Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis

The heptahelical G protein-coupled receptor (GPCR) family includes approximately 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.     

G protein-coupled receptor endocytosis in ADP-ribosylation factor 6-depleted cells

The internalization of G protein-coupled receptors is regulated by several important proteins that act in concert to finely control this complex cellular process. Here, we have applied the RNA interference approach to demonstrate that ADP-ribosylation factor 6 (ARF6) is essential for the endocytosis of a broad variety of receptors. Reduction of endogenous expression of ARF6 in HEK 293 cells resulted in a correlated inhibition of the beta(2) -adrenergic receptor internalization previously characterized as being sequestered via the clathrin-coated vesicle pathway. Furthermore, other receptors internalizing via this endocytic route, namely the angiotensin type 1 receptor and the vasopressin type 2 receptor, were also impaired in their ability to be sequestered when levels of endogenous ARF6 in cells were reduced. Interestingly, endocytosis of the endothelin type B receptor, characterized as being internalized via the caveolae pathway, was also markedly inhibited in ARF6-depleted cells. In contrast, internalization of the vasoactive intestinal peptide receptor was unaffected by reduced levels of ARF6. Finally, internalization of the acetylcholine-muscarinic type 2 receptor via the non-clathrin-coated vesicle pathway was also inhibited in ARF6-depleted cells. Taken together, our results demonstrate that ARF6 proteins play an essential role in the internalization process of most G protein-coupled receptors regardless of the endocytic route being utilized. However, this phenomenon is not general. In some cases, another ARF isoform or other proteins may be essential to regulate the endocytic process.     

Arrestin binding to the G protein-coupled N-formyl peptide receptor is regulated by the conserved "DRY" sequence

Following activation by ligand, the N-formyl peptide receptor (FPR) undergoes processing events initiated by phosphorylation that lead to receptor desensitization and internalization. Our previous results have shown that FPR internalization can occur in the absence of receptor desensitization, suggesting that FPR desensitization and internalization are controlled by distinct mechanisms. More recently, we have provided evidence that internalization of the FPR occurs via a mechanism that is independent of the actions of arrestin, dynamin, and clathrin. In the present report, we demonstrate that stimulation of the FPR with agonist leads to a significant translocation of arrestin-2 from the cytosol to the membrane. Fluorescence microscopy revealed that the translocated arrestin-2 is highly colocalized with the ligand-bound FPR. A D71A mutant FPR, which does not undergo activation or phosphorylation in response to ligand, did not colocalize with arrestin-2. Surprisingly, an R123G mutant FPR, which does not bind G protein but does become phosphorylated and subsequently internalized, also did not bind arrestin. These results indicate that arrestin binding is not required for FPR internalization and demonstrate for the first time that a common motif, the conserved "DRY" domain of G protein-coupled receptors, is essential for phosphorylation-dependent arrestin binding, as well as G protein activation.     

Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases

G protein-coupled receptors (GPCRs) are integral membrane proteins that, in response to activation by extracellular stimuli, regulate intracellular second messenger levels via their coupling to heterotrimeric G proteins. GPCR activation also initiates a series of molecular events that leads to G protein-coupled receptor kinase-mediated receptor phosphorylation and the binding of beta-arrestin proteins to the intracellular face of the receptor. beta-Arrestin binding not only contributes to the G protein-uncoupling of GPCRs, but also mediates the targeting of many GPCRs for endocytosis in clathrin-coated pits. Several GPCRs internalize as a stable complex with beta-arrestin and the stability of this complex appears to regulate, at least in part, whether the receptors are dephosphorylated in early endosomes and recycled back to the cell surface as fully functional receptors, retained in early endosomes or targeted for degradation in lysosomes. More recently, it has become appreciated that the movement of GPCRs through functionally distinct intracellular membrane compartments is regulated by a variety of Rab GTPases and that the activity of these Rab GTPases may influence GPCR function. Moreover, it appears that GPCRs are not simply passive cargo molecules, but that GPCR activation may directly influence Rab GTPase activity and as such, GPCRs may directly control their own targeting between intracellular compartments. This review provides a synopsis of the current knowledge regarding the role of beta-arrestins and Rab GTPases in regulating the intracellular trafficking and function of GPCRs.     

Regulation of G protein-coupled receptor endocytosis by ARF6 GTP-binding proteins.

The function of G protein-coupled receptors is regulated by a broad variety of membrane-bound and intracellular proteins. These act in concert to activate signaling pathways that will lead to the desensitization of activated receptors and, for most receptor types, their trafficking to intracellular compartments. This review focuses mainly on the endocytic pathways used by a G protein-coupled receptor and on the proteins that play an essential role in the regulation of the internalization process, most specifically the ADP-ribosylation factors. This family of proteins has been shown to be important for vesicle trafficking between different cellular membranes. The latest findings regarding the molecular mechanisms that regulate internalization of an agonist-stimulated receptor are presented here. Finally, a perspective on how ARF6 proteins might regulate the internalization process is also proposed.     

Sustained diacylglycerol accumulation resulting from prolonged G protein-coupled receptor agonist-induced phosphoinositide breakdown in hepatocytes

Studies in various cells have led to the idea that agonist-stimulated diacylglycerol (DAG) generation results from an early, transient phospholipase C (PLC)-catalyzed phosphoinositide breakdown, while a more sustained elevation of DAG originates from phosphatidylcholine (PC). We have examined this issue further, using cultured rat hepatocytes, and report here that various G protein-coupled receptor (GPCR) agonists, including vasopressin (VP), angiotensin II (Ang.II), prostaglandin F2alpha, and norepinephrine (NE), may give rise to a prolonged phosphoinositide hydrolysis. Preincubation of hepatocytes with 1-butanol to prevent conversion of phosphatidic acid (PA) did not affect the agonist-induced DAG accumulation, suggesting that phospholipase D-mediated breakdown of PC was not involved. In contrast, the GPCR agonists induced phosphoinositide turnover, assessed by accumulation of inositol phosphates, that was sustained for up to 18 h, even under conditions where PLC was partially desensitized. Pretreatment of hepatocytes with wortmannin, to inhibit synthesis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP2), prevented agonist-induced inositol phosphate and DAG accumulation. Upon VP stimulation the level of PIP) declined, but only transiently, while increases in inositol 1,4,5-trisphosphate (InsP3) and DAG mass were sustained, suggesting that efficient resynthesis of PIP2 allowed sustained PLC activity. This was confirmed when cells were pretreated with wortmannin to prevent resynthesis of PIP2. Furthermore, metabolism of InsP3 was rapid, compared to that of DAG, with a more than 20-fold difference in half-life. Thus, rapid metabolism of InsP3 and efficient resynthesis of PIP2 may account for the larger amount of DAG generated and the more sustained time course, compared to InsP3. The results suggest that DAG accumulation that is sustained for many hours in response to VP, Ang.II, NE, and prostaglandin F2alpha in hepatocytes is mainly due to phosphoinositide breakdown.     

Arrestin specificity for G protein-coupled receptors in human airway smooth muscle.

Despite a widely accepted role of arrestins as "uncouplers" of G protein-coupled receptor (GPCR) signaling, few studies have demonstrated the ability of arrestins to affect second messenger generation by endogenously expressed receptors in intact cells. In this study we demonstrate arrestin specificity for endogenous GPCRs in primary cultures of human airway smooth muscle (HASM). Expression of arrestin-green fluorescent protein (ARR2-GFP or ARR3-GFP) chimeras in HASM significantly attenuated isoproterenol (beta(2)-adrenergic receptor (beta(2)AR)-mediated)- and 5'-(N-ethylcarboxamido)adenosine (A2b adenosine receptor-mediated)-stimulated cAMP production, with fluorescent microscopy demonstrating agonist-promoted redistribution of cellular ARR2-GFP into a punctate formation. Conversely, prostaglandin E(2) (PGE(2))-mediated cAMP production was unaffected by arrestin-GFP, and PGE(2) had little effect on arrestin-GFP distribution. The pharmacological profile of various selective EP receptor ligands suggested a predominantly EP2 receptor population in HASM. Further analysis in COS-1 cells revealed that ARR2-GFP expression increased agonist-promoted internalization of wild type beta(2)AR and EP4 receptors, whereas EP2 receptors remained resistant to internalization. However, expression of an arrestin whose binding to GPCRs is largely independent of receptor phosphorylation (ARR2(R169E)-GFP) enabled substantial agonist-promoted EP2 receptor internalization, increased beta(2)AR internalization to a greater extent than did ARR2-GFP, yet promoted EP4 receptor internalization to the same degree as did ARR2-GFP. Signaling via endogenous EP4 receptors in CHO-K1 cells was attenuated by ARR2-GFP expression, whereas ARR2(R169E)-GFP expression in HASM inhibited EP2 receptor-mediated cAMP production. These findings demonstrate differential effects of arrestins in altering endogenous GPCR signaling in a physiologically relevant cell type and reveal a variable dependence on receptor phosphorylation in dictating arrestin-receptor interaction.     

Heterologous inhibition of G protein-coupled receptor endocytosis mediated by receptor-specific trafficking of beta-arrestins

We have observed an unexpected type of nonreciprocal "cross-regulation" of the agonist-induced endocytosis of G protein-coupled receptors by clathrin-coated pits. Isoproterenol-dependent internalization of beta2-adrenergic receptors in stably transfected HEK293 cells was specifically blocked (>65% inhibition) by vasopressin-induced activation of V2 vasopressin receptors co-expressed at similar levels. In contrast, activation of beta2 receptors caused no detectable effect on V2 receptor internalization in the same cells. Several pieces of evidence suggest that this nonreciprocal inhibition of endocytosis is mediated by receptor-specific intracellular trafficking of beta-arrestins. First, previous studies showed that the activation of V2 but not beta2 receptors caused pronounced recruitment of beta-arrestins to endocytic membranes (Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron, M. G. (1999) J. Biol. Chem. 274, 32248-32257). Second, overexpression of arrestin 2 or 3 (beta-arrestin 1 or 2) abolished the V2 receptor-mediated inhibition of beta2 receptor internalization. Third, mutations of the V2 receptor that block endomembrane recruitment of beta-arrestins eliminated the V2 receptor-dependent blockade of beta2 receptor internalization. These results identify a novel type of heterologous regulation of G protein-coupled receptors, define a new functional role of receptor-specific intracellular trafficking of beta-arrestins, and suggest an experimental method to rapidly modulate the functional activity of beta-arrestins in intact cells.     

Type-specific sorting of G protein-coupled receptors after endocytosis

The beta(2)-adrenergic receptor (B2AR) and delta-opioid receptor (DOR) are structurally distinct G protein-coupled receptors (GPCRs) that undergo rapid, agonist-induced internalization by clathrin-coated pits. We have observed that these receptors differ substantially in their membrane trafficking after endocytosis. B2AR expressed in stably transfected HEK293 cells exhibits negligible (<10%) down-regulation after continuous incubation of cells with agonist for 3 h, as assessed both by radioligand binding (to detect functional receptors) and immunoblotting (to detect total receptor protein). In contrast, DOR exhibits substantial (>/=50%) agonist-induced down-regulation when examined by similar means. Degradation of internalized DOR is sensitive to inhibitors of lysosomal proteolysis. Flow cytometric and surface biotinylation assays indicate that differential sorting of B2AR and DOR between distinct recycling and non-recycling pathways (respectively) can be detected within approximately 10 min after endocytosis, significantly before the onset of detectable proteolytic degradation of receptors ( approximately 60 min after endocytosis). Studies using pulsatile application of agonist suggest that after this sorting event occurs, later steps of membrane transport leading to lysosomal degradation of receptors do not require the continued presence of agonist in the culture medium. These observations establish that distinct GPCRs differ significantly in endocytic membrane trafficking after internalization by the same membrane mechanism, and they suggest a mechanism by which brief application of agonist can induce substantial down-regulation of receptors.     

Signaling through Disabled 1 requires phosphoinositide binding

The Reelin signaling pathway plays a critical role in the correct positioning of neurons within the developing brain. Within this pathway, Disabled 1 (Dab1) serves as an intracellular adaptor that is tyrosine phosphorylated when Reelin, a secreted glycoprotein, binds to the lipoprotein receptors VLDLR and ApoER2 on the surface of neurons. The phosphotyrosine-binding (PTB) domain within its amino terminus enables Dab1 to recognize and bind to a conserved sequence motif within the cytoplasmic tails of the receptors. In addition, the PTB contains a Pleckstrin Homology-like subdomain that binds to phosphoinositides. Here, we show that the phosphoinositide-binding region within Dab1 PTB domain is required for membrane localization and basal tyrosine phosphorylation of Dab1 independently of VLDLR and ApoER2. Furthermore, receptor-independent membrane targeting of Dab1 is required for its interaction with Src and Crk, and disruption of phosphoinositide binding also blocks subsequent Reelin-induced tyrosine phosphorylation of Dab1.     

Probing the binding pocket and endocytosis of a G protein-coupled receptor in live cells reported by a spin-labeled substance P agonist

To probe the molecular nature of the binding pocket of a G protein-coupled receptor and the events immediately following the binding and activation, we have modified the substance P peptide, a potent agonist for the neurokinin-1 receptor, with a nitroxide spin probe specifically attached at Lys-3. The agonist properties and binding affinity of the spin-labeled substance P are similar to the native peptide. Using electron paramagnetic resonance (EPR) spectroscopy, the substance P analogue is capable of reporting the microenvironment found in the binding pocket of the receptor. The EPR spectrum of bound peptide indicates that the Lys-3 portion of the agonist is highly flexible. In addition, we detect a slight increase in the mobility of the bound peptide in the presence of a non-hydrolyzable analogue of GTP, indicative of the alternate conformational states described for this class of receptor. The down-regulation of neurokinin-tachykinin receptors is accomplished by a rapid internalization of the activated protein. Thus, it was also of interest to establish whether spin-labeled substance P could serve as a real time reporter for endocytosis. Our findings show the receptor agonist is efficiently endocytosed and the loss of EPR signal upon internalization provides a real time monitor of endocytosis. The rapid loss of signal suggests that endosomal trafficking vesicles maintain a reductive environment. Whereas the reductive capacity of the lysosome has been established, our findings indicate this capacity in early endosomes as well.     

The ubiquitin-like protein PLIC-2 is a negative regulator of G protein-coupled receptor endocytosis

The activity of many signaling receptors is regulated by their endocytosis via clathrin-coated pits (CCPs). For G protein-coupled receptors (GPCRs), recruitment of the adaptor protein arrestin to activated receptors is thought to be sufficient to drive GPCR clustering in CCPs and subsequent endocytosis. We have identified an unprecedented role for the ubiquitin-like protein PLIC-2 as a negative regulator of GPCR endocytosis. Protein Linking IAP to Cytoskeleton (PLIC)-2 overexpression delayed ligand-induced endocytosis of two GPCRs: the V2 vasopressin receptor and β-2 adrenergic receptor, without affecting endocytosis of the transferrin or epidermal growth factor receptor. The closely related isoform PLIC-1 did not affect receptor endocytosis. PLIC-2 specifically inhibited GPCR concentration in CCPs, without affecting membrane recruitment of arrestin-3 to activated receptors or its cellular levels. Depletion of cellular PLIC-2 accelerated GPCR endocytosis, confirming its regulatory function at endogenous levels. The ubiquitin-like domain of PLIC-2, a ligand for ubiquitin-interacting motifs (UIMs), was required for endocytic inhibition. Interestingly, the UIM-containing endocytic adaptors epidermal growth factor receptor protein substrate 15 and Epsin exhibited preferential binding to PLIC-2 over PLIC-1. This differential interaction may underlie PLIC-2 specific effect on GPCR endocytosis. Identification of a negative regulator of GPCR clustering reveals a new function of ubiquitin-like proteins and highlights a cellular requirement for exquisite regulation of receptor dynamics.     

植物G蛋白偶联受体及其在信号转导中的作用

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质超家族.GPCRs能调控细胞周期,参与多种植物信号通路以及影响一系列的代谢和分化活动.简要介绍了GPCR和G蛋白介导的信号转导机制,GPCRs的结构和植物GPCR及其在植物跨膜信号转导中的作用,并对GPCR的信号转导机制及植物抗病反应分子机制的研究提出展望.     

Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways

Agonist-induced phosphorylation of beta-adrenergic receptors (beta ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of beta ARs, particularly the beta 1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type beta 1AR (WT beta 1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA-beta 1AR), the putative GRK phosphorylation sites (GRK-beta 1AR), and both sets of phosphorylation sites (PKA-/GRK-beta 1AR). Following agonist stimulation, both PKA-beta 1AR and GRK-beta 1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WT beta 1AR and PKA-beta 1AR, sequestration of the GRK-beta 1AR and PKA-/GRK-beta 1AR was independent of beta-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WT beta 1AR and PKA-beta 1AR, whereas caveolae inhibitors prevented internalization only of the GRK-beta 1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the beta 1AR and 2) the pathway selected for beta 1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits.     

G protein-coupled receptor dimerisation: Molecular basis and relevance to function

The belief that G protein-coupled receptors exist and function as monomeric, non-interacting species has been largely supplanted in recent years by evidence, derived from a range of approaches, that indicate they can form dimers and/or higher-order oligomeric complexes. Key roles for receptor homo-dimerisation include effective quality control of protein folding prior to plasma membrane delivery and interactions with hetero-trimeric G proteins. Growing evidence has also indicated the potential for many co-expressed G protein-coupled receptors to form hetero-dimers/oligomers. The relevance of this to physiology and function is only beginning to be unravelled but may offer great potential for more selective therapeutic intervention.     

G protein-coupled receptor dimerisation: molecular basis and relevance to function

The belief that G protein-coupled receptors exist and function as monomeric, non-interacting species has been largely supplanted in recent years by evidence, derived from a range of approaches, that indicate they can form dimers and/or higher-order oligomeric complexes. Key roles for receptor homo-dimerisation include effective quality control of protein folding prior to plasma membrane delivery and interactions with hetero-trimeric G proteins. Growing evidence has also indicated the potential for many co-expressed G protein-coupled receptors to form hetero-dimers/oligomers. The relevance of this to physiology and function is only beginning to be unravelled but may offer great potential for more selective therapeutic intervention.     

G protein coupled receptor endocytosis

Most G protein-coupled receptors (GPCR) are rapidly internalized upon agonist stimulation. From the large number of studies available to date on receptor endocytosis, substantial differences seem to exist among GPCRs in terms of both the molecular pathways of receptor internalization and the biological significance of this process. The aim of this review is to outline common themes in GPCR endocytosis and to delineate true phenotypic variations, which reflect specific necessities for receptor's function.