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.     

Characterization of interactions of Na+/H+ exchanger regulatory factor-1 with the parathyroid hormone receptor and phospholipase C.

The Na(+)/H(+) exchanger regulatory factor-1 (NHERF1) is a molecular scaffold important for the signaling of the G-protein coupled receptor for the parathyroid hormone (PTH1R). The two PDZ (PSD-95, Discs-large, ZO1) domains of NHERF1 through association with the C-termini of PTH1R and phospholipase C enhance the signaling pathway associated with PTH. To examine these interactions, we have produced the individual PDZ1 and PDZ2 domains as well as the tandem PDZ1-PDZ2 domains (PDZ12) of NHERF1 and have characterized the binding affinities of the C-terminal motifs of PTH1R and PLCbeta using fluorescence anisotropy. Circular dichroism indicates that the PDZ1 and PDZ2 are properly folded. Based on fluorescence anisotropy we find that the C-terminus of PTH1R, containing ETVM, has similar affinities (approximately 10 microm) for both PDZ1 and PDZ2. The PTH1R displayed reduced binding affinity for the tandem PDZ12 (16 microm) compared with the individual domains or a solution of equal molar concentrations of PDZ1 and PDZ2 (5.8 microm), suggesting negative cooperativity between the PDZ domains or intervening region. The C-termini of PLCbeta (both beta1 and beta2 isozymes were examined, containing DTPL and ESRL, respectively) displayed a diminished affinity for PDZ2 (approximately 30 microm) over that of PDZ1 (approximately 8 microm). Finally, we demonstrate trans PDZ1-PDZ2 association that is enhanced in the presence of the C-terminus of PTH1R or PLCbeta, suggesting oligomerization of NHERF as a mode for enhancing the signaling associated with PTH.     

Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate.

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internalization upon activation. The parathyroid hormone (PTH) receptor undergoes endocytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investigate compartmentalization of the PTH receptor, we have established a stable cell line expressing a PTH receptor-green fluorescent protein (PTHR-GFP) conjugate and an algorithm to quantify PTH receptor internalization. HEK 293 cells expressing the PTHR-GFP were compared with cells expressing the wild-type PTH receptor in whole-cell binding and functional assays. 125I-PTH binding studies revealed similar Bmax and kD values in cells expressing either the PTHR-GFP or the wild-type PTH receptor. PTH-induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the cytoplasmic tail of the PTH receptor does not alter the ligand binding or G-protein coupling properties of the receptor. Using confocal fluorescence microscopy, we demonstrated that PTH treatment of cells expressing the PTHR-GFP conjugate produced a time-dependent redistribution of the receptor to the endosomal compartment which was blocked by pretreatment with PTH antagonist peptides. Treatment with hypertonic sucrose prevented PTH-induced receptor internalization, suggesting that the PTH receptor internalizes via a clathrin-dependent mechanism. Moreover, co-localization with internalized transferrin showed that PTHR-GFP trafficking utilized the endocytic recycling compartment. Experiments using cycloheximide to inhibit protein synthesis demonstrated that recycling of the PTHR-GFP back to the plasma membrane was complete within 1-2 h of ligand removal and was partially blocked by pretreatment with cytochalasin D, but not nocodazole. We also demonstrated that the PTH receptor, upon recycling to the plasma membrane, is capable of undergoing a second round of internalization, a finding consistent with a role for receptor recycling in functional resensitization.     

G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domain-mediated interaction.

The Na(+)/H(+) exchanger regulatory factor (NHERF) is constitutively phosphorylated in cells, but the site(s) of this phosphorylation and the kinase(s) responsible for it have not been identified. We show here that the primary site of constitutive NHERF phosphorylation in human embryonic kidney 293 (HEK-293) cells is Ser(289), and that the stoichiometry of phosphorylation is near 1 mol/mol. NHERF contains two PDZ domains that recognize the sequence S/T-X-L at the carboxyl terminus of target proteins, and thus we examined the possibility that kinases containing this motif might associate with and phosphorylate NHERF. Overlay experiments and co-immunoprecipitation studies revealed that NHERF binds with high affinity to a splice variant of the G protein-coupled receptor kinase 6, GRK6A, which terminates in the motif T-R-L. NHERF does not associate with GRK6B or GRK6C, alternatively spliced variants that differ from GRK6A at their extreme carboxyl termini. GRK6A phosphorylates NHERF efficiently on Ser(289) in vitro, whereas GRK6B, GRK6C, and GRK2 do not. Furthermore, the endogenous "NHERF kinase" activity in HEK-293 cell lysates is sensitive to treatments that alter the activity of GRK6A. These data suggest that GRK6A phosphorylates NHERF via a PDZ domain-mediated interaction and that GRK6A is the kinase in HEK-293 cells responsible for the constitutive phosphorylation of NHERF.     

The Na+/H+ exchanger regulatory factor stabilizes epidermal growth factor receptors at the cell surface

Ligand binding to cell surface receptors initiates both signal transduction and endocytosis. Although signaling may continue within the endocytic compartment, down-regulation is the major mechanism that controls the concentration of cell surface receptors, their ability to receive environmental signals, and the ultimate strength of biological signaling. Internalization, recycling, and trafficking of receptor tyrosine kinases (RTKs) within the endosome compartment are each regulated to control the overall process of down-regulation. We have identified the Na(+)/H(+) exchanger regulatory factor (NHERF) as an important molecular component that stabilizes epidermal growth factor receptors (EGFRs) at the cell surface to restrict receptor down-regulation. The NH(2)-terminal PDZ domain (PDZ 1) of NHERF specifically binds to an internal peptide motif located within the COOH-terminal regulatory domain of EGFR. Expression of NHERF slows the rate of EGF-induced receptor degradation. A point mutation that abolishes the PDZ 1 recognition sequence of EGFR enhances the rate of ligand-induced endocytosis and down-regulation of EGFR. Similarly, expression of a dominant negative mutant of NHERF enhances EGF-induced receptor down-regulation. In contrast to beta-adrenergic receptors where NHERF enhances recycling of internalized receptors, NHERF stabilizes EGFR at the cell surface and slows the rate of endocytosis without affecting recycling. Although the mechanisms differ, for both RTKs and G protein-coupled receptors, the overall effect of NHERF is to enhance the fraction of receptors present at the cell surface.     

Parathyroid hormone receptor trafficking contributes to the activation of extracellular signal-regulated kinases but is not required for regulation of cAMP signaling

Agonist-mediated activation of the type 1 parathyroid hormone receptor (PTH1R) results in several signaling events and receptor endocytosis. It is well documented that arrestins contribute to desensitization of both G(s)- and G(q)-mediated signaling and mediate PTH1R internalization. However, whether PTH1R trafficking directly contributes to signaling remains unclear. To address this question, we investigated the role of PTH1R trafficking in cAMP signaling and activation of extracellular signal-regulated kinases ERK1/2 in HEK-293 cells. Dominant negative forms of dynamin (K44A-dynamin) and beta-arrestin1 (beta-arrestin1-(319-418)) abrogated PTH1R internalization but had no effect on cAMP signaling; neither acute cAMP production by PTH nor desensitization and resensitization of cAMP signaling were affected. Therefore, PTH1R trafficking is not necessary for regulation of cAMP signaling. PTH-(1-34) induced rapid and robust activation of ERK1/2. A PTHrP-based analog ([p-benzoylphenylalanine1, Ile5,Arg(11,13),Tyr36]PTHrP-(1-36)NH2), which selectively activates the G(s)/cAMP pathway without inducing PTH1R endocytosis, failed to stimulate ERK1/2 activity. Inhibition of PTH1R endocytosis by K44A-dynamin dampened ERK1/2 activation in response to PTH-(1-34) by 69%. Incubation with the epidermal growth factor receptor inhibitor AG1478 reduced ERK1/2 phosphorylation further. In addition, ERK1/2 phosphorylation occurred following internalization of a PTH1R mutant induced by PTH-(7-34) in the absence of G protein signaling. Collectively, these data indicate that PTH1R trafficking and G(q) (but not G(s)) signaling independently contribute to ERK1/2 activation, predominantly via transactivation of the epidermal growth factor receptor.     

Arrestin scaffolds NHERF1 to the P2Y12 receptor to regulate receptor internalization

We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y(12) receptor (P2Y(12)R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y(12)R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y(12)R internalization. In vitro and prior to agonist stimulation P2Y(12)R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization.     

Type I PDZ ligands are sufficient to promote rapid recycling of G Protein-coupled receptors independent of binding to N-ethylmaleimide-sensitive factor

Molecular sorting of G protein-coupled receptors (GPCRs) between divergent recycling and lysosomal pathways determines the functional consequences of agonist-induced endocytosis. The carboxyl-terminal cytoplasmic domain of the beta2 adrenergic receptor (beta2AR) mediates both PDZ binding to Na+/H+ exchanger regulatory factor/ezrin/radixin/moesin-binding phosphoprotein of 50 kDa (NHERF/EBP50) family proteins and non-PDZ binding to the N-ethylmaleimide-sensitive factor (NSF). We have investigated whether PDZ interaction(s) are actually sufficient to promote rapid recycling of endocytosed receptors and, if so, whether PDZ-mediated sorting is restricted to the beta2AR tail or to sequences that bind NHERF/EBP50. The trafficking effects of short (10 residue) sequences differing in PDZ and NSF binding properties were examined using chimeric mutant receptors. The recycling activity of the beta2AR-derived tail sequence was not blocked by a point mutation that selectively disrupts binding to NSF, and naturally occurring PDZ ligand sequences were identified that do not bind detectably to NSF yet function as strong recycling signals. The carboxyl-terminal cytoplasmic domain of the beta1-adrenergic receptor, which does not bind either to NSF or NHERF/EBP50 and interacts selectively with a distinct group of PDZ proteins, promoted rapid recycling of chimeric mutant receptors with efficiency similarly high as that of the beta2AR tail. These results indicate that PDZ domain-mediated protein interactions are sufficient to promote rapid recycling of GPCRs, independent of binding to NSF. They also suggest that PDZ-directed recycling is a rather general mechanism of GPCR regulation, which is not restricted to a single GPCR, and may involve additional PDZ domain-containing protein(s) besides NHERF/EBP50.     

Protein interacting with C kinase 1 (PICK1) reduces reinsertion rates of interaction partners sorted to Rab11-dependent slow recycling pathway

The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β(2)-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.     

Cellular distribution of constitutively active mutant parathyroid hormone (PTH)/PTH-related protein receptors and regulation of cyclic adenosine 3',5'-monophosphate signaling by beta-arrestin2

PTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating protein kinase C and recruiting beta-arrestin2. We examined the role of beta-arrestin2 in regulating the cellular distribution and cAMP signaling of two constitutively active PTH1Rc mutants, H223R and T410P. Overexpression of a beta-arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells inhibited constitutive cAMP accumulation by H223R and T410P in a dose-dependent manner, as well as the response to PTH of both mutant and wild-type PTH1Rcs. The cellular distribution of PTH1Rc-GFP conjugates, fluorescent ligands, and ssarrestin2-GFP was analyzed by fluorescence microscopy in HEK-293T cells. In cells expressing either receptor mutant, a ligand-independent mobilization of beta-arrestin2 to the cell membrane was observed. In the absence of ligand, H223R and wild-type PTH1Rcs were mainly localized on the cell membrane, whereas intracellular trafficking of T410P was also observed. While agonists promoted beta-arrestin2-mediated endocytosis of bot PTH1Rc mutants, antagonists were rapidly internalized only with T410P. The protein kinases inhibitor, staurosporine, significantly decreased internalization of ligand-PTH1Rc mutant complexes, although the recruitment of beta-arrestin2 to the cell membrane was unaffected. Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the C-terminal cytoplasmic domain, agonists stimulated translocation of beta-arrestin2 to the cell membrane followed by ligand-receptor complex internalization without associated beta-arrestin2. In conclusion, cAMP signaling by constitutively active mutant and wild-type PTH1Rcs is inhibited by a receptor interaction with beta-arrestin2 on the cell membrane, possibly leading to uncoupling from G(s)alpha. This phenomenon is independent from protein kinases activity and the receptor C-terminal cytoplasmic domain. In addition, there are differences in the cellular localization and internalization features of constitutively active PTH1Rc mutants H223R and T410P.     

Regulation of parathyroid hormone type 1 receptor dynamics, traffic, and signaling by the Na+/H+ exchanger regulatory factor-1 in rat osteosarcoma ROS 17/2.8 cells

The effects of the expression of the Na+/H+ exchanger regulatory factor-1 (NHERF1) on the distribution, dynamics, and signaling properties of the PTH type 1 receptor (PTH1R) were studied in rat osteosarcoma cells ROS 17/2.8. NHERF1 had a dramatic effect on the subcellular distribution of PTH1R, promoting a substantial relocation of the receptor to regions of the plasma membrane located in very close proximity to cytoskeletal fibers. Direct interactions of NHERF1 with the PTH1R and the cytoskeleton were required for these effects, because they were abolished by 1) PTH1R mutations that impair NHERF1 binding, and 2) NHERF1 mutations that impair binding to the PTH1R or the cytoskeleton. NHERF1 reduced significantly the diffusion of the PTH1R by a mechanism that was also dependent on a direct association of NHERF1 with the PTH1R and the cytoskeleton. NHERF1 increased ligand-dependent production of cAMP and induced ligand-dependent rises in intracellular calcium. These effects on calcium were due to increased calcium uptake, as they were blocked by calcium channel inhibitors and by the addition of EGTA to the medium. These calcium effects were abolished by protein kinase A inhibition but phospholipase C inhibition was without effect. Based on these analyses, we propose that, in ROS cells, the presence of NHERF1 induces PTH-dependent calcium signaling by a cAMP-mediated mechanism that involves local protein kinase A-dependent activation of calcium channels.     

Alteration of intracellular histamine H2 receptor cycling precedes antagonist-induced upregulation

Long-term administration of a histamine H2 receptor (H2R) antagonist (inverse agonist) induces upregulation of H2R in parietal cells, which may be relevant to the rebound hypersecretion of gastric acid that occurs after withdrawal of treatment. The mechanisms underlying this effect are unknown. We hypothesized that the H2R upregulation could be related to receptor trafficking and used H2R-green fluorescent protein (H2R-GFP) to test the hypothesis. Human H2R-GFP was generated and functionally expressed in HEK-293 cells. Binding of the H2R antagonist [3H]tiotidine was performed to quantify H2R expression, and H2R-GFP was imaged in living cells by confocal and evanescent wave microscopy. The binding affinity of [3H]tiotidine was not significantly different between H2R-GFP- and wild-type H2R-expressing HEK-293 cells, both of which had constitutive activity of adenylate cyclase. Visualization of H2R-GFP revealed that the agonist-induced H2R internalization and the antagonist-induced recycling of the internalized H2R from the recycling endosome within 2 h. Long exposure to the antagonist increased GFP fluorescence in the plasma membrane and also induced upregulation of H2R-GFP estimated by the binding assay, whereas long exposure to the agonist enhanced degradative trafficking of H2R-GFP. We examined whether the upregulation reflected an increase in receptor synthesis. Treatment with antagonist did not augment H2R mRNA, and subsequent inhibition of protein synthesis by cycloheximide had no effect on H2R upregulation. These findings suggested that upon exposure to an antagonist (inverse agonist), the equilibrium between receptor endocytosis and recycling is altered before H2R upregulation, probably via suppressing H2R degradation.     

Constitutive cholesterol-dependent endocytosis of melanocortin-4 receptor (MC4R) is essential to maintain receptor responsiveness to α-melanocyte-stimulating hormone (α-MSH)

Melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor expressed in the hypothalamus where it controls feeding behavior. MC4R cycles constitutively and is internalized at the same rate in the presence or absence of stimulation by the agonist, melanocyte-stimulating hormone (α-MSH). This is different from other G-protein-coupled receptors, such as β(2)-adrenergic receptor (β(2)AR), which internalizes more rapidly in response to agonist stimulation. Here, it is found that in immortalized neuronal Neuro2A cells expressing exogenous receptors, constitutive endocytosis of MC4R and agonist-dependent internalization of β(2)AR were equally sensitive to clathrin depletion. Inhibition of MC4R endocytosis by clathrin depletion decreased the number of receptors at the cell surface that were responsive to the agonist, α-MSH, by 75%. Mild membrane cholesterol depletion also inhibited constitutive endocytosis of MC4R by ～5-fold, while not affecting recycling of MC4R or agonist-dependent internalization of β(2)AR. Reduced cholesterol did not change the MC4R dose-response curve to α-MSH, but it decreased the amount of cAMP generated per receptor number indicating that a population of MC4R at the cell surface becomes nonfunctional. The loss of MC4R function increased over time (25-50%) and was partially reversed by mutations at putative phosphorylation sites (T312A and S329A). This was reproduced in hypothalamic GT1-7 cells expressing endogenous MC4R. The data indicate that constitutive endocytosis of MC4R is clathrin- and cholesterol-dependent. MC4R endocytosis is required to maintain MC4R responsiveness to α-MSH by constantly eliminating from the plasma membrane a pool of receptors modified at Thr-312 and Ser-329 that have to be cycled to the endosomal compartment to regain function.     

Ezrin-radixin-moesin-binding phosphoprotein-50/Na+/H+ exchanger regulatory factor (EBP50/NHERF) blocks U50,488H-induced down-regulation of the human kappa opioid receptor by enhancing its recycling rate

We have investigated whether Ezrin-radixin-moesin (ERM)-binding phosphoprotein-50/Na(+)/H(+) exchanger regulatory factor (EBP50/NHERF), a PDZ domain-containing phosphoprotein, is associated with the human kappa opioid receptor (hkor) and whether it regulates the trafficking and signaling of the hkor. When expressed in CHO cells stably transfected with the FLAG-tagged hkor (FLAG-hkor), EBP50/NHERF co-immunoprecipitated with FLAG-hkor, and the PDZ domain I, but not the PDZ domain II, of EBP50/NHERF was involved in the interaction. Treatment with the agonist (-)-(trans)-3,4- dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclohexyl]benzeneacetamide (U50,488H) enhanced the association of EBP50/NHERF with FLAG-hkor. Expression of EBP50/NHERF, but not a truncated form lacking the ERM-binding domain, abolished U50,488H-induced down-regulation of FLAG-hkor, which was apparently due to an increase in the recycling rate of internalized receptors. However, expression of EBP50/NHERF did not affect U50,488H binding affinity and U50,488H-stimulated [(35)S]guanosine 5'-3-O-(thio)triphosphate binding and p42/p44 MAP kinase activation, nor did it affect U50,488H-induced desensitization and internalization of FLAG-hkor. To determine the motif of FLAG-hkor involved in EBP50/NHERF binding, we generated two mutants, FLAG-hkor-A and FLAG-hkor-EE, in which one Ala or two Glu residues were added to the C terminus, respectively. Neither FLAG-hkor-A nor FLAG-hkor-EE co-immunoprecipitated with EBP50/NHERF, and U50,488H-induced down-regulation of FLAG-hkor-A and FLAG-hkor-EE were not affected by expression of EBP50/NHERF. Thus, EBP50/NHERF binds to the C terminus of FLAG-hkor and blocks the down-regulation of FLAG-hkor. The C-terminal sequence of the hkor, NKPV, is distinctly different from the sequence D(S/T)XL, the optimal C-terminal motif in the beta(2)-adrenergic receptor for EBP50/NHERF binding. EBP50/NHERF may have a broader binding specificity and may interact with a subset of G protein-coupled receptors to serve as a recycling signal for these receptors.     

Role of the carboxyl-terminal region, di-leucine motif and cysteine residues in signalling and internalization of vasopressin V1a receptor.

The structural requirements for internalization and signalling of the vasopressin V1a receptor were investigated in stably transfected HEK-293 cells. Removal of the 51 C-terminal amino acids did not affect vasopressin binding, calcium signalling, heterologous desensitization or internalization of the receptor. Deletion of 14 additional amino acids reduced vasopressin-dependent calcium increase and impaired receptor internalization. Substitution of cysteines 371-372 did not affect intracellular signalling, but decreased endocytosis by 26%. Substitution of the 361-362 leucine by alanine residues reduced by 56% V1a receptor sequestration without affecting calcium signalling. These results indicate that di-cysteine and mostly di-leucine motifs present in the C-terminal region of the V1a receptor are involved in its internalization.     

Glucagon receptor recycling: role of carboxyl terminus, beta-arrestins, and cytoskeleton

Glucagon receptor (GR) activity and expression are altered in several diseases, including Type 2 diabetes. Previously, we investigated the mechanism of GR desensitization and internalization. The present study focused on the fate of internalized GR. Using both hamster hepatocytes and human embryonic kidney (HEK)-293 cells, we showed that internalized GR recycled to the plasma membrane within 30-60 min following stimulation of the cells with 100 nM glucagon. In HEK-293 cells and during recycling, GR colocalized with Rab4, Rab11, beta-arrestin1, beta-arrestin2, and actin filaments, in the cytosolic and/or perinuclear domains. Glucagon treatment triggered redistribution of actin filaments from the plasma membrane to the cytosol. GR coimmunoprecipitated with beta-actin in both hepatocytes and HEK-293 cells. Downregulation of beta-arrestin1 and beta-arrestin2 or disruption of the cytoskeleton inhibited recycling, but not internalization of GR. Deletion of the GR carboxyl-terminal 70 amino acids abolished internalization of GR in response to glucagon while deletion of the last 40 amino acids only did not affect GR internalization and recycling. After exposure of the cells to either high concentrations or prolonged duration of glucagon, GR colocalized with lysosomes. GR degradation was inhibited by lysosomal, but not proteosomal, inhibitors. In conclusion, GR recycles through Rab4- and Rab11- positive vesicles. The actin cytoskeleton, beta-arrestin1, beta-arrestin2, and the receptor's carboxyl terminus are involved in recycling. Prolonged stimulation with glucagon targets GR for degradation in lysosomes. Therefore, the present study provides a better understanding of the GR recycling mechanism, which could become useful in the treatment of certain diseases, including diabetes.     

GDC-0152-induced autophagy promotes apoptosis in HL-60 cells

Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), have pivotal roles in renal hemodynamics, neuroendocrine signaling, blood pressure regulation, and cardiovascular homeostasis. Binding of ANP and BNP to the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) induces rapid internalization and trafficking of the receptor via endolysosomal compartments, with concurrent generation of cGMP. However, the mechanisms of the endocytotic processes of NPRA are not well understood. The present study, using ¹²⁵I-ANP binding assay and confocal microscopy, examined the function of dynamin in the internalization of NPRA in stably transfected human embryonic kidney-293 (HEK-293) cells. Treatment of recombinant HEK-293 cells with ANP time-dependently accelerated the internalization of receptor from the cell surface to the cell interior. However, the internalization of ligand–receptor complexes of NPRA was drastically decreased by the specific inhibitors of clathrin- and dynamin-dependent receptor internalization, almost 85% by monodansylcadaverine, 80% by chlorpromazine, and 90% by mutant dynamin, which are specific blockers of endocytic vesicle formation. Visualizing the internalization of NPRA and enhanced GFP-tagged NPRA in HEK-293 cells by confocal microscopy demonstrated the formation of endocytic vesicles after 5 min of ANP treatment; this effect was blocked by the inhibitors of clathrin and by mutant dynamin construct. Our results suggest that NPRA undergoes internalization via clathrin-mediated endocytosis as part of its normal itinerary, including trafficking, signaling, and metabolic degradation.     

Na+/H+ Exchanger Regulatory Factor-1 Is Involved in Chemokine Receptor Homodimer CCR5 Internalization and Signal Transduction but Does Not Affect CXCR4 Homodimer or CXCR4-CCR5 Heterodimer

Chemokine receptors are members of the G protein-coupled receptor (GPCR) family. CCR5 is also the principal co-receptor for macrophage-tropic strains of human immunodeficiency virus, type 1 (HIV-1), and efforts have been made to develop ligands to inhibit HIV-1 infection by promoting CCR5 receptor endocytosis. Given the nature of GPCRs and their propensity to form oligomers, one can consider ligand-based therapies as unselective in terms of the oligomeric composition of complexes. For example, a ligand targeting a CCR5 homomer could likely induce signal transduction on a heteromeric CCR5-CXCR4. Other avenues could therefore be explored. We identified a receptor adaptor interacting specifically with one receptor complex but not others. NHERF1, an adaptor known for its role in desensitization, internalization, and regulation of the ERK signaling cascade for several GPCRs, interacts via its PDZ2 domain with the CCR5 homodimer but not with the CXCR4-CCR5 heterodimer or CXCR4 homodimer. To further characterize this interaction, we also show that NHERF1 increases the CCR5 recruitment of arrestin2 following stimulation. NHERF1 is also involved in CCR5 internalization, as we demonstrate that co-expression of constructs bearing the PDZ2 domain can block CCR5 internalization. We also show that NHERF1 potentiates RANTES (regulated on activation normal T cell expressed and secreted)-induced ERK1/2 phosphorylation via CCR5 activation and that this activation requires NHERF1 but not arrestin2. Taken together, our results suggest that oligomeric receptor complexes can associate specifically with partners and that in this case NHERF1 could represent an interesting new target for the regulation of CCR5 internalization and potentially HIV infection.     

The interaction between megalin and ClC-5 is scaffolded by the Na⁺-H⁺ exchanger regulatory factor 2 (NHERF2) in proximal tubule cells

Albumin endocytosis in the proximal tubule is mediated by a number of proteins, including the scavenger receptor megalin/cubilin and the PSD-95/Dlg/ZO-1 (PDZ) scaffolds NHERF1 and NHERF2. In addition, in a number of in vitro and in vivo models, the loss of ClC-5 results in a decreased cell surface expression and whole cell level of megalin, suggesting an interaction between these two proteins in vivo. We investigated if ClC-5 and megalin interact directly, and as ClC-5 binds to NHERF2, we investigated if this PDZ scaffold was required for a megalin/ClC-5 complex. GST-pulldown and immunoprecipitation experiments using rat kidney lysate demonstrated an interaction between ClC-5 and megalin, which was mediated by their C-termini. As this interaction may be controlled by a scaffold protein, we characterised any interaction between megalin and NHERF2. Immunoprecipitation experiments indicated that megalin interacts with NHERF2 in vivo, and that this interaction was via an internal NHERF binding domain in the C-terminus of megalin and PDZ2 and the C-terminus of NHERF2. Silencing NHERF2 had no effect on megalin protein levels in the whole cell or plasma membrane. Using siRNA against NHERF2, we demonstrated that NHERF2 was required to facilitate the interaction between megalin and ClC-5. Using fusion proteins, we characterised a protein complex containing ClC-5 and megalin, which is scaffolded by NHERF2, in the absence of any other proteins. Importantly, these observations are the first to describe an interaction between megalin and ClC-5, which is scaffolded by NHERF2 in proximal tubule cells.