During telomere replication in yeast, chromosome ends acquire an S-phase-specific overhang of the guanosine-rich strand. Here it is shown that in cells lacking Ku, a heterodimeric protein involved in nonhomologous DNA end joining, these overhangs are present throughout the cell cycle. In vivo cross-linking experiments demonstrated that Ku is bound to telomeric DNA. These results show that Ku plays a direct role in establishing a normal DNA end structure on yeast chromosomes, conceivably by functioning as a terminus-binding factor. Because Ku-mediated DNA end joining involving telomeres would result in chromosome instability, our data also suggest that Ku has a distinct function when bound to telomeres.

The strand of telomeric DNA that runs 5'-3' toward a chromosome end is typically G rich. Telomerase-generated G tails are expected at one end of individual DNA molecules. Saccharomyces telomeres acquire TG1-3 tails late in S phase. Moreover, the telomeres of linear plasmids can interact when the TG1-3 tails are present. Molecules that mimic the structures predicted for telomere replication intermediates were generated in vitro. These in vitro generated molecules formed telomere-telomere interactions similar to those on molecules isolated from yeast, but only if both ends that interacted had a TG1-3 tail. Moreover, TG1-3 tails were generated in vivo in cells lacking telomerase. These data suggest a new step in telomere maintenance, cell cycle-regulated degradation of the C1-3A strand, which can generate a potential substrate for telomerase and telomere-binding proteins at every telomere.

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...

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...