Protease-activated receptors 1-3 (PAR1, PAR2, and PAR3) are members of a unique G protein-coupled receptor family. They are characterized by a tethered peptide ligand at the extracellular amino terminus that is generated by minor proteolysis. A partial cDNA sequence of a fourth member of this family (PAR4) was identified in an expressed sequence tag database, and the full-length cDNA clone has been isolated from a lymphoma Daudi cell cDNA library. The ORF codes for a seven transmembrane domain protein of 385 amino acids with 33% amino acid sequence identity with PAR1, PAR2, and PAR3. A putative protease cleavage site (Arg-47͞Gly-48) was identified within the extracellular amino terminus. COS cells transiently transfected with PAR4 resulted in the formation of intracellular inositol triphosphate when treated with either thrombin or trypsin. A PAR4 mutant in which the Arg-47 was replaced with Ala did not respond to thrombin or trypsin. A hexapeptide (GYPGQV) representing the newly exposed tethered ligand from the amino terminus of PAR4 after proteolysis by thrombin activated COS cells transfected with either wild-type or the mutant PAR4. Northern blot showed that PAR4 mRNA was expressed in a number of human tissues, with high levels being present in lung, pancreas, thyroid, testis, and small intestine. By f luorescence in situ hybridization, the human PAR4 gene was mapped to chromosome 19p12.

The development of blood cells including expansion of megakaryocyte progenitor cells requires the interplay of marrow stromal cells and polypeptide cytokines. Recently, characterization of c-Mpl, the receptor encoded by the proto-oncogene c-mpl, revealed structural homology with the haematopoietic cytokine receptor family, and its involvement in megakaryocyte development. We report here that the ligand for c-Mpl is relatively lineage specific, works both alone and synergistically with early acting cytokines to support megakaryocyte colony formation, and acts at a late stage of development to increase megakaryocyte size, polyploidization and expression of differentiation markers. In vivo, c-Mpl ligand stimulates platelet production by greatly expanding marrow and splenic megakaryocytes and their progenitors, and by shifting the distribution of megakaryocyte ploidy to higher values. Thus, as c-Mpl ligand has the expected characteristics of the major regulator of megakaryocyte development, we propose that it be termed thrombopoietin.

The major regulator of circulating platelet levels is believed to be a cytokine termed thrombopoietin. It is thought to be a lineage-specific cytokine affecting the proliferation and maturation of committed cells resulting in the production of megakaryocytes and platelets. Despite considerable efforts by a number of laboratories, the unequivocal identification of thrombopoietin has proven elusive. Here we report the functional cloning of a murine complementary DNA encoding a ligand for the receptor encoded by the c-mpl proto-oncogene (c-Mpl). The encoded polypeptide has a predicted molecular mass of 35,000 (M(r) 35K). The protein has a novel two-domain structure with an amino-terminal domain homologous with erythropoietin and a carboxy-terminal domain rich in serine, threonine and proline residues and containing seven potential N-linked glycosylation sites. Intraperitoneal injections of mice with recombinant protein increase circulating platelet levels by greater than fourfold after 7 days. These results along with those presented in the accompanying report strongly suggest that the ligand for c-Mpl is thrombopoietin.

Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z-DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin.

Correction of the obese state induced by genetic leptin deficiency reduces elevated levels of both blood glucose and hypothalamic neuropeptide Y (NPY) mRNA in ob/ob mice. To determine whether these responses are due to a specific action of leptin or to the reversal of the obese state, we investigated the specificity of the effect of systemic leptin administration to ob/ob mice (n = 8) on levels of plasma glucose and insulin and on hypothalamic expression of NPY mRNA. Saline-treated controls were either fed ad libitum (n = 8) or pair-fed to the intake of the leptin-treated group (n = 8) to control for changes of food intake induced by leptin. The specificity of the effect of leptin was further assessed by 1) measuring NPY gene expression in db/db mice (n = 6) that are resistant to leptin, 2) measuring NPY gene expression in brain areas outside the hypothalamus, and 3) measuring the effect of leptin administration on hypothalamic expression of corticotropin-releasing hormone (CRH) mRNA. Five daily intraperitoneal injections of recombinant mouse leptin (150 micrograms) in ob/ob mice lowered food intake by 56% (P < 0.05), body weight by 4.1% (P < 0.05), and levels of NPY mRNA in the hypothalamic arcuate nucleus by 42.3% (P < 0.05) as compared with saline-treated controls. Pair-feeding of ob/ob mice to the intake of leptin-treated animals produced equivalent weight loss, but did not alter expression of NPY mRNA in the arcuate nucleus. Leptin administration was also without effect on food intake, body weight, or NPY mRNA levels in the arcuate nucleus of db/db mice. In ob/ob mice, leptin did not alter NPY mRNA levels in cerebral cortex or hippocampus or the expression of CRH mRNA in the hypothalamic paraventricular nucleus (PVN). Leptin administration to ob/ob mice also markedly reduced serum glucose (8.3 +/- 1.2 vs. 24.5 +/- 3.8 mmol/l; P < 0.01) and insulin levels (7,263 +/- 1,309 vs. 3,150 +/- 780 pmol/l), but was ineffective in db/db mice. Pair-fed mice experienced reductions of glucose and insulin levels that were < 60% of the reduction induced by leptin. The results suggest that in ob/ob mice, systemic administration of leptin inhibits NPY gene overexpression through a specific action in the arcuate nucleus and exerts a hypoglycemic action that is partly independent of its weight-reducing effects. Furthermore, both effects occur before reversal of the obesity syndrome. Defective leptin signaling due to either leptin deficiency (in ob/ob mice) or leptin resistance (in db/db mice) therefore leads directly to hyperglycemia and the overexpression of hypothalamic NPY that is implicated in the pathogenesis of the obesity syndrome.

A human genomic DNA library was screened for the gene for protein C by using a cDNA probe coding for the human protein. Three different overlapping A Charon 4A phage were isolated that contain inserts for the gene for protein C. The complete sequence of the gene was determined by the dideoxy method and shown to span about 11 kilobases of DNA. The coding and 3' noncoding portion of the gene consists of eight exons and seven introns. The eight exons code for a preproleader sequence of 42 amino acids, a light chain of 155 amino acids, a connecting dipeptide of Lys-Arg, and a heavy chain of 262 amino acids. The preproleader sequence and the connecting dipeptide are removed during processing, resulting in the mature protein composed of a heavy and a light chain held together by a disulfide bond. The heavy chain also contains the catalytic region for the serine protease. Two Alu sequences and two homologous repeats of about 160 nucleotides were found in intron E. The seven introns in the gene for protein C are located in essentially the same positions in the amino acid sequence as the seven introns in the gene for human factor IX, while the first three introns in protein C are located in the same positions as the first three in the gene for human prothrombin.Protein C is a precursor to a serine protease present in plasma that plays an important physiological role in the regulation of blood coagulation (1,2). Human protein C is a vitamin K-dependent glycoprotein containing nine residues ofcarboxyglutamic acid and one equivalent of p-hydroxyaspartic acid. Protein C shows considerable structural homology with the other vitamin K-dependent plasma proteins involved in blood coagulation, including prothrombin, factor VII, factor IX, and factor X. Protein C is synthesized as a single-chain polypeptide that undergoes considerable processing to give rise to a two-chain molecule held together by a disulfide bond. The two-chain form is converted to activated protein C by thrombin by the cleavage of a 12-residue peptide from the amino terminus of the heavy chain (2). This reaction is greatly accelerated by the presence of thrombomodulin (3). Activated protein C regulates the coagulation process by the inactivation of factor Va (4, 5) and factor VIIIa (4, 6) by minor proteolysis. Consequently, individuals lacking protein C often have a history of thrombotic disease (7,8).Studies from our laboratory (9) and that of others (10) have led to the isolation and characterization of the cDNA coding for human and bovine protein C. In the present investigation, the cDNA for human protein C has been used for the isolation of overlapping genomic clones from a X Charon 4A phage library. The nucleotide sequence of the gene was then determined and compared with the genes for human factor LX (11, 12) and prothrombin (13).MATERIALS AND METHODS Screening of the Genomic Library. A human genomic library in X Charon 4A phage (14) was screened for genomic clones of human protein C by the plaque hybridization procedure ofBenton and Davis as modi...

To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 jpg reduced 24-h chow consumption in a dose-dependent manner from values of 81±6.8% of control (10-pmg dose, P = 0.04) to 29±7.7% of control (100-pug dose, P < 0.0001). Daily injections of 80 pzg of ob protein into six ob/ob mice for 2 wk led to an 11±1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26±4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content.(J. Clin.

Interleukin 21 (IL-21) has recently been identified as a multifunctional cytokine that induces the proliferation of T cells and B cells and differentiation of natural killer cells. To determine whether IL-21 regulates IL-4–mediated immune responses, we examined the effect of IL-21 on antigen-specific IgE production in mice. We also examined the effect of IL-21 on IL-4–induced IgE production from B cells and antigen-induced T-helper 2 (Th2) cell differentiation. The in vivo injection of IL-21 prevented antigen-specific IgE but not IgG2a production on immunization. IL-21 did not affect Th2 cell differentiation or IL-4 production from CD4+ T cells but directly inhibited IL-4–induced IgE production from B cells at single-cell levels. Moreover, IL-21 inhibited IL-4–induced germ line Cε transcription in B cells without the inhibition of signal transducer and activator of transcription 6 (Stat6) activation. Taken together, these results indicate that IL-21 down-regulates IgE production from IL-4–stimulated B cells through the inhibition of germ line Cε transcription and thus suggest that IL-21 may be useful for the treatment of IgE-dependent allergic diseases.