Regulatory influence of the G-quadruplex or G4 motif present within the nuclease hypersensitive element (NHE) in the promoter of c-MYC has been noted. On the other hand, association of NM23-H2 to the NHE leads to c-MYC activation. Therefore, NM23-H2 interaction with the G4 motif within the c-MYC NHE presents an interesting mechanistic possibility. Herein, using luciferase reporter assay and chromatin immunoprecipitation we show NM23-H2 mediated c-MYC activation involves NM23-H2-G4 motif binding within the c-MYC NHE. G4 motif complex formation with recombinant NM23-H2 was independently confirmed using fluorescence energy transfer, which also indicated that the G4 motif was resolved to an unfolded state within the protein-bound complex. Taken together, this supports transcriptional role of NM23-H2 via a G4 motif.

Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes ␤-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 ␤-arrestin-independent but dynamindependent 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.Extracellular calcium homeostasis in vertebrate animals is primarily under the endocrine control of the parathyroid hormone (PTH) 1 /type I PTH receptor (PTH1R). The PTH1R, predominantly expressed in kidney and bone cells, belongs to class B of the large superfamily of G protein-coupled receptors (GPCRs) that consists of receptors for peptide hormones and neuropeptides (1). Class B GPCRs are characterized by a common topology and by their ability to couple to multiple signaling pathways via distinct G proteins.PTH is synthesized by the parathyroid glands as a mature peptide of 84 amino acids that is stored in secretory vesicles and dense core granules. Reductions of extracellular calcium levels are detected by the calcium-sensing receptor on parathyroid chief cells and promote the release of PTH, which acts on bone (to increase resorption) and kidney (to augment reabsorption), thereby restoring serum calcium levels. PTH-(1-84) is usually the major form of PTH secreted by the parathyroid glands. However, recent analyses reveal that PTH fragments that are likely to be PTH-(7-84) are also secreted by the parathyroid glands and generated by peripheral metabolism (2, 3). These PTH fragments or their synthetic a...

The thromboxane A 2 receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, ␣ (343 residues) and ␤ (407 residues) that share the first 328 residues. We have previously shown that TP␤, but not TP␣, undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestindependent manner. In the present report, we demonstrate that TP␤, but not TP␣, also undergoes tonic internalization. Tonic internalization of TP␤ was temperature-and dynamin-dependent and was inhibited by sucrose and NH 4 Cl treatment but unaffected by wildtype or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX 3 motif (where X is any residue and is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TP␤ was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX 2 or YX 3 motif in the carboxyl-terminal tail of TP␣ induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TP␤ undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TP␤. Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TP␤ and represent the first report of a YX 3 motif involved in tonic internalization of a cell surface receptor.Cell surface receptors provide a primary mechanism by which cells perceive their environment. Many cell surface receptors are dynamically regulated and often undergo a process of endocytic sorting (1). For some receptors (e.g. G proteincoupled and growth factor), sorting is often initiated by hormone binding, whereas for others (e.g. low density lipoprotein and transferrin), the receptors undergo continuous or tonic internalization and recycling. Recent studies have demonstrated that several GPCRs 1 including the CXCR4, thyrotropin, M 2 muscarinic, and thrombin receptors also undergo tonic internalization (2-5). Although no particular motif responsible for tonic internalization of GPCRs has been identified, tyrosine-containing (YXX and NPXY) and dileucine motifs have been shown to be determinants for a number of other receptor types (1). Various studies have demonstrated direct interaction between YXX motifs and the chain of the clathrin-associated proteins AP-1, AP-2 (Ref. 6 and references therein), and AP-3 (7, 8), allowing the efficient targeting of transmembrane proteins containing these motifs to clathrin-coated vesicles.Thromboxane has been implicated in a number of cardiovascular, bronchial, and kidney diseases (9, 10). It is produced by the sequential metabolism of arachidonic acid by cyclooxygenase and thromboxane synthase following activation of a variety of cell types including platelets, macrophages, and vascular smooth muscle cells (11). Thromboxane is a strong activator of platelet aggregation and smooth muscle cell proliferation and mediates its effects v...

The G protein-coupled chemokine receptor CXCR4 serves as the primary coreceptor for entry of T-cell tropic human immunodeficiency virus. CXCR4 undergoes tonic internalization as well as internalization in response to stimulation with phorbol esters and ligand (SDF-1␣). We investigated the trafficking of this receptor, and we attempted to define the residues of CXCR4 that were critical for receptor internalization. In both COS-1 and HEK-293 cells transiently overexpressing CXCR4, SDF-1␣ and phorbol esters (PMA) promoted rapid internalization of cell surface receptors as assessed by both enzyme-linked immunosorbent assay and immunofluorescence analysis. Expression of GRK2 and/or arrestins promoted modest additional CXCR4 internalization in response to both PMA and SDF. Both PMA-and SDF-mediated CXCR4 internalization was inhibited by coexpression of dominant negative mutants of dynamin-1 and arrestin-3. Arrestin was also recruited to the plasma membrane and appeared to colocalize with internalized receptors in response to SDF but not PMA. We then evaluated the ability of CXCR4 receptors containing mutations of serines and threonines, as well as a dileucine motif, within the C-terminal tail to be internalized and phosphorylated in response to either PMA or SDF-1␣. This analysis showed that multiple residues within the CXCR4 C-terminal tail appear to mediate both PMA-and SDF-1␣-mediated receptor internalization. The ability of coexpressed GRK2 and arrestins to promote internalization of the CXCR4 mutants revealed distinct differences between respective mutants and suggested that the integrity of the dileucine motif (Ile-328 and Leu-329) and serines 324, 325, 338, and 339 are critical for receptor internalization.

Thromboxane A 2 (TXA 2 ) potently stimulates platelet aggregation and smooth muscle constriction and is thought to play a role in myocardial infarction, atherosclerosis, and bronchial asthma. The TXA 2 receptor (TXA 2 R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, ␣ (343 residues) and ␤ (407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent assay and immunofluorescence microscopy that the TXA 2 R␤, but not the TXA 2 R␣, undergoes agonist-induced internalization when expressed in HEK293 cells as well as several other cell types. Various dominant negative mutants were used to demonstrate that the internalization of the TXA 2 R␤ is dynamin-, GRK-, and arrestin-dependent in HEK293 cells, suggesting the involvement of receptor phosphorylation and clathrin-coated pits in this process. Interestingly, the agonist-stimulated internalization of both the ␣ and ␤ isoforms, but not of a mutant truncated after residue 328, can be promoted by overexpression of arrestin-3, identifying the C-tails of both receptors as necessary in arrestin-3 interaction. Simultaneous mutation of two dileucine motifs in the C-tail of TXA 2 R␤ did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between residues 355 and 366 of the TXA 2 R␤ is essential for agonist-promoted internalization. These data demonstrate that alternative splicing of the TXA 2 R plays a critical role in regulating arrestin binding and subsequent receptor internalization.Thromboxane A 2 (TXA 2 ) 1 has a variety of pharmacologic effects which modulate the physiological responses of several cells and tissues (1). It is a product of the sequential metabolism of arachidonic acid by the cyclooxygenases and TXA 2 synthase (2). TXA 2 formation can result from activation of various cell types, including platelets, macrophages, and vascular smooth muscle cells (1). Binding of TXA 2 to its receptor (TXA 2 R) induces platelet aggregation, constriction of vascular and bronchiolar smooth muscle cells, as well as mitogenesis and hypertrophy of vascular smooth muscle cells. TXA 2 has been implicated in a wide variety of cardiovascular diseases (1).While pharmacological studies have suggested the existence of TXA 2 R subtypes (3), the receptor appears to be encoded by a single gene that can be alternatively spliced in the carboxylterminal tail (C-tail) leading to two variants, TXA 2 R␣ and -␤, that share the first 328 amino acids. Complementary DNAs for the 343-amino acid TXA 2 R␣ were cloned from placental and megakaryocytic sources (4), whereas a cDNA for the 407-amino acid TXA 2 R␤ was isolated from a vascular endothelial library (5). The TXA 2 Rs have been shown to couple to the G proteins G q , G i2 , G 11 , G 12 , G 13 , G 16 , and an 85-kDa unidentified G protein, explaining the multiplicity of TXA 2 R-mediated signal transduction (6 -11). While no isoform-specific biological functions have been a...

No abstract

The beta2ARs (beta(2)-adrenergic receptors) undergo ligand-induced internalization into early endosomes, but then are rapidly and efficiently recycled back to the plasma membrane, restoring the numbers of functional cell-surface receptors. Gathering evidence suggests that, during prolonged exposure to agonist, some beta2ARs also utilize a slow recycling pathway through the perinuclear recycling endosomal compartment regulated by the small GTPase Rab11. In the present study, we demonstrate by co-immunoprecipitation studies that there is a beta2AR-Rab11 association in HEK-293 cells (human embryonic kidney cells). We show using purified His(6)-tagged Rab11 protein and beta2AR intracellular domains fused to GST (glutathione transferase) that Rab11 interacts directly with the C-terminal tail of beta2AR, but not with the other intracellular domains of the receptor. Pull-down and immunoprecipitation assays revealed that the beta2AR interacts preferentially with the GDP-bound form of Rab11. Arg(333) and Lys(348) in the C-terminal tail of the beta2AR were identified as crucial determinants for Rab11 binding. A beta2AR construct with these two residues mutated to alanine, beta2AR RK/AA (R333A/K348A), was generated. Analysis of cell-surface receptors by ELISA revealed that the recycling of beta2AR RK/AA was drastically reduced when compared with wild-type beta2AR after agonist washout, following prolonged receptor stimulation. Confocal microscopy demonstrated that the beta2AR RK/AA mutant failed to co-localize with Rab11 and recycle to the plasma membrane, in contrast with the wild-type receptor. To our knowledge, the present study is the first report of a direct interaction between the beta2AR and a Rab GTPase, which is required for the accurate intracellular trafficking of the receptor.

Intracellular trafficking pathways of cell surface receptors following their internalization are the subject of intense research efforts. However, the mechanisms by which they recycle back to the cell surface are still poorly defined. We have recently demonstrated that the small Rab11 GTPase protein is a determinant factor in controlling the recycling to the cell surface of the ␤-isoform of the thromboxane A 2 receptor (TP␤) following its internalization. Here, we demonstrate with co-immunoprecipitation studies in HEK293 cells that there is a Rab11-TP␤ association occurring in the absence of agonist, which is not modulated by stimulation of TP␤. We show with purified TP␤ intracellular domains fused to GST and HISRab11 proteins that Rab11 interacts directly with the first intracellular loop and the C-tail of TP␤. Amino acids 335-344 of the TP␤ C-tail were determined to be essential for the interaction of Rab11 with this receptor domain. This identified sequence appears to be important in directing the intracellular trafficking of the receptor from the Rab5-positive intracellular compartment to the perinuclear recycling endosome. Interestingly, our data indicate that TP␤ interacts with the GDP-bound form, and not the GTP-bound form, of Rab11 which is necessary for recycling of the receptor back to the cell surface. To our knowledge, this is the first demonstration of a direct interaction between Rab11 and a transmembrane receptor.