Platelets have a crucial role in the maintenance of normal haemostasis, and perturbations of this system can lead to pathological thrombus formation and vascular occlusion, resulting in stroke, myocardial infarction and unstable angina. ADP released from damaged vessels and red blood cells induces platelet aggregation through activation of the integrin GPIIb-IIIa and subsequent binding of fibrinogen. ADP is also secreted from platelets on activation, providing positive feedback that potentiates the actions of many platelet activators. ADP mediates platelet aggregation through its action on two G-protein-coupled receptor subtypes. The P2Y1 receptor couples to Gq and mobilizes intracellular calcium ions to mediate platelet shape change and aggregation. The second ADP receptor required for aggregation (variously called P2Y(ADP), P2Y(AC), P2Ycyc or P2T(AC)) is coupled to the inhibition of adenylyl cyclase through Gi. The molecular identity of the Gi-linked receptor is still elusive, even though it is the target of efficacious antithrombotic agents, such as ticlopidine and clopidogrel and AR-C66096 (ref. 9). Here we describe the cloning of this receptor, designated P2Y12, and provide evidence that a patient with a bleeding disorder has a defect in this gene. Cloning of the P2Y12 receptor should facilitate the development of better antiplatelet agents to treat cardiovascular diseases.

The eukaryotic upstream binding factor (UBF), recognizes the ribosomal RNA gene promoter and activates transcription mediated by RNA polymerase I through cooperative interactions with the species-specific factor, SL1. Isolation of complementary DNA clones and sequence analysis reveals similarities between DNA binding domains of human UBF (hUBF) and high mobility group (HMG) protein 1. Expression, cellular localization and in vitro transcription studies establish that cloned hUBF encodes a nucleolar factor that binds specifically to the upstream control element and core of the rRNA gene promoter to activate transcription in a binding site-dependent manner.

The toxicity of DNA-damaging agents is widely believed to result from the formation of lesions that block polymerases or disrupt the integrity of the genome. A mechanism heretofore not addressed is that DNA damage may titrate essential DNA-binding proteins away from their natural sites of action. This report shows that the ribosomal RNA (rRNA) transcription factor hUBF (human upstream binding factor) binds with striking affinity (Kd(app) (Kd(pp) 18 pM) to that measured for the c isplati n adduct. In addiion, we observe that the hUBF-promoter interaction is hly sensitive to the antagonistic effects of cisplathi-DNA adducts. These results suggest that a cisplatin-mediated transcription-factor-hiacking mechanism could disrupt rRNA synthesis, which is stimulated in proliferating cells.cis-Diamminedichloroplatinum(II) (cisplatin) is a widely used anticancer drug that is remarkably effective as a cure for testicular tumors (1). Cytotoxicity is believed to be mediated by cisplatin-DNA adducts, which include mainly 1,2-intrastrand d(GpG) (65%) and d(ApG) (25%) crosslinks and also 1,3-d(GpNpG) (6%) intrastrand crosslinks (2). Cisplatin-DNA adducts may exert their effects by inhibiting DNA and RNA synthesis (2) and by inducing programmed cell death (3). Despite this knowledge, an adequate mechanistic rationale for the significant chemotherapeutic efficacy of this drug remains elusive.Of possible importance to the cytotoxic mechanism of cisplatin is a family of cisplatin adduct-binding proteins (4, 5) that contain high mobility group (HMG) boxes (6-9). The HMG box is an 80-amino acid region that has conserved basic and aromatic residues and is the structural motif of a novel class of DNA-binding proteins (10-12). An unusual feature ofthe HMG domain is its affinity for noncanonical DNA structures with sharp angles, such as four-wayjunctions (13). Of interest is the observation that only the adducts ofclinically effective platinum anticancer drugs bind HMG box proteins (HMG-BPs) (7,14). It is believed that DNA duplex bending and unwinding induced by these cisplatin adducts provide the recognition cues for HMGBPs (6, 7). HMG1 binds selectively to 1,2 intrastrand cis-[Pt(NH3)212+-d(GpG) (GAG) and -d(ApG) crosslinks but lacks specificity for -1,3-d(GpNpG) crosslinks, indicating that the HMG box does not bind to all DNA structures bent by platinum coordination (7). The clinically inactive isomer of cisplatin, trans-daminedichloroplatinum(II), forms 1,3-but not 1,2-intrastrand crosslinks; consequently, DNA modified by this compound is not recognized by HMG-BPs (7,14). In addition to providing a useful system for studying structure-specific DNA recognition, the selective affinity of HMG-BPs for therapeutically effective cisplatin adducts has suggested a possible role for these proteins in the clinical efficacy of the drug.Most proteins that interact specifically with damaged DNA play a role in DNA repair. By contrast, HMG-BPs appear to function in processes unrelated to repair, such as transcriptional regulation and ...

Recent evidence suggests that transcription initiation by all three eukaryotic RNA polymerases involves a complex of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). Here, we map the functional domains of the nucleolar HMG box protein hUBF, which binds to the human rRNA promoter and stimulates transcription by RNA polymerase I through cooperative interactions with a distinct TBP-TAF complex, hSL1. DNase I footprint analysis of mutant hUBF proteins and of a synthetic peptide of 84 amino acids reveals that HMG box 1 is necessary and sufficient for DNA sequence specificity, whereas other HMG boxes and the amino terminus modulate the binding efficiency, hUBF contains multiple activation domains that include the acidic carboxyl terminus and three HMG boxes. HMG boxes 3 and 4 and the acidic tail contribute significantly to an extended footprinting pattern in the presence of hSL1, suggestive of specific protein-protein interactions. Moreover, the inability of xUBF from Xenopus laevis to form an initiation complex with hSL1 can be overcome by hybrid proteins containing human HMG box 4 and the acidic carboxyl terminus. These results strongly suggest an important role of transcription activation domains of hUBF in mediating interactions with the TBP-TAF complex hSL1. In recent years considerable progress has been made toward elucidating the molecular mechanisms of transcription initiation. In particular, a multitude of sequence-specific transcription factors that bind to cis-acting promoter and enhancer elements have been identified and their structure and function have been analyzed (Johnson and McKnight 1989;Mitchell and Tjian 1989). Moreover, a number of components of the basal transcriptional apparatus have been implicated as targets for activation (Sawadogo and Sentenac 1990;Greenblatt 1991;Roeder 1991; Pugh and Tjian 1992). Although domains involved in transcriptional activation by sequence-specific transcription factors have been identified, their targets in the basal transcriptional machinery remained elusive.The recent identification of coactivators and TATAbinding protein-associated factors (TAFs) that are associated with the TATA-binding protein (TBP) in the TFIID fraction of the RNA polymerase II (Pol II) system focused our attention on a novel class of transcription factors. At least some of these TAFs are required for activation by sequence-specific regulatory proteins Tjian 1990, 1991;Dynlacht et al. 1991;Tanese et al. 1991;Gill and Tjian 1992). Thus, there appear to be adaptor molecules that link the basal transcriptional apparatus to the activators. Several recent reports indicate that TBP is not only involved in transcription initiation by RNA Pol II but is also necessary for transcription by RNA polymerase III (Pol III) as well as RNA polymerase I (Pol I) (Lobo et al. 1991;Margottin et al. 1991;Simmen et al. 1991;Comai et al. 1992;Cormack and Struhl 1992;Schultz et al. 1992;White et al. 1992). These studies strongly support the unifying principle that in spite of vast differenc...

How can trans-activators with the same DNA binding specificity direct different transcriptional programs? The rRNA transcriptional apparatus offers a useful model system to address this question and to dissect the mechanisms that generate alternative transcription complexes. Here, we compare the mouse and human transcription factors that govern species-specific RNA polymerase I promoter recognition. We find that both human and mouse rRNA transcription is mediated by a specific multiprotein complex. One component of this complex is the DNA-binding transcription factor, UBF. Paradoxically, human and mouse UBF display identical DNA binding specificities even though transcription of rRNA is species specific. Promoter selectivity is conferred by a second essential factor, SL1, which, for humans, does not bind DNA independently but, instead, cooperates with UBF in the formation of high-affinity DNA-binding complexes. In contrast, mouse SLI can selectively interact with DNA in the absence of UBF. Reconstituted transcription experiments establish that UBF and RNA polymerase I from the two species are functionally interchangeable, whereas mouse and human SL1 exhibit distinct DNA binding and transcription activities. Together, these results suggest a critical role for a specific multiprotein assembly in RNA polymerase I promoter recognition and reveal distinct mechanisms through which such complexes can generate functional diversity.[Key Words: RNA pol I; rRNA transcription; species specificity; transcription factors; UBF; SL1] Received February 7, 1990; revised version accepted March 14, 1990.Transcriptional initiation in eukaryotic cells is a highly regulated process requiring the correct association of numerous proteins into a specific complex with RNA polymerase {Mitchell and Tjian 1989; Saltzman and Weinmann 1989). Although mapping of essential promoter elements has identified multiple DNA sequences important for template recognition, how the different transcription factors work together with RNA polymerase to select these sequences as sites of initiation is poorly understood. Current results suggest strongly that the DNA sequence specificity of a single protein cannot account for promoter recognition by any of the three cellular RNA polymerases {Yoshinaga et al. 1987; Murphy et al. 1989; Smale and Baltimore 19891.Instead, it seems that the interactions of multiple proteins, both with the DNA and with one another, are required to generate the observed selectivity of initiation.The specificity of RNA polymerase I (RNA pol I} transcription is well suited for studies of promoter recognition. Unlike RNA pol II and RNA pol III, which recognize a wide variety of promoters, RNA pol I apparently initiates from only a single type of promoter in the cell, that of the large rRNA gene . This limited promoter range is reflected in the stringent species specificity of RNA pol I transcription.Studies using either intact cells or in vitro transcription extracts indicate that whereas very closely related species [e.g., mouse and rat)...