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.

Protease-activated receptors (PAR) are G protein-coupled receptors that function as cell-surface sensors for coagulant proteases, as well as other proteases associated with the tumor microenvironment. PAR1 is activated by thrombin whereas the upstream coagulant protease VIIa bound to tissue factor and Xa can activate both PAR1 and PAR2. PAR1 has been implicated in tumor cell growth, migration, and invasion whereas the function of PAR2 in these processes is largely unknown. Towards defining the functional importance of PAR2 in cancer cells, we used small interfering RNAs to deplete highly invasive breast cancer cells of endogenous PAR proteins. Our findings strongly suggest that PAR2 is critical for MDA-MB-231 and BT549 breast cancer cell migration and invasion towards NIH 3T3 fibroblast conditioned medium. To define the relative importance of PAR1 versus PAR2 in mediating factor VIIa and Xa responses, we assessed signaling in cancer cells lacking either endogenous PAR1 or PAR2 proteins. Strikingly, in MDA-MB-231 cells depleted of PAR2, we observed a marked inhibition of VIIa and Xa signaling to phosphoinositide hydrolysis and extracellular signalregulated kinase 1/2 activation whereas signaling by VIIa and Xa remained intact in PAR1-deficient cells. Factor VIIa and Xa-induced cellular migration was also impaired in MDA-MB-231 cells deficient in PAR2 but not in cells lacking PAR1. Together, these studies reveal the novel findings that PAR2, a second protease-activated G protein-coupled receptor, has a critical role in breast cancer cell migration and invasion and functions as the endogenous receptor for coagulant proteases

Protease-activated receptor-1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is irreversibly activated by proteolysis. Consequently, PAR1 trafficking is critical for the fidelity of thrombin signaling. PAR1 displays constitutive and agonist-induced internalization, which are clathrin and dynamin dependent but are independent of arrestins. The clathrin adaptor AP2 (adaptor protein complex-2) is critical for constitutive but not for activated PAR1 internalization. In this study, we show that ubiquitination negatively regulates PAR1 constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 is basally ubiquitinated and deubiquitinated after activation. A PAR1 lysineless mutant signaled normally but was not ubiquitinated. Constitutive internalization of ubiquitin (Ub)-deficient PAR1 was markedly increased and inhibited by the fusion of Ub to the cytoplasmic tail. Ub-deficient PAR1 constitutive internalization was AP2 dependent like the wild-type receptor. However, unlike wild-type PAR1, AP2 was required for the internalization of activated Ub-deficient receptor, suggesting that the internalization of ubiquitinated PAR1 requires different endocytic machinery. These studies reveal a novel function for ubiquitination in the regulation of GPCR internalization.

Sorting nexin 1 (SNX1) and SNX2 are the mammalian homologues of the yeast Vps5p retromer component that functions in endosome-to-Golgi trafficking. SNX1 is also implicated in endosome-to-lysosome sorting of cell surface receptors, although its requirement in this process remains to be determined. To assess SNX1 function in endocytic sorting of protease-activated receptor-1 (PAR1), we used siRNA to deplete HeLa cells of endogenous SNX1 protein. PAR1, a G-protein-coupled receptor, is proteolytically activated by thrombin, internalized, sorted predominantly to lysosomes, and efficiently degraded. Strikingly, depletion of endogenous SNX1 by siRNA markedly inhibited agonist-induced PAR1 degradation, whereas expression of a SNX1 siRNA-resistant mutant protein restored agonist-promoted PAR1 degradation in cells lacking endogenous SNX1, indicating that SNX1 is necessary for lysosomal degradation of PAR1. SNX1 is known to interact with components of the mammalian retromer complex and Hrs, an early endosomal membrane-associated protein. However, activated PAR1 degradation was not affected in cells depleted of retromer Vps26/Vps35 subunits, Hrs or Tsg101, an Hrs-interacting protein. We further show that SNX2, which dimerizes with SNX1, is not essential for lysosomal sorting of PAR1, but rather can regulate PAR1 degradation by disrupting endosomal localization of endogenous SNX1 when ectopically expressed. Together, our findings establish an essential role for endogenous SNX1 in sorting activated PAR1 to a distinct lysosomal degradative pathway that is independent of retromer, Hrs, and Tsg101.

The irreversible proteolytic nature of protease-activated receptor-2 (PAR2) activation suggests that mechanism(s) responsible for termination of receptor signaling are critical determinants of the magnitude and duration of PAR2-elicited cellular responses. Rapid desensitization of activated G-protein-coupled receptors (GPCRs) involves both phosphorylation and binding of arrestins. Arrestins also function as scaffolds and transducers of mitogen-activated protein (MAP) kinase signaling cascades. The PAR2 cytoplasmic tail (C-tail) contains multiple sites of phosphorylation and may be an important determinant for arrestin interaction. Desensitization and internalization of activated PAR2 were markedly impaired in arrestin-deficient cells compared with wild-type control cells. PAR2 C-tail truncation mutants displayed normal agonist-induced internalization, caused rapid distribution of ␤arr2-GFP to the plasma membrane, and desensitized in an arrestin-dependent manner similar to that of wild-type PAR2. It is interesting that PAR2 C-tail mutants lost the capacity to stably associate with arrestins and consequently, redistributed to endocytic vesicles without ␤arr2-GFP, whereas internalized wild-type PAR2 remained stably associated with ␤arr2-GFP in endosomes. Moreover, activated PAR2 caused rapid and prolonged activation of endogenous extracellular signal-regulated kinase (ERK1/2). It was striking that in arrestin-deficient cells, activated PAR2 induced an initial peak in ERK1/2 activity that rapidly declined. The inability of internalized PAR2 C-tail mutants to stably associate with arrestins also resulted in loss of prolonged ERK2 activation. Thus, the PAR2 C-tail regulates the stability of arrestin interaction and kinetics of ERK1/2 activation but is not essential for desensitization or internalization. These findings further suggest that the diverse functions of arrestins in regulating PAR2 signaling and trafficking are controlled by multiple independent interactions involving both the intracellular loops and the C-tail.Protease-activated receptor-2 (PAR2) is activated by multiple trypsin-like serine proteases, including trypsin, tryptase, and coagulation proteases factors VIIa and Xa, but not by thrombin (Coughlin and Camerer, 2003). Because of the irreversible proteolytic nature of PAR2 activation and the generation of a tethered ligand that cannot diffuse away, mechanisms that contribute to the termination of signaling are critical determinants of the magnitude and kinetics of protease-elicited cellular responses. The regulation of PAR1 signaling has been extensively studied (Trejo, 2003), whereas considerably less is known about PAR2. G-protein-coupled receptors (GPCRs) are initially desensitized by rapid phosphorylation of agonist-occupied receptors by GPCR serine/ threonine kinases (GRKs) and other kinases (Krupnick and Benovic, 1998). In many cases, phosphorylation enhances receptor affinity for arrestin, and arrestin binding prevents receptor-G-protein interaction, thereby uncoupling the receptor fro...