Endosomal trafficking of the G protein-coupled receptor somatostatin receptor 3

Intracellular trafficking of G protein-coupled receptors (GPCRs) regulates their surface availability and determines cellular response to agonists. Rab GTPases regulate membrane trafficking and identifying Rab networks controlling GPCR trafficking is essential for understanding GPCR signaling. We used real time imaging to show that somatostatin receptor 3 (SSTR3) traffics through Rab4-, Rab21-, and Rab11-containing endosomes, but largely bypasses Rab5 and Rab7 endosomes. We show that SSTR3 rapidly traffics through Rab4 endosomes but moves slower through Rab21 and Rab11 endosomes. SSTR3 passage through each endosomal compartment is regulated by the cognate Rab since expression of the inactive Rab4/S22N, Rab21/T33N, and Rab11/S25N inhibits SSTR3 trafficking. Thus, Rab4, Rab21, and Rab11 may represent therapeutic targets to modulate surface availability of SSTR3 for agonist binding. Our novel finding that Rab21 regulates SSTR3 trafficking suggests that Rab21 may play a role in trafficking of other GPCRs.     

Cellular trafficking of G protein-coupled receptor/beta-arrestin endocytic complexes

beta-Arrestins are multifunctional proteins identified on the basis of their ability to bind and uncouple G protein-coupled receptors (GPCR) from heterotrimeric G proteins. In addition, beta-arrestins play a central role in mediating GPCR endocytosis, a key regulatory step in receptor resensitization. In this study, we visualize the intracellular trafficking of beta-arrestin2 in response to activation of several distinct GPCRs including the beta2-adrenergic receptor (beta2AR), angiotensin II type 1A receptor (AT1AR), dopamine D1A receptor (D1AR), endothelin type A receptor (ETAR), and neurotensin receptor (NTR). Our results reveal that in response to beta2AR activation, beta-arrestin2 translocation to the plasma membrane shares the same pharmacological profile as described for receptor activation and sequestration, consistent with a role for beta-arrestin as the agonist-driven switch initiating receptor endocytosis. Whereas redistributed beta-arrestins are confined to the periphery of cells and do not traffic along with activated beta2AR, D1AR, and ETAR in endocytic vesicles, activation of AT1AR and NTR triggers a clear time-dependent redistribution of beta-arrestins to intracellular vesicular compartments where they colocalize with internalized receptors. Activation of a chimeric AT1AR with the beta2AR carboxyl-terminal tail results in a beta-arrestin membrane localization pattern similar to that observed in response to beta2AR activation. In contrast, the corresponding chimeric beta2AR with the AT1AR carboxyl-terminal tail gains the ability to translocate beta-arrestin to intracellular vesicles. These results demonstrate that the cellular trafficking of beta-arrestin proteins is differentially regulated by the activation of distinct GPCRs. Furthermore, they suggest that the carboxyl-tail of the receptors might be involved in determining the stability of receptor/betaarrestin complexes and cellular distribution of beta-arrestins.     

Differential regulation of corticotropin releasing factor 1alpha receptor endocytosis and trafficking by beta-arrestins and Rab GTPases

The corticotropin releasing factor (CRF) type 1alpha receptor, a member of the G protein-coupled receptor (GPCR) subfamily B, is involved in the aetiology of anxiety and depressive disorders. In the present study, we examined the internalization and trafficking of the CRF1alpha receptor in both human embryonic kidney (HEK)293 cells and primary cortical neurons. We found that CRF1alpha receptor activation leads to the selective recruitment of beta-arrestin2 in both HEK293 cells and neurons. We observed distinct distribution patterns of CRF1alpha receptor and beta-arrestin2 in HEK293 cells and cortical neurons. In HEK293 cells, beta-arrestin2-green fluorescent protein (GFP) co-localized with CRF1alpha receptor in vesicles at the plasma membrane but was dissociated from the receptor in endosomes. In contrast, in primary cortical neurons, beta-arrestin2 and CRF1alpha receptor were internalized in distinct endocytic vesicles. By bioluminescence resonance energy transfer, we demonstrated that beta-arrestin2 association with CRF1alpha receptor was increased in cells transfected with G protein-coupled receptor kinase (GRK)3 and GRK6 and decreased in cells transfected with GRK2 and GRK5. In both HEK293 cells and cortical neurons, internalized CRF1alpha receptor transited from Rab5-positive early endosomes to Rab4-positive recycling endosomes and was not targeted to lysosomes. However, CRF1alpha receptor resensitization was blocked by the overexpression of wild-type, but not dominant-negative, Rab5 and Rab4 GTPases. Taken together, our results suggest that beta-arrestin trafficking differs between HEK293 cells and neurons, and that CRF1alpha receptor resensitization is regulated in an atypical manner by Rab GTPases.     

Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization.

Resensitization of G protein-coupled receptors (GPCRs) following agonist-mediated desensitization is a necessary step for maintaining physiological responsiveness. However, the molecular mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of beta-arrestin in the resensitization of the beta(2) adrenergic receptor (beta(2)AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit beta-arrestin to the plasma membrane and internalize in a beta-arrestin- and clathrin-dependent manner. However, whereas beta-arrestin dissociates from the beta(2)AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of beta-arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of beta-arrestin to remain associated with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of beta-arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which beta-arrestins regulate the reestablishment of GPCR responsiveness.     

A beta-arrestin binding determinant common to the second intracellular loops of rhodopsin family G protein-coupled receptors

beta-Arrestins have been shown to inhibit competitively G protein-dependent signaling and to mediate endocytosis for many of the hundreds of nonvisual rhodopsin family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology, when considered in toto, can all recruit and activate the two highly conserved beta-arrestin proteins as part of their signaling/desensitization process. Although the serine and threonine residues that form GPCR kinase phosphorylation sites are common beta-arrestin-associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of a GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. Here we identified a beta-arrestin binding determinant common to the rhodopsin family GPCRs formed from the proximal 10 residues of the second intracellular loop. We demonstrated by both gain and loss of function studies for the serotonin 2C, beta2-adrenergic, alpha2a)adrenergic, and neuropeptide Y type 2 receptors that the highly conserved amino acids, proline and alanine, naturally occurring in rhodopsin family receptors six residues distal to the highly conserved second loop DRY motif regulate beta-arrestin binding and beta-arrestin-mediated internalization. In particular, as demonstrated for the beta2 AR, this occurs independently of changes in GPCR kinase phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning beta-arrestin from its inactive form to its active receptor-binding state.     

The interaction of beta-arrestin with the AP-2 adaptor is required for the clustering of beta 2-adrenergic receptor into clathrin-coated pits

Beta-arrestins are cytosolic proteins that regulate the signaling and the internalization of G protein-coupled receptors (GPCRs). Although termination of receptor coupling requires beta-arrestin binding to agonist-activated receptors, GPCR endocytosis involves the coordinate interactions between receptor-beta-arrestin complexes and other endocytic proteins such as adaptor protein 2 (AP-2) and clathrin. Clathrin interacts with a conserved motif in the beta-arrestin C-terminal tail; however, the specific molecular determinants in beta-arrestin that bind AP-2 have not been identified. Moreover, the respective contributions of the interactions of beta-arrestin with AP-2 and clathrin toward the targeting of GPCRs to clathrin-coated vesicles have not been established. Here, we identify specific arginine residues (Arg(394) and Arg(396)) in the beta-arrestin 2 C terminus that mediate beta-arrestin binding to AP-2 and show, in vitro, that these domains in beta-arrestin 1 and 2 interact equally well with AP-2 independently of clathrin binding. We demonstrate in HEK 293 cells by fluorescence microscopy that beta(2)-adrenergic receptor-beta-arrestin complexes lacking the beta-arrestin-clathrin binding motif are still targeted to clathrin-coated pits. In marked contrast, receptor-beta-arrestin complexes lacking the beta-arrestin/AP-2 interactions are not effectively compartmentalized in punctated areas of the plasma membrane. These results reveal that the binding of a receptor-beta-arrestin complex to AP-2, not to clathrin, is necessary for the initial targeting of beta(2)-adrenergic receptor to clathrin-coated pits.     

Dissociation of beta-arrestin from internalized bradykinin B2 receptor is necessary for receptor recycling and resensitization

Beta-arrestins are multifunctional adaptors that bind agonist-activated G protein-coupled receptors (GPCRs), mediate their desensitization and internalization, and control the rate at which receptors recycle back at the plasma membrane ready for subsequent stimulation. The activation of the bradykinin (BK) type 2 receptor (B2R) results in the rapid desensitization and internalization of the receptor. Little is known, however, about the role of beta-arrestin in regulating the intracellular trafficking and the resensitization of the B2R. Using confocal microscopy, we show that BK stimulation of COS-7 cells expressing B2R induces the colocalization of the agonist-activated receptor with beta-arrestin into endosomes. Fluorescent imaging and ligand binding experiments also reveal that upon agonist removal, beta-arrestin rapidly dissociates from B2R into endosomes, and that receptors return back to the plasma membrane, fully competent for reactivating B2R signaling as measured by NO production upon a second BK challenge. However, when the receptor is mutated in its C-terminal domain to increase its avidity for beta-arrestin, B2R remains associated with beta-arrestin into endosomes, and receptors fail to recycle to the plasma membrane postagonist wash. Similarly, the recycling of receptors is prevented when a beta-arrestin mutant exhibiting increased avidity for agonist-bound GPCRs is expressed with B2R. Stabilizing receptor/beta-arrestin complexes into endosomes results in the dampening of the BK-mediated NO production. These results provide evidence for the involvement of beta-arrestin in the intracellular trafficking of B2R, and highlight the importance of receptor recycling in reestablishing B2R signaling.     

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.     

Keratinocyte-releasable factors increased the expression of MMP1 and MMP3 in co-cultured fibroblasts under both 2D and 3D culture conditions

G protein-coupled receptors (GPCRs) are known to modulate intracellular effectors involved in cardiac function. We recently reported homocysteine (Hcy)-induced ERK-phosphorylation was suppressed by pertussis toxin (PTX), which suggested the involvement of GPCRs in initiating signal transduction. An activated GPCR undergoes down regulation via a known mechanism involving ERK, GRK2, beta-arrestin1: ERK activity increases; GRK2 activity increases; beta-arrestin1 is degraded. We hypothesized that Hcy treatment leads to GPCR activation and down regulation. Microvascular endothelial cells were treated with Hcy. Expression of phospho-ERK1 and phospho-GRK2 was determined using Western blot, standardized to ERK1, GRK2, and beta-actin. Hcy was shown to dephosphorylate GRK2, thereby enhancing the activity. The results provided further evidence that Hcy acts as an agonist to activate GPCRs, followed by their down regulation. Hcy was also shown to decrease the content of the following G proteins and other proteins: beta-arrestin1, Galpha(q/11), Galpha(12/13), G(i/o).     

From sorting endosomes to exocytosis: association of Rab4 and Rab11 GTPases with the Fc receptor, FcRn, during recycling

A longstanding question in cell biology is how is the routing of intracellular organelles within cells regulated? Although data support the involvement of Rab4 and Rab11 GTPases in the recycling pathway, the function of Rab11 in particular is uncertain. Here we have analyzed the association of these two Rab GTPases with the Fc receptor, FcRn, during intracellular trafficking. This Fc receptor is both functionally and structurally distinct from the classical Fcgamma receptors and transports immunoglobulin G (IgG) within cells. FcRn is therefore a recycling receptor that sorts bound IgG from unbound IgG in sorting endosomes. In the current study we have used dual color total internal reflection fluorescence microscopy (TIRFM) and wide-field imaging of live cells to analyze the events in human endothelial cells that are involved in the trafficking of FcRn positive (FcRn(+)) recycling compartments from sorting endosomes to exocytic sites at the plasma membrane. Our data are consistent with the following model for this pathway: FcRn leaves sorting endosomes in Rab4(+)Rab11(+) or Rab11(+) compartments. For Rab4(+)Rab11(+) compartments, Rab4 depletion occurs by segregation of the two Rab proteins into discrete domains that can separate. The Rab11(+)FcRn(+) vesicle or tubule subsequently fuses with the plasma membrane in an exocytic event. In contrast to Rab11, Rab4 is not involved in exocytosis.     

Targeted construction of phosphorylation-independent beta-arrestin mutants with constitutive activity in cells

Arrestin proteins play a key role in the desensitization of G protein-coupled receptors (GPCRs). Recently we proposed a molecular mechanism whereby arrestin preferentially binds to the activated and phosphorylated form of its cognate GPCR. To test the model, we introduced two different types of mutations into beta-arrestin that were expected to disrupt two crucial elements that make beta-arrestin binding to receptors phosphorylation-dependent. We found that two beta-arrestin mutants (Arg169 --> Glu and Asp383 --> Ter) (Ter, stop codon) are indeed "constitutively active." In vitro these mutants bind to the agonist-activated beta2-adrenergic receptor (beta2AR) regardless of its phosphorylation status. When expressed in Xenopus oocytes these beta-arrestin mutants effectively desensitize beta2AR in a phosphorylation-independent manner. Constitutively active beta-arrestin mutants also effectively desensitize delta opioid receptor (DOR) and restore the agonist-induced desensitization of a truncated DOR lacking the critical G protein-coupled receptor kinase (GRK) phosphorylation sites. The kinetics of the desensitization induced by phosphorylation-independent mutants in the absence of receptor phosphorylation appears identical to that induced by wild type beta-arrestin + GRK3. Either of the mutations could have occurred naturally and made receptor kinases redundant, raising the question of why a more complex two-step mechanism (receptor phosphorylation followed by arrestin binding) is universally used.     

The regulatory mechanisms of export trafficking of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are a superfamily of cell-surface receptors that regulate a variety of cell functions by responding to a myriad of ligands. The magnitude of the response elicited by a ligand is dictated by the level of receptor available at the plasma membrane. GPCR expression levels at the cell surface are a balance of three highly regulated, dynamic intracellular trafficking processes, namely export, internalization and degradation. This review will cover recent advances in understanding the mechanism underlying GPCR export trafficking by focusing on specific motifs required for ER export and the role of the Ras-like Rab1 GTPase and glycosylation in regulating ER–Golgi-cell-surface transport. The manifestation of diseases due to the disruption of GPCR export is also discussed.     

A robust antibody discovery platform for difficult-to-express G protein-coupled receptors

G protein-coupled receptors (GPCRs) are in the spotlight as drug targets due to the fact that multiple research results have verified the correlation between the activation of GPCRs and disease indications. This is because the GPCRs are present across the cell membranes, which interact with either extracellular ligands or other types of compartments and simultaneously mediate intracellular signaling. Despite the importance of the GPCRs as drug targets, they are too difficult to express in soluble forms. Currently, the difficulty of preparing functional GPCRs and the lack of efficient antibody screening methods are the most challenging steps in the discovery of antibodies targeting GPCRs. In this study, we developed a powerful platform that facilitates isolating GPCR-specific antibodies by obviating difficulties in GPCR preparation. The strategies include (i) conjugation of the P9 peptide, an envelope protein of Pseudomonas phi6, to the N-terminus of GPCRs to improve the expression level of the GPCRs in Escherichia coli, (ii) stabilization of the GPCRs in their active forms with amphiphilic poly-γ-glutamate (APG) to shield the seven hydrophobic transmembrane domains, and (iii) further limiting the size of the APG complex to improve the chance to isolate antibodies targeting the proteins-of-interest. Capitalizing on the above strategies, we could prepare GPCR proteins in their active forms as facile as other general-soluble antigen proteins. Furthermore, this protocol was validated to be successful in discovering three individual GPCR-specific antibodies targeting glucagon-like peptide-1 receptor, C-X-C chemokine receptor type 4, and prostaglandin E2 receptor 4 in this study.     

The role of beta-arrestins in the formyl peptide receptor-like 1 internalization and signaling

The N-formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor (GPCR) that transmits intracellular signals in response to a variety of agonists, many of them being clearly implicated in human pathology. beta-arrestins are adaptor proteins that uncouple GPCRs from G protein and regulate receptor internalization. They can also function as signal transducers through the scaffolding of signaling molecules, such as components of the extracellular signal-regulated kinase (ERK) cascade. We investigated the role of beta-arrestins in ligand-induced FPRL1 internalization and signaling. In HEK293 cells expressing FPRL1, fluorescence microscopy revealed that agonist-stimulated FPRL1 remained co-localized with beta-arrestins during endocytosis. Internalization of FPRL1, expressed in a mouse embryonic fibroblast (MEF) cell line lacking endogenous beta-arrestins, was highly compromised. This distinguishes FPRL1 from the prototypical formyl peptide receptor FPR that is efficiently internalized in the absence of beta-arrestins. In both HEK293 and MEF cells, FPRL1-mediated ERK1/2 activation was a rapid and transient event. The kinetics and extent of ERK1/2 activation were not significantly modified by beta-arrestin overexpression. The pattern of FPRL1-mediated ERK1/2 activation was similar whether cells express or not beta-arrestins. Furthermore, treatment of the FPRL1 expressing cells with pertussis toxin inhibited ERK1/2 activation in MEF and in HEK293 cells. These results led us to conclude that activation of ERK1/2 mediated by FPRL1 occurs primarily through G protein signaling. Since beta-arrestin-mediated signaling has been observed essentially for receptors coupled to G proteins other than G(i), this may be a characteristic of G(i) protein-coupled chemoattractant receptors.     

Heterotrimeric G Proteins and the Single-Transmembrane Domain IGF-II/M6P Receptor: Functional Interaction and Relevance to Cell Signaling

The G protein-coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors. Classical GPCR signaling constitutes ligand binding to a seven-transmembrane domain receptor, receptor interaction with a heterotrimeric G protein, and the subsequent activation or inhibition of downstream intracellular effectors to mediate a cellular response. However, recent reports on direct, receptor-independent G protein activation, G protein-independent signaling by GPCRs, and signaling of nonheptahelical receptors via trimeric G proteins have highlighted the intrinsic complexities of G protein signaling mechanisms. The insulin-like growth factor-II/mannose-6 phosphate (IGF-II/M6P) receptor is a single-transmembrane glycoprotein whose principal function is the intracellular transport of lysosomal enzymes. In addition, the receptor also mediates some biological effects in response to IGF-II binding in both neuronal and nonneuronal systems. Multidisciplinary efforts to elucidate the intracellular signaling pathways that underlie these effects have generated data to suggest that the IGF-II/M6P receptor might mediate transmembrane signaling via a G protein-coupled mechanism. The purpose of this review is to outline the characteristics of traditional and nontraditional GPCRs, to relate the IGF-II/M6P receptor’s structure with its role in G protein-coupled signaling and to summarize evidence gathered over the years regarding the putative signaling of the IGF-II/M6P receptor mediated by a G protein.     

S-nitrosylation of beta-arrestin regulates beta-adrenergic receptor trafficking

Signal transduction through G protein-coupled receptors (GPCRs) is regulated by receptor desensitization and internalization that follow agonist stimulation. Nitric oxide (NO) can influence these processes, but the cellular source of NO bioactivity and the effects of NO on GPCR-mediated signal transduction are incompletely understood. Here, we show in cells and mice that beta-arrestin 2, a central element in GPCR trafficking, interacts with and is S-nitrosylated at a single cysteine by endothelial NO synthase (eNOS), and that S-nitrosylation of beta-arrestin 2 is promoted by endogenous S-nitrosogluthathione. S-nitrosylation after agonist stimulation of the beta-adrenergic receptor, a prototypical GPCR, dissociates eNOS from beta-arrestin 2 and promotes binding of beta-arrestin 2 to clathrin heavy chain/beta-adaptin, thereby accelerating receptor internalization. The agonist- and NO-dependent shift in the affiliations of beta-arrestin 2 is followed by denitrosylation. Thus, beta-arrestin subserves the functional coupling of eNOS and GPCRs, and dynamic S-nitrosylation/denitrosylation of beta-arrestin 2 regulates stimulus-induced GPCR trafficking.     

Spatially Restricted G Protein-coupled Receptor Activity via Divergent Endocytic Compartments

Postendocytic sorting of G protein-coupled receptors (GPCRs) is driven by their interactions between highly diverse receptor sequence motifs with their interacting proteins, such as postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (zo-1) (PDZ) domain proteins. However, whether these diverse interactions provide an underlying functional specificity, in addition to driving sorting, is unknown. Here we identify GPCRs that recycle via distinct PDZ ligand/PDZ protein pairs that exploit their recycling machinery primarily for targeted endosomal localization and signaling specificity. The luteinizing hormone receptor (LHR) and β2-adrenergic receptor (B2AR), two GPCRs sorted to the regulated recycling pathway, underwent divergent trafficking to distinct endosomal compartments. Unlike B2AR, which traffics to early endosomes (EE), LHR internalizes to distinct pre-early endosomes (pre-EEs) for its recycling. Pre-EE localization required interactions of the LHR C-terminal tail with the PDZ protein GAIP-interacting protein C terminus, inhibiting its traffic to EEs. Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling. Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment. We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.     

Novel roles for arrestins in the post-endocytic trafficking of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling molecules in the human genome. As such, they interact with numerous intracellular molecules, which can act either to propagate or curtail signaling from the receptor. Their primary mode of cellular activation occurs through heterotrimeric G proteins, which in turn can activate a wide spectrum of effector molecules, including phosphodiesterases, phospholipases, adenylyl cyclases and ion channels. Active GPCRs are also the target of G protein-coupled receptor kinases, which phosphorylate the receptors culminating in the binding of the protein arrestin. This results in rapid desensitization through inhibition of G protein binding, as well as novel mechanisms of cellular activation that involve the scaffolding of cellular kinases to GPCR-arrestin complexes. Arrestins can also serve to mediate the internalization of certain GPCRs, a process which plays an important role in regulating cellular activity both by mediating long-term desensitization through down regulation (degradation) of receptors and by recycling desensitized receptors back to the cell surface to initiate additional rounds of signaling. The mechanisms that regulate the subsequent intracellular trafficking of GPCRs following internalization are largely unknown. Recently however, it has become clear that the pattern of receptor phosphorylation and subsequent binding of arrestin play a critical role in the intracellular trafficking of internalized receptors, thereby dictating the ultimate fate of the receptor. In addition, arrestins have now been shown to be required for the recycling of GPCRs that are capable of internalizing through arrestin-independent mechanisms. This review will summarize recent advances in our understanding of the roles of arrestins in post-endocytic GPCR trafficking.     

Expression patterns of the regulatory proteins G protein-coupled receptor kinase 2 and beta-arrestin 1 during rat postnatal brain development: effect of hypothyroidism.

G protein-coupled receptor kinase 2 (GRK2) and beta-arrestin 1 are key regulatory proteins that modulate the desensitization and resensitization of a wide variety of G protein-coupled receptors (GPCRs) involved in brain functions. In this report, we describe the postnatal developmental profile of the mRNA and protein levels of GRK2 and beta-arrestin 1 in rat brain. The expression levels of GRK2 and beta-arrestin 1 display a marked increase at the second and third week after birth, respectively, consistent with an involvement of these proteins in brain maturation processes. However, the expression attained at birth and during the first postnatal week with respect to adult values (45-70% for GRK2, approximately 30% for beta-arrestin 1) is relatively high compared to that reported for several GPCRs, indicating the existence of changes in the ratio of receptors to their regulatory proteins during brain development. On the other hand, we report that experimental hypothyroidism results in changes in the patterns of expression of GRK2 and beta-arrestin 1 in cerebral cortex, leading to a 25-30% reduction in GRK2 levels at several stages of development. Such changes could help to explain the alterations in GPCR signaling that occur during this pathophysiological condition.     

Receptor/beta-arrestin complex formation and the differential trafficking and resensitization of beta2-adrenergic and angiotensin II type 1A receptors

Beta-arrestins target G protein-coupled receptors (GPCRs) for endocytosis via clathrin-coated vesicles. Beta-arrestins also become detectable on endocytic vesicles in response to angiotensin II type 1A receptor (AT1AR), but not beta2-adrenergic receptor (beta2AR), activation. The carboxyl-terminal tails of these receptors contribute directly to this phenotype, since a beta2AR bearing the AT1AR tail acquired the capacity to stimulate beta-arrestin redistribution to endosomes, whereas this property was lost for an AT1AR bearing the beta2AR tail. Using beta2AR/AT1AR chimeras, we tested whether the beta2AR and AT1AR carboxyl-terminal tails, in part via their association with beta-arrestins, might regulate differences in the intracellular trafficking and resensitization patterns of these receptors. In the present study, we find that beta-arrestin formed a stable complex with the AT1AR tail in endocytic vesicles and that the internalization of this complex was dynamin dependent. Internalization of the beta2AR chimera bearing the AT1AR tail was observed in the absence of agonist and was inhibited by a dominant-negative beta-arrestin1 mutant. Agonist-independent AT1AR internalization was also observed after beta-arrestin2 overexpression. After internalization, the beta2AR, but not the AT1AR, was dephosphorylated and recycled back to the cell surface. However, the AT1AR tail prevented beta2AR dephosphorylation and recycling. In contrast, although the beta2AR-tail promoted AT1AR recycling, the chimeric receptor remained both phosphorylated and desensitized, suggesting that receptor dephosphorylation is not a property common to all receptors. In summary, we show that the carboxyl-terminal tails of GPCRs not only contribute to regulating the patterns of receptor desensitization, but also modulate receptor intracellular trafficking and resensitization patterns.