mice. Our results indicate that the tissue factor/thrombin/PAR-1 pathway enhances IFN-β expression and contributes to the innate immune response during single-stranded RNA viral infection.

The widely expressed ␤-arrestin isoforms 1 and 2 bind phosphorylated G protein-coupled receptors (GPCRs) and mediate desensitization and internalization. Phosphorylation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, is important for desensitization and internalization, however, the role of ␤-arrestins in signaling and trafficking of PAR1 remains unknown. To assess ␤-arrestin function we examined signaling and trafficking of PAR1 in mouse embryonic fibroblasts (MEFs) derived from ␤-arrestin (␤arr) knockouts. Desensitization of PAR1 signaling was markedly impaired in MEFs lacking both ␤arr1 and ␤arr2 isoforms compared with wild-type cells. Strikingly, in cells lacking only ␤arr1 PAR1 desensitization was also significantly impaired compared with ␤arr2-lacking or wild-type cells. In wild-type MEFs, activated PAR1 was internalized through a dynamin-and clathrin-dependent pathway and degraded. Surprisingly, in cells lacking both ␤arr1 and ␤arr2 activated PAR1 was similarly internalized through a dynamin-and clathrin-dependent pathway and degraded, whereas the ␤ 2 -adrenergic receptor (␤ 2 -AR) failed to internalize. A PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation failed to internalize in both wild-type and ␤-arrestin knockout cells. Thus, PAR1 appears to utilize a distinct phosphorylation-dependent but ␤-arrestin-independent pathway for internalization through clathrin-coated pits. Together, these findings strongly suggest that the individual ␤-arrestin isoforms can differentially regulate GPCR desensitization and further reveal a novel mechanism by which GPCRs can internalize through a dynamin-and clathrin-dependent pathway that is independent of arrestins.

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.

The chemokine receptor CXCR4 is rapidly targeted for lysosomal degradation by the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4). Although it is known that AIP4 mediates ubiquitination and degradation of CXCR4 and that perturbations in these events contribute to disease, the mechanisms mediating AIP4-dependent regulation of CXCR4 degradation remain poorly understood. Here we show that AIP4 directly interacts with the amino-terminal half of nonvisual arrestin-2 via its WW domains. We show that depletion of arrestin-2 by small interfering RNA blocks agonist-promoted degradation of CXCR4 by preventing CXCR4 trafficking from early endosomes to lysosomes. Surprisingly, CXCR4 internalization and ubiquitination remain intact, suggesting that the interaction between arrestin-2 and AIP4 is not required for ubiquitination of the receptor at the plasma membrane but perhaps for a later post-internalization event. Accordingly, we show that activation of CXCR4 promotes the interaction between AIP4 and arrestin-2 that is consistent with a time when AIP4 co-localizes with arrestin-2 on endocytic vesicles. Taken together, our data suggest that the AIP4⅐arrestin-2 complex functions on endosomes to regulate sorting of CXCR4 into the degradative pathway.The chemokine receptor CXCR4, a G protein-coupled receptor (GPCR), 3 together with its cognate ligand, stromal cell-derived factor-1␣, also termed CXCL12, play an important role in several biological processes such as development of the heart and brain, leukocyte chemotaxis, and stem cell homing (1-3). Although CXCR4 dysregulation has been linked to several pathologies, especially cancer, the molecular mechanisms regulating CXCR4 remain poorly understood (4, 5). Activated CXCR4 is targeted for lysosomal degradation through a pathway involving ubiquitination of carboxyl-terminal tail lysine residues mediated by the E3 ubiquitin ligase atrophininteracting protein 4 (AIP4) (6, 7). AIP4 belongs to the neural precursor cell-expressed developmentally down-regulated gene 4-like family of homologous to E6-AP carboxyl-terminal domain E3 ubiquitin ligases, which interact with their target proteins either directly or indirectly via their WW domains or possibly other domains (8, 9).In addition, AIP4 regulates endosomal sorting of activated CXCR4 by targeting the receptor to the endosomal sorting complex required for transport pathway (7), which is a complex network of proteins that recognize and sort ubiquitinated cargo into the multivesicular body (10). Entry into this pathway requires the action of HRS, a ubiquitin-binding protein localized to flat clathrin lattices on endosomes adjacent to invaginating domains, where it sequesters ubiquitinated cargo destined for entry into the multivesicular body for subsequent degradation (11, 12). CXCR4 and AIP4 localize to HRS-positive microdomains on endosomes (7). In addition, AIP4 mediates CXCR4-dependent ubiquitination of HRS, an action that likely plays a role in the sorting function of HRS (7). Whether additional proteins play a role in ...

These results demonstrate that hPAK1 is a GTPase effector controlling a downstream MAP kinase pathway in mammalian cells, as Ste20 does in yeast. Thus, PAK and Ste20 kinases play key parts in linking extracellular signals from membrane components, such as receptor-associated G proteins and Rho-related GTPases, to nuclear responses, such as transcriptional activation.

The heptahelical G protein‐coupled receptor (GPCR) family includes ∼900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein‐coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane and some internalized receptors continue to signal from intracellular compartments. In addition to rapid desensitization, receptor trafficking is critical for regulation of the temporal and spatial aspects of GPCR signaling. Indeed, GPCR internalization functions to control signal termination and propagation as well as receptor resensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin‐ and dynamin‐dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin‐mediated endocytosis of diverse cargo. Given the vast number and diversity of GPCRs, the complexity of clathrin‐mediated endocytosis and the discovery of multiple clathrin adaptors, a single universal mechanism controlling endocytosis of all mammalian GPCRs is unlikely. Indeed, several recent studies now suggest that endocytosis of different GPCRs is regulated by distinct mechanisms and clathrin adaptors. In this review, we discuss the diverse mechanisms that regulate clathrin‐dependent GPCR endocytosis.