Agonist-biased signaling via proteinase activated receptor-2: differential activation of calcium and mitogen-activated protein kinase pathways.

We evaluated the ability of different trypsin-revealed tethered ligand (TL) sequences of rat proteinase-activated receptor 2 (rPAR(2)) and the corresponding soluble TL-derived agonist peptides to trigger agonist-biased signaling. To do so, we mutated the proteolytically revealed TL sequence of rPAR(2) and examined the impact on stimulating intracellular calcium transients and mitogen-activated protein (MAP) kinase. The TL receptor mutants, rPAR(2)-Leu(37)Ser(38), rPAR(2)-Ala(37-38), and rPAR(2)-Ala(39-42) were compared with the trypsin-revealed wild-type rPAR(2) TL sequence, S(37)LIGRL(42)-. Upon trypsin activation, all constructs stimulated MAP kinase signaling, but only the wt-rPAR(2) and rPAR(2)-Ala(39-42) triggered calcium signaling. Furthermore, the TL-derived synthetic peptide SLAAAA-NH2 failed to cause PAR(2)-mediated calcium signaling but did activate MAP kinase, whereas SLIGRL-NH2 triggered both calcium and MAP kinase signaling by all receptors. The peptides AAIGRL-NH2 and LSIGRL-NH2 triggered neither calcium nor MAP kinase signals. Neither rPAR(2)-Ala(37-38) nor rPAR(2)-Leu(37)Ser(38) constructs recruited beta-arrestins-1 or -2 in response to trypsin stimulation, whereas both beta-arrestins were recruited to these mutants by SLIGRL-NH2. The lack of trypsin-triggered beta-arrestin interactions correlated with impaired trypsin-activated TL-mutant receptor internalization. Trypsin-stimulated MAP kinase activation by the TL-mutated receptors was not blocked by inhibitors of Galpha(i) (pertussis toxin), Galpha(q) [N-cyclohexyl-1-(2,4-dichlorophenyl)-1,4-dihydro-6-methylindeno[1,2-c]pyrazole-3-carboxamide (GP2A)], Src kinase [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], or the epidermal growth factor (EGF) receptor [4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478)], but was inhibited by the Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27362). The data indicate that the proteolytically revealed TL sequence(s) and the mode of its presentation to the receptor (tethered versus soluble) can confer biased signaling by PAR(2), its arrestin recruitment, and its internalization. Thus, PAR(2) can signal to multiple pathways that are differentially triggered by distinct proteinase-revealed TLs or by synthetic signal-selective activating peptides.

Differential regulation of the signaling and trafficking of the two prostaglandin D2 receptors, prostanoid DP receptor and CRTH2.

Prostaglandin D2 (PGD2) exerts its actions on two G protein-coupled receptors, the prostanoid DP receptor and CRTH2 (chemoattractant homologous receptor expressed on TH2 cells). Here, we characterize the regulation of the signaling and trafficking of the prostanoid DP receptor and CRTH2. Time-course and dose-response curves showed that both receptors expressed in HEK293 cells internalized maximally after 2 h of stimulation with 1 microM PGD2. Co-expression of the G protein-coupled receptor kinases GRK2, GRK5 or GRK6 increased agonist-induced internalization of CRTH2, while only GRK2 had an effect on the internalization of the prostanoid DP receptor. Protein kinase C (PKC) activation stimulated the internalization of both receptors. Interestingly, only PGD2-induced internalization of CRTH2, and not of prostanoid DP receptor, was decreased by inhibition of PKC or protein kinase A (PKA). Our data also indicate that CRTH2 is subjected to basal phosphorylation by PKA, which appears to be involved in CRTH2 internalization. Prostanoid DP receptor internalization was promoted by co-expression of arrestin-2 and -3, while the internalization of CRTH2 was increased by co-expression of arrestin-3 only. The detection of prostanoid DP receptor and CRTH2 internalization was reduced by the co-expression of Rab4 and Rab11, respectively, suggesting differential regulation of receptor recycling. Moreover, immunofluorescence microscopy experiments showed that the prostanoid DP receptor specifically co-localized with Rab4, and CRTH2 with Rab11. The signaling of the prostanoid DP receptor was regulated by GRK2 overexpression, while that of CRTH2 was modulated by overexpression of GRK2, -5 and -6. Our results show a differential regulation of the prostanoid DP receptor and CRTH2, two receptors for PGD2.

ARF6 activation by Galpha q signaling: Galpha q forms molecular complexes with ARNO and ARF6.

G protein-coupled receptors (GPCRs) are widely expressed hepta-helical receptors with tightly regulated pleiotropic effects. ADP-Ribosylation Factor 6 (ARF6) plays an important role in GPCR trafficking and is the subject of intense research. However, the mechanisms underlying activation and regulation of ARF6 by GPCRs are poorly characterized. Here we report that Galpha(q) signaling leads to the activation of ARF6. Stimulation of the TPbeta receptor triggered ARF6 activation which was completely inhibited by the RGS domain of GRK2 known to specifically bind and sequester Galpha(q). Co-immunoprecipitation studies revealed that ARNO (a guanine nucleotide exchange factor for ARF6) and ARF6 formed complexes preferentially with activated Galpha(q) compared to non-activated Galpha(q). Formation of the Galpha(q) complexes with ARNO and ARF6 was detected early and was optimal after 30 min of receptor stimulation corresponding with the profile of ARF6 activation. Interestingly, binding experiments using purified proteins showed that Galpha(q) interacted directly with ARNO. Galpha(q)-dependent TPbeta receptor-mediated activation of ARF6 resulted in phosphoinositol-4,5-bisphosphate production which was potently inhibited by dominant negative mutants of ARNO and ARF6. Furthermore, our data show that the expression of ARNO and ARF6 promoted, whereas dominant negative mutants of these proteins inhibited the internalization of the TPbeta receptor. This further elucidates our previous data on the PLCbeta- and PKC-independent mechanism involved in Galpha(q)-mediated internalization of the TPbeta receptor. Taken altogether, our results support a novel model where activated Galpha(q) forms molecular complexes with ARNO and ARF6, possibly through a direct interaction with ARNO, leading to ARF6 activation.

Distinct motifs of neuropeptide Y receptors differentially regulate trafficking and desensitization.

Activated human neuropeptide Y Y(1) receptors rapidly desensitize and internalize through clathrin-coated pits and recycle from early and recycling endosomes, unlike Y(2) receptors that neither internalize nor desensitize. To identify motifs implicated in Y(1) receptor desensitization and trafficking, mutants with varying C-terminal truncations or a substituted Y(2) C-terminus were constructed. Point mutations of key putative residues were made in a C-terminal conserved motif [phi-H-(S/T)-(E/D)-V-(S/T)-X-T] that we have identified and in the second intracellular i2 loop. Receptors were analyzed by functional assays, spectrofluorimetric measurements on living cells, flow cytometry, confocal imaging and bioluminescence resonance energy transfer assays for beta-arrestin activation and adaptor protein (AP-2) complex recruitment. Inhibitory GTP-binding protein-dependent signaling of Y(1) receptors to adenylyl cyclase and desensitization was unaffected by C-terminal truncations or mutations, while C-terminal deletion mutants of 42 and 61 amino acids no longer internalized. Substitutions of Thr357, Asp358, Ser360 and Thr362 by Ala in the C-terminus abolished both internalization and beta-arrestin activation but not desensitization. A Pro145 substitution by His in an i2 consensus motif reported to mediate phosphorylation-independent recruitment of beta-arrestins affected neither desensitization, internalization or recycling kinetics of activated Y(1) receptors nor beta-arrestin activation. Interestingly, combining Pro145 substitution by His and C-terminal substitutions significantly attenuates Y(1) desensitization. In the Y(2) receptor, replacement of His155 with Pro at this position in the i2 loop motif promotes agonist-mediated desensitization, beta-arrestin activation, internalization and recycling. Overall, our results indicate that beta-arrestin-mediated desensitization and internalization of Y(1) and Y(2) receptors are differentially regulated by the C-terminal motif and the i2 loop consensus motif.

Unraveling G protein-coupled receptor endocytosis pathways using real-time monitoring of agonist-promoted interaction between beta-arrestins and AP-2.

The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves beta-arrestin and clathrin-coated pits. However, both beta-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that beta-arrestin binding to the beta2 subunit of the clathrin adaptor AP-2 (beta2-adaptin) is needed for the beta-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted beta-arrestin 1 and 2 interaction with beta2-adaptin, indicating a beta-arrestin- and clathrin-dependent endocytic process. Detailed analyses of beta-arrestin interactions with both the receptor and beta2-adaptin also allowed us to demonstrate that recruitment of beta-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between beta-arrestins and beta2-adaptin. However, both receptors recruited beta-arrestins upon agonist stimulation, suggesting a beta-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based beta-arrestin/beta2-adaptin interaction assay represents a novel biosensor to assess receptor activation.

Involvement of actin in agonist-induced endocytosis of the G protein-coupled receptor for thromboxane A2: overcoming of actin disruption by arrestin-3 but not arrestin-2.

The role of actin in endocytosis of G protein-coupled receptors is poorly defined. In the present study, we demonstrate that agents that depolymerize (latrunculin B and cytochalasin D) or stabilize (jasplakinolide) the actin cytoskeleton blocked agonist-induced endocytosis of the beta isoform of the thromboxane A(2) receptor (TPbeta) in HEK293 cells. This suggests that endocytosis of TPbeta requires active remodeling of the actin cytoskeleton. On the other hand, disruption of microtubules with colchicine did not affect endocytosis of the receptor. Expression of wild-type and mutant forms of the small GTPases RhoA and Cdc42 potently inhibited endocytosis of TPbeta, further indicating a role for the dynamic regulation of the actin cytoskeleton in this pathway. Agonist treatment of TPbeta in HEK293 cells resulted in the formation of actin stress fibers through Galpha(q/11) signaling. Because we previously showed that endocytosis of TPbeta is dependent on arrestins, we decided to explore the relation between arrestin-2 and -3 and actin in endocytosis of this receptor. Interestingly, we show that the inhibition of TPbeta endocytosis by the actin toxins in HEK293 cells was overcome by the overexpression of arrestin-3, but not of arrestin-2. These results indicate that the actin cytoskeleton is not essential in arrestin-3-mediated endocytosis of TPbeta. However, arrestin-3 could not promote endocytosis of the TPbetaY339A and TPbetaI343A carboxyl-terminal mutants when the actin cytoskeleton was disrupted. Our data provide new evidence that the actin cytoskeleton plays an essential role in TPbeta endocytosis. Furthermore, our work suggests the existence of actin-dependent and -independent arrestin-mediated pathways of endocytosis.

Galphaq-coupled receptor internalization specifically induced by Galphaq signaling. Regulation by EBP50.

In the present report, we investigated the effect of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) expression on the agonist-induced internalization of the thromboxane A(2) beta receptor (TPbeta receptor). Interestingly, we found that EBP50 almost completely blocked TPbeta receptor internalization, which could not be reversed by overexpression of G protein-coupled receptor (GPCR) kinases and arrestins. Because we recently demonstrated that EBP50 can bind to and inhibit Galpha(q), we next studied whether Galpha(q) signaling could induce TPbeta receptor internalization, addressing the long standing question about the relationship between GPCR signaling and their internalization. Expression of a constitutively active Galpha(q) mutant (Galpha(q)-R183C) resulted in a robust internalization of the TPbeta receptor, which was unaffected by expression of dominant negative mutants of arrestin-2 and -3, but inhibited by expression of EBP50 or dynamin-K44A, a dominant negative mutant of dynamin. Phospholipase Cbeta and protein kinase C did not appear to significantly contribute to internalization of the TPbeta receptor, suggesting that Galpha(q) induces receptor internalization through a phospholipase Cbeta- and protein kinase C-independent pathway. Surprisingly, there appears to be specificity in Galpha protein-mediated GPCR internalization. Galpha(q)-R183C also induced the internalization of CXCR4 (Galpha(q)-coupled), whereas it failed to do so for the beta(2)-adrenergic receptor (Galpha(s)-coupled). Moreover, Galpha(s)-R201C, a constitutively active form of Galpha(s), had no effect on internalization of the TPbeta, CXCR4, and beta(2)-adrenergic receptors. Thus, we showed that Galpha protein signaling can lead to internalization of GPCRs, with specificity in both the Galpha proteins and GPCRs that are involved. Furthermore, a new function has been described for EBP50 in its capacity to inhibit receptor endocytosis.

Role of the Rab11-associated intracellular pool of receptors formed by constitutive endocytosis of the beta isoform of the thromboxane A2 receptor (TP beta).

Intracellular trafficking pathways of G protein-coupled receptors (GPCRs), following their agonist-induced endocytosis and their consequences on receptor function, are the subject of intense research efforts. However, less is known regarding their constitutive endocytosis. We previously demonstrated that the beta isoform of the thromboxane A(2) receptor (TPbeta) undergoes constitutive and agonist-induced endocytosis. Constitutive endocytosis of GPCRs can lead to the formation of an intracellular pool of receptors from which they can recycle back to the cell surface. In the present report, we show with the help of two TPbeta mutants (TPbeta-Y339A and TPbeta-I343A) specifically deficient in constitutive endocytosis that this intracellular pool of receptors serves to maintain agonist sensitivity over prolonged receptor stimulation in HEK293 cells. Second messenger generation by the TPbeta-Y339A and TPbeta-I343A mutants was drastically reduced compared to the wild-type receptor as suggested by dose-response and time-course experiments of inositol phosphates production following agonist treatment, despite normal coupling between the receptors and the Galpha(q) protein. Moreover, second messenger production after receptor activation was dramatically reduced when cells were pretreated with monensin, a recycling inhibitor. Receptor cell surface expression and endocytosis experiments further revealed that the small GTPase Rab11 protein is a determinant factor in controlling TPbeta recycling back to the cell surface. Co-localization experiments performed by immunofluorescence microscopy indicated that both constitutive and agonist-triggered endocytosis resulted in targeting of TPbeta to the Rab11-positive recycling endosome. Thus, we provide evidence that constitutive endocytosis of TPbeta forms a pool of receptors in the perinuclear recycling endosome from which they recycle to the cell surface, a process involved in preserving receptor sensitivity to agonist stimulation.

Regulation of GTP-binding protein alpha q (Galpha q) signaling by the ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50).

Although ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ domain-containing protein known to bind to various channels, receptors, cytoskeletal elements, and cytoplasmic proteins, there is still very little evidence for a role of EBP50 in the regulation of receptor signal transduction. In this report, we show that EBP50 inhibits the phospholipase C (PLC)-beta-mediated inositol phosphate production of a Galpha(q)-coupled receptor as well as PLC-beta activation by the constitutively active Galpha(q)-R183C mutant. Coimmunoprecipitation experiments revealed that EBP50 interacts with Galpha(q) and to a greater extent with Galpha(q)-R183C. Agonist stimulation of the thromboxane A(2) receptor (TP receptor) resulted in an increased interaction between EBP50 and Galpha(q), suggesting that EBP50 preferentially interacts with activated Galpha(q). We also demonstrate that EBP50 inhibits Galpha(q) signaling by preventing the interaction between Galpha(q) and the TP receptor and between activated Galpha(q) and PLC-beta1. Investigation of the EBP50 regions involved in Galpha(q) binding indicated that its two PDZ domains are responsible for this interaction. This study constitutes the first demonstration of an interaction between a G protein alpha subunit and another protein through a PDZ domain, with broad implications in the regulation of diverse physiological systems.

Nm23-H2 interacts with a G protein-coupled receptor to regulate its endocytosis through an Rac1-dependent mechanism.

G protein-coupled receptors (GPCRs) represent a vast family of transmembrane proteins involved in the regulation of several physiological responses. The thromboxane A2 receptor (present as two isoforms: TP alpha and TP beta) is a GPCR displaying diverse pharmacological effects. As seen for many other GPCRs, TP beta is regulated by agonist-induced internalization. In the present study, we report the identification by yeast two-hybrid screening of Nm23-H2, a nucleoside diphosphate kinase, as a new interacting molecular partner with the C-terminal tail of TP beta. This interaction was confirmed in a cellular context when Nm23-H2 was co-immunoprecipitated with TP beta in HEK293 cells, a process dependent on agonist stimulation of the receptor. We observed that agonist-induced internalization of TP beta was regulated by Nm23-H2 through modulation of Rac1 signaling. Immunofluorescence microscopy in HEK293 cells revealed that Nm23-H2 had a cytoplasmic and nuclear localization but was induced to translocate to the plasma membrane upon stimulation of TP beta to show extensive co-localization with the receptor. Our findings represent the first demonstration of an interaction of an Nm23 protein with a membrane receptor and constitute a novel molecular regulatory mechanism of GPCR endocytosis.

Activation-independent parathyroid hormone receptor internalization is regulated by NHERF1 (EBP50).

Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes beta-arrestin/dynamin-mediated endocytosis in response to the biologically active forms of PTH, PTH-(1-34), and PTH-(1-84). We now show that amino-truncated forms of PTH that do not activate the PTH1R nonetheless induce PTH1R internalization in a cell-specific pattern. Activation-independent PTH1R endocytosis proceeds through a distinct arrestin-independent mechanism that is operative in cells lacking the adaptor protein Na/H exchange regulatory factor 1 (NHERF1) (ezrin-binding protein 50). Using a combination of radioligand binding experiments and quantitative, live cell confocal microscopy of fluorescently tagged PTH1Rs, we show that in kidney distal tubule cells and rat osteosarcoma cells, which lack NHERF1, the synthetic antagonist PTH-(7-34) and naturally circulating PTH-(7-84) induce internalization of PTH1R in a beta-arrestin-independent but dynamin-dependent manner. Expression of NHERF1 in these cells inhibited antagonist-induced endocytosis. Conversely, expression of dominant-negative forms of NHERF1 conferred internalization sensitivity to PTH-(7-34) in cells expressing NHERF1. Mutation of the PTH1R PDZ-binding motif abrogated interaction of the receptor with NHERF1. These mutated receptors were fully functional but were now internalized in response to PTH-(7-34) even in NHERF1-expressing cells. Removing the NHERF1 ERM domain or inhibiting actin polymerization allowed otherwise inactive ligands to internalize the PTH1R. These results demonstrate that NHERF1 acts as a molecular switch that legislates the conditional efficacy of PTH fragments. Distinct endocytic pathways are determined by NHERF1 that are operative for the PTH1R in kidney and bone cells.