Internalization determinants of the parathyroid hormone receptor differentially regulate beta-arrestin/receptor association.

beta-Arrestins have been implicated in regulating internalization of the parathyroid hormone receptor (PTHR), but the structural features in the receptor required for this effect are unknown. In the present study performed in HEK-293 cells, we demonstrated that different topological domains of PTHR are implicated in agonist-dependent receptor internalization; truncation of the cytoplasmic tail (PTHR-TR), selective mutations of the cytoplasmic tail to remove the sites of parathyroid hormone (PTH)-stimulated phosphorylation (PTHR-PD), and mutations in the third transmembrane helix (N289A) or in the third cytoplasmic loop (K382A) resulted in a 30-60% reduction in (125)I-PTH-related protein internalization. To better define the role of these internalization determinants, we have tested the ability of these mutant PTHRs to associate with beta-arrestins by using three different methodological approaches: 1) ability of overexpression of beta-arrestins to restore the internalization of (125)I-PTH-related protein for the mutant PTHRs; 2) visualization of PTH-mediated trafficking of beta-arrestin1 and -2 fused to the green fluorescent protein with receptors by confocal microscopy; 3) quantification of beta-arrestin1-green fluorescent protein translocation by Western blot. Our data reveal that the receptor' cytoplasmic tail contains determinants of beta-arrestin interaction that are distinct from the phosphorylation sites and are sufficient for transient association of beta-arrestin2, but stable association requires receptor phosphorylation. Determinants in the receptor's core (Asn-289 and Lys-382) appear to regulate internalization of the receptor/beta-arrestin complex toward early endocytic endosomes during the initial step of endocytosis.     

Formyl peptide receptor-mediated ERK1/2 activation occurs through G(i) and is not dependent on beta-arrestin1/2

Formyl peptide receptor (FPR) and C5a receptor (C5aR) are chemoattractant G protein-coupled receptors (GPCRs) involved in the innate immune response against bacterial infections and tissue injury. Like other GPCRs, they recruit beta-arrestin1/2 to the plasma membrane and activate the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Previous studies with several GPCRs have suggested that beta-arrestins play an important role as signal transducers by scaffolding signaling molecules such as ERK1/2. This function of the beta-arrestins was not discovered until several years after their role in desensitization and endocytosis had been reported. In this study, we investigated the role of the beta-arrestins in the activation of ERK1/2 and receptor endocytosis. We took advantage of previously described mutants of FPR that have defects in G(i) coupling or beta-arrestin recruitment. The results obtained with the mutant FPRs, as well as experiments using an inhibitor of G(i) and cells overexpressing beta-arrestin2, showed that activation of ERK1/2 takes place through G(i) and is not affected by beta-arrestins. However, overexpression of beta-arrestin2 does enhance FPR sequestration from the cell surface, suggesting a role in desensitization, as shown for many other GPCRs. Experiments with CHO C5aR cells showed similar sensitivity to the G(i) inhibitor as CHO FPR cells, suggesting that the predominant activation of ERK1/2 through G protein may be a common characteristic among chemoattractant receptors.     

Beta-arrestin mediates desensitization and internalization but does not affect dephosphorylation of the thyrotropin-releasing hormone receptor

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.     

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.     

Differential beta-arrestin trafficking and endosomal sorting of somatostatin receptor subtypes

The physiological responses of somatostatin are mediated by five different G protein-coupled receptors. Although agonist-induced endocytosis of the various somatostatin receptor subtypes (sst(1)-sst(5)) has been studied in detail, little is known about their postendocytic trafficking. Here we show that somatostatin receptors profoundly differ in patterns of beta-arrestin mobilization and endosomal sorting. The beta-arrestin-dependent trafficking of the sst(2A) somatostatin receptor resembled that of a class B receptor in that upon receptor activation, beta-arrestin and the receptor formed stable complexes and internalized together into the same endocytic vesicles. This pattern was dependent on GRK2 (G protein-coupled receptor kinase 2)-mediated phosphorylation of a cluster of phosphate acceptor sites within the cytoplasmic tail of the sst(2A) receptor. Unlike other class B receptors, however, the sst(2A) receptor was rapidly resensitized and recycled to the plasma membrane. The beta-arrestin mobilization of the sst(3) and the sst(5) somatostatin receptors resembled that of a class A receptor in that upon receptor activation, beta-arrestin and the receptor formed relatively unstable complexes that dissociated at or near the plasma membrane. Consequently, beta-arrestin was excluded from sst(3)-containing vesicles. Unlike other class A receptors, a large proportion of sst(3) receptors was subject to ubiquitin-dependent lysosomal degradation and did not rapidly recycle to the plasma membrane. The sst(4) somatostatin receptor is unique in that it did not exhibit agonist-dependent receptor phosphorylation and beta-arrestin recruitment. Together, these findings may provide important clues about the regulation of receptor responsiveness during long-term administration of somatostatin analogs.     

Endocytosis of the AT1A angiotensin receptor is independent of ubiquitylation of its cytoplasmic serine/threonine-rich region

Agonist-induced internalisation of the rat type 1A (AT(1A)) angiotensin II receptor is associated with phosphorylation of a serine/threonine-rich region in its cytoplasmic tail. In yeast, hyperphosphorylation of the alpha-factor pheromone receptor regulates endocytosis of the receptor by facilitating the monoubiquitylation of its cytoplasmic tail on lysine residues. The role of receptor ubiquitylation in AT(1A) receptor internalisation was evaluated by deletion or replacement of lysine residues in its agonist-sensitive serine/threonine-rich region. Expression of such receptor mutants in CHO cells showed that these modifications had no detectable effect on the angiotensin II-induced endocytosis of the AT(1A) receptor. Furthermore, fusion of ubiquitin in-frame to an internalisation-deficient AT(1A) receptor mutant with a truncated carboxyl-terminal tail did not restore the endocytosis of the resulting chimeric receptor. No impairment of receptor internalisation was observed after substitution of all lysine residues in the serine/threonine-rich region at saturating angiotensin II concentrations, where endocytosis occurs by a beta-arrestin and dynamin independent mechanism. Taken together, these data demonstrate that ubiquitylation of the cytoplasmic serine/threonine-rich region of the AT(1A) receptor on lysine residues is not required for its agonist-induced internalisation, and suggest that endocytosis of mammalian G protein-coupled receptors (GPCRs) occurs by a different mechanism than that of yeast 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.     

The amino terminus of the fourth cytoplasmic loop of rhodopsin modulates rhodopsin-transducin interaction

Rhodopsin is a seven-transmembrane helix receptor that binds and catalytically activates the heterotrimeric G protein transducin (G(t)). This interaction involves the cytoplasmic surface of rhodopsin, which comprises four putative loops and the carboxyl-terminal tail. The fourth loop connects the carboxyl end of transmembrane helix 7 with Cys(322) and Cys(323), which are both modified by membrane-inserted palmitoyl groups. Published data on the roles of the fourth loop in the binding and activation of G(t) are contradictory. Here, we attempt to reconcile these conflicts and define a role for the fourth loop in rhodopsin-G(t) interactions. Fluorescence experiments demonstrated that a synthetic peptide corresponding to the fourth loop of rhodopsin inhibited the activation of G(t) by rhodopsin and interacted directly with the alpha subunit of G(t). A series of rhodopsin mutants was prepared in which portions of the fourth loop were replaced with analogous sequences from the beta(2)-adrenergic receptor or the m1 muscarinic receptor. Chimeric receptors in which residues 310-312 were replaced could not efficiently activate G(t). The defect in G(t) interaction in the fourth loop mutants was not affected by preventing palmitoylation of Cys(322) and Cys(323). We suggest that the amino terminus of the fourth loop interacts directly with G(t), particularly with Galpha(t), and with other regions of the intracellular surface of rhodopsin to support G(t) binding.     

Mechanisms and functions of AT(1) angiotensin receptor internalization

The type 1 (AT(1)) angiotensin receptor, which mediates the known physiological and pharmacological actions of angiotensin II, activates numerous intracellular signaling pathways and undergoes rapid internalization upon agonist binding. Morphological and biochemical studies have shown that agonist-induced endocytosis of the AT(1) receptor occurs via clathrin-coated pits, and is dependent on two regions in the cytoplasmic tail of the receptor. However, it is independent of G protein activation and signaling, and does not require the conserved NPXXY motif in the seventh transmembrane helix. The dependence of internalization of the AT(1) receptor on a cytoplasmic serine-threonine-rich region that is phosphorylated during agonist stimulation suggests that endocytosis is regulated by phosphorylation of the AT(1) receptor tail. beta-Arrestins have been implicated in the desensitization and endocytosis of several G protein-coupled receptors, but the exact nature of the adaptor protein required for association of the AT(1) receptor with clathrin-coated pits, and the role of dynamin in the internalization process, are still controversial. There is increasing evidence for a role of internalization in sustained signal generation from the AT(1) receptor. Several aspects of the mechanisms and specific function of AT(1) receptor internalization, including its precise mode and route of endocytosis, and the potential roles of cytoplasmic and nuclear receptors, remain to be elucidated.     

Palmitoylation of the V2 vasopressin receptor carboxyl tail enhances beta-arrestin recruitment leading to efficient receptor endocytosis and ERK1/2 activation

A large number of G protein-coupled receptors are palmitoylated on cysteine residues located in their carboxyl tail, but the general role of this post-translational modification remains poorly understood. Here we show that preventing palmitoylation of the V2 vasopressin receptor, by site-directed mutagenesis of cysteines 341 and 342, significantly delayed and decreased both agonist-promoted receptor endocytosis and mitogen-activated protein kinase activation. Pharmacological blockade of receptor endocytosis is without effect on the vasopressin-stimulated mitogen-activated protein kinase activity, excluding the possibility that the reduced kinase activation mediated by the palmitoylation-less mutant could result from altered receptor endocytosis. In contrast, two dominant negative mutants of beta-arrestin which inhibit receptor endocytosis also attenuated vasopressin-stimulated mitogen-activated protein kinase activity, suggesting that the scaffolding protein, beta-arrestin, represents the common link among receptor palmitoylation, endocytosis, and kinase activation. Coimmunoprecipitation and bioluminescence resonance energy transfer experiments confirmed that inhibiting receptor palmitoylation considerably reduced the vasopressin-stimulated recruitment of beta-arrestin to the receptor. Interestingly, the changes in beta-arrestin recruitment kinetics were similar to those observed for vasopressin-stimulated receptor endocytosis and mitogen-activated protein kinase activation. Taken together the results indicate that palmitoylation enhances the recruitment of beta-arrestin to the activated V2 vasopressin receptor thus facilitating processes requiring the scaffolding action of beta-arrestin.     

Regulation of corticotropin-releasing hormone receptor type 1alpha signaling: structural determinants for G protein-coupled receptor kinase-mediated phosphorylation and agonist-mediated desensitization

Attenuation of CRH receptor type 1 (CRH-R1) signaling activity might involve desensitization and uncoupling of CRH-R1 from intracellular effectors. We investigated the desensitization of native CRH-R in human myometrial cells from pregnant women and recombinant CRH-R1alpha stably overexpressed in human embryonic kidney (HEK) 293 cells. In both cell types, CRH-R1-mediated adenylyl cyclase activation was susceptible to homologous desensitization induced by pretreatment with high concentrations of CRH. Time course studies showed half-maximal desensitization occurring after approximately 40 min of pretreatment and full recovery of CRH-R1alpha functional response within 2 h of removal of CRH pretreatment. In HEK 293 cells, desensitization of CRH-R1alpha was associated with receptor phosphorylation and subsequent endocytosis. To analyze the mechanism leading to CRH-R1alpha desensitization, we overexpressed a truncated beta-arrestin (319-418) and performed coimmunoprecipitation and G protein-coupled receptor kinase (GRK) translocation studies. We found that GRK3 and GRK6 are the main isoforms that interact with CRH-R1alpha, and that recruitment of GRK3 requires Gbetagamma-subunits as well as beta-arrestin. Site-directed mutagenesis of Ser and Thr residues in the CRH-R1alpha C terminus, identified Thr399 as important for GRK-induced receptor phosphorylation and desensitization.We conclude that homologous desensitization of CRH-R1alpha involves the coordinated action of multiple GRK isoforms, Gbeta gamma dimers and beta-arrestin. Based on our identification of key amino acid(s) for GRK-dependent phosphorylation, we demonstrate the importance of the CRH-R1alpha carboxyl tail for regulation of receptor activity.     

Structure and function in rhodopsin: effects of disulfide cross-links in the cytoplasmic face of rhodopsin on transducin activation and phosphorylation by rhodopsin kinase.

Six rhodopsin mutants containing disulfide cross-links between different cytoplasmic regions were prepared: disulfide bond 1, between Cys65 (interhelical loop I-II) and Cys316 (end of helix VII); disulfide bond 2, between Cys246 (end of helix VI) and Cys312 (end of helix VII); disulfide bond 3, between Cys139 (end of helix III) and Cys248 (end of helix VI); disulfide bond 4, between Cys139 (end of helix III) and Cys250 (end of helix VI); disulfide bond 5, between Cys135 (end of helix III) and Cys250 (end of helix VI); and disulfide bond 6, between Cys245 (end of helix VI) and Cys338 (C-terminus). The effects of local restrictions caused by the cross-links on transducin (G(T)) activation and phosphorylation by rhodopsin kinase (RK) following illumination were studied. Disulfide bond 1 showed little effect on either G(T) activation or phosphorylation by RK, suggesting that the relative motion between interhelical loop I-II and helix VII is not crucial for recognition by G(T) or by RK. In contrast, disulfide bonds 2-5 abolished both G(T) activation and phosphorylation by RK. Disulfide bond 6 resulted in enhanced G(T) activation but abolished phosphorylation by RK, suggesting the structure recognized by G(T) was stabilized in this mutant by cross-linking of the C-terminus to the cytoplasmic end of helix VI. Thus, the consequences of the disulfide cross-links depended on the location of the restriction. In particular, relative motions of helix VI, with respect to both helices III and VII upon light activation, are required for recognition of rhodopsin by both G(T) and RK. Further, the conformational changes in the cytoplasmic face that are necessary for protein-protein interactions need not be cooperative, and may be segmental.     

Bradykinin-induced internalization of the human B2 receptor requires phosphorylation of three serine and two threonine residues at its carboxyl tail

The binding of bradykinin (BK) to B2 receptor triggers the internalization of the agonist-receptor complex. To investigate the mechanisms and the receptor structures involved in this fundamental process of receptor regulation, the human B2 receptor was mutated within its cytoplasmic tail by complementary strategies of truncation, deletion, and amino acid substitution. Ligand binding, signal transduction, internalization as well as phosphorylation were studied for the mutated receptors expressed in COS, CHO, and HEK 293 cells. Truncation of 44 out of 55 amino acid residues of the receptor's cytoplasmic tail corresponding to positions 321-364 did not alter the kinetics of BK binding and the receptor coupling to phospholipase C and phospholipase A2. By contrast, truncations after positions 320 and 334, deletions within the segment covering positions 335-351, as well as alanine substitution of serine and threonine residues within segment 335-351 diminished the internalization capacity of the mutant receptors. Mutants with a markedly reduced internalization potential failed to produce BK-induced receptor phosphorylation suggesting that phosphorylation may be involved in receptor internalization. The mutagenesis approaches converged at the conclusion that three serines in positions 339, 346, and 348 and two threonines in positions 342 and 345, contained in a sequence segment that is highly conserved between species, have a critical role in the ligand-dependent internalization and phosphorylation of kinin receptors and can intervene in these processes in an alternative manner. However, mutants lacking these residues were still sensitive to dominant-negative forms of beta-arrestin and dynamin, suggesting the existence of additional receptor structure(s) involved in the receptor sequestration through clathrin-coated vesicles.     

Arrestin regulates MAPK activation and prevents NADPH oxidase-dependent death of cells expressing CXCR2

Activation of CXCR2 IL-8 receptor leads to activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and rapid receptor endocytosis. Co-immunoprecipitation and co-localization experiments showed that arrestin and CXCR2 form complexes with components of the ERK1/2 cascade following ligand stimulation. However, in contrast to the activation of the beta2-adrenergic receptor, arrestin was not necessary for ERK1/2 phosphorylation or receptor endocytosis. In contrast, beta-arrestin 1/2 double knockout cells showed greatly enhanced phosphorylation of ERK1/2, as well as phosphorylation of the stress kinases p38 and c-Jun N-terminal protein kinase. The stimulation of stress kinases in arrestin double knockout cells could be attenuated in the presence of diphenylene iodonium (DPI), an inhibitor of the NADPH oxidase, suggesting that reactive oxidant species (ROS) participated in mitogen-activated protein kinase (MAPK) activation. ROS could indeed be detected in IL-8-stimulated beta-arrestin 1/2 knockout cells, and cytoplasmic Rac was translocated to the membrane fraction, which is a prerequisite for oxidant formation. The oxidative burst induced cell death within 6 h of IL-8 stimulation of these cells, which could be prevented in the presence of DPI. These results indicate a novel function for arrestin, which is protection from an excessive oxidative burst, resulting from the sustained stimulation of G-protein-coupled receptors that cause Rac translocation.     

c-Src is involved in regulating signal transmission from PDGFbeta receptor-GPCR(s) complexes in mammalian cells

We have reported that the platelet-derived growth factor receptor-beta (PDGFbeta) forms a novel signaling complex with G protein-coupled receptors (GPCR) (e.g. S1P(1) receptor) that enables more efficient activation of p42/p44 mitogen-activated protein kinase (MAPK) in response to PDGF and sphingosine 1-phosphate (S1P). We now demonstrate that c-Src participates in regulating the endocytosis of PDGFbeta receptor-GPCR complexes in response to PDGF. This leads to association of cytoplasmic p42/p44 MAPK with the receptor complex in endocytic vesicles. c-Src is regulated by G protein betagamma subunits and can interact with beta-arrestin. Indeed, the PDGF-dependent activation of p42/p44 MAPK was reduced by over-expression of the C-terminal domain of GRK2 (sequesters Gbetagamma subunits), the clathrin-binding domain of beta-arrestin and by inhibitors of c-Src and clathrin-mediated endocytosis. Moreover, PDGF and S1P induce the recruitment of c-Src to the PDGFbeta receptor-S1P(1) receptor complex. This leads to a G protein/c-Src-dependent tyrosine phosphorylation of Gab1 and accumulation of dynamin II at the plasma membrane, a step required for endocytosis of the PDGFbeta receptor-GPCR complex. These findings provide important information concerning the molecular organisation of novel receptor tyrosine kinase (RTK)-GPCR signal relays in mammalian cells.     

Importance of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor

The ghrelin receptor (GhrelinR) and its related orphan GPR39 each display constitutive signaling, but only GhrelinRs undergo basal internalization. Here we investigate these differences by considering the roles of the C tail receptor domains for constitutive internalization and activity. Furthermore the interaction between phosphorylated receptors and beta-arrestin adaptor proteins has been examined. Replacement of the FLAG-tagged GhrelinR C tail with the equivalent GPR39 domain (GhR-39 chimera) preserved G(q) signaling. However in contrast to the GhrelinR, GhR-39 receptors exhibited no basal and substantially decreased agonist-induced internalization in transiently transfected HEK293 cells. Internalized GhrelinR and GhR-39 were predominantly localized to recycling compartments, identified with transferrin and the monomeric G proteins Rab5 and Rab11. Both the inverse agonist [d-Arg(1), d-Phe(5), d-Trp(7,9), Leu(11)] substance P and a naturally occurring mutant GhrelinR (A204E) with eliminated constitutive activity inhibited basal GhrelinR internalization. Surprisingly, we found that noninternalizing GPR39 was highly phosphorylated and that basal and agonist-induced phosphorylation of the GhR-39 chimera was elevated compared with GhrelinRs. Moreover, basal GhrelinR endocytosis occurred without significant phosphorylation, and it was not prevented by cotransfection of a dominant-negative beta-arrestin1(319-418) fragment or by expression in beta-arrestin1/2 double-knockout mouse embryonic fibroblasts. In contrast, agonist-stimulated GhrelinRs recruited the clathrin adaptor green fluorescent protein-tagged beta-arrestin2 to endosomes, coincident with increased receptor phosphorylation. Thus, GhrelinR internalization to recycling compartments depends on C-terminal motifs and constitutive activity, but the high levels of GPR39 phosphorylation, and of the GhR-39 chimera, are not sufficient to drive endocytosis. In addition, basal GhrelinR internalization occurs independently of beta-arrestins.     

The adaptor protein beta-arrestin2 enhances endocytosis of the low density lipoprotein receptor

Endocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor beta-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of beta-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.05), but had no effect on high density lipoprotein cholesterol levels. We therefore tested whether beta-arrestins could affect the constitutive endocytosis of the LDLR. When overexpressed in cells, beta-arrestin1 and beta-arrestin2 each associated with the LDLR, as judged by co-immunoprecipitation, and augmented LDLR endocytosis by approximately 70%, as judged by uptake of fluorescent LDL. However, physiologic expression levels of only beta-arrestin2, and not beta-arrestin1, enhanced endogenous LDLR endocytosis (by 65%) in stably transfected beta-arrestin1/beta-arrestin2 double-knockout mouse embryonic fibroblasts (MEFs). Concordantly, when RNA interference was used to suppress expression of beta-arrestin2, but not beta-arrestin1, LDLR endocytosis was reduced. Moreover, beta-arrestin2-/- MEFs demonstrated LDLR endocytosis that was 50% less than cognate wild type MEFs. In fusion protein pull-down assays, beta-arrestin2 bound to the LDLR cytoplasmic tail stoichiometrically, and binding was abolished by mutation of LDLR Tyr807 to Ala. Mutation of LDLR cytoplasmic tail Ser833 to Asp enhanced both the affinity of LDLR fusion protein binding to beta-arrestin2, and the efficiency of LDLR endocytosis in cells expressing beta-arrestin2 physiologically. We conclude that beta-arrestin2 can bind to and enhance endocytosis of the LDLR, both in vitro and in vivo, and may thereby influence lipoprotein metabolism.     

Activation and molecular recognition of the GPCR rhodopsin - insights from time-resolved fluorescence depolarisation and single molecule experiments

The cytoplasmic surface of the G-protein coupled receptor (GPCR) rhodopsin is a key element in membrane receptor activation, molecular recognition by signalling molecules, and receptor deactivation. Understanding of the coupling between conformational changes in the intramembrane domain and the membrane-exposed surface of the photoreceptor rhodopsin is crucial for the elucidation of the molecular mechanism in GPCR activation. As little is known about protein dynamics, particularly the conformational dynamics of the cytoplasmic surface elements on the nanoseconds timescale, we utilised time-resolved fluorescence anisotropy experiments and site-directed fluorescence labelling to provide information on both, conformational space and motion. We summarise our recent advances in understanding rhodopsin dynamics and function using time-resolved fluorescence depolarisation and single molecule fluorescence experiments, with particular focus on the amphipathic helix 8, lying parallel to the cytoplasmic membrane surface and connecting transmembrane helix 7 with the long C-terminal tail.     

Different mechanisms regulate lysophosphatidic acid (LPA)-dependent versus phorbol ester-dependent internalization of the LPA1 receptor

Lysophosphatidic acid (LPA) stimulates cells by activation of five G-protein-coupled receptors, termed LPA 1-5. The LPA 1 receptor is the most widely expressed and is a major regulator of cell migration. In this study, we show that phorbol ester (PMA)-induced internalization of the LPA(1) receptor requires clathrin AP-2 complexes, protein kinase C, and a distal dileucine motif (amino acids 352 and 353) in the cytoplasmic tail but not beta-arrestin. Agonist-dependent internalization of LPA 1, however, requires a cluster of serine residues (amino acids 341-347) located proximal to the dileucine motif, beta-arrestin, and to a lesser extent clathrin AP-2. The serine cluster of LPA 1 is required for beta-arrestin2-GFP translocation to the plasma membrane and signal desensitization. In contrast, the dileucine motif (IL) is required for both basal and PMA-induced internalization. Evidence for the beta-arrestin independence of PMA-induced internalization of LPA 1 comes from the observations that beta-arrestin2-GFP is not recruited to the plasma membrane upon PMA treatment and that LPA 1 is readily internalized in beta-arrestin1/2 knock-out mouse embryonic fibroblasts. These results indicate that distinct molecular mechanisms regulate agonist-dependent and PMA-dependent internalization of the LPA 1 receptor.