The signal transducer and activator of transcription-3 (Stat3) protein is activated by the interleukin 6 (IL-6) family of cytokines, epidermal growth factor, and leptin. A protein named PIAS3 (protein inhibitor of activated STAT) that binds to Stat3 was isolated and characterized. The association of PIAS3 with Stat3 in vivo was only observed in cells stimulated with ligands that cause the activation of Stat3. PIAS3 blocked the DNA-binding activity of Stat3 and inhibited Stat3-mediated gene activation. Although Stat1 is also phosphorylated in response to IL-6, PIAS3 did not interact with Stat1 or affect its DNA-binding or transcriptional activity. The results indicate that PIAS3 is a specific inhibitor of Stat3.

The binding of cytokines to their cell surface receptors activates, by tyrosine phosphorylation, a family of latent cytoplasmic transcription factors termed STATs (signal transducer and activator of transcription). After cytokine receptor activation, STATs dimerize and translocate into the nucleus to activate genes. Seven members of the STAT family, activated by a variety of cytokines, have been cloned (1-5). Genetic knockout studies indicate that STATs have highly specific functions. Stat1, the founding member of the STAT family, is essential for innate response to either viral or bacterial infection (6, 7). Stat1 is phosphorylated on a single residue, Tyr-701, in response to stimulation by a number of ligands including interferons (IFNs), interleukin 6 (IL-6), and epidermal growth factor (8-11). The phosphorylation on the Tyr-701 residue of Stat1 is required for its nuclear translocation, dimerization, DNA binding, and gene activation (8,12).Although great progress has been made toward the understanding of STAT activation, little is known about how STAT signals are down-regulated. Several mechanisms to downregulate STAT signaling have been proposed. (i) Because the activities of STATs depend on tyrosine phosphorylation, the precise recognition and dephosphorylation of STATs by their protein tyrosine phosphatases (PTPases) is expected to be crucial for gene regulation (13-16). However, it is not known what and how PTPases can dephosphorylate STATs. (ii) Inhibitors of proteosome activity are shown to prolong the activation of Stat1, implying the involvement of ubiquitination in the degradation of Stat1 (17). However, because these inhibitors also affect the half-life of IFN receptor, the importance of the degradation of Stat1 through ubiquitination pathway remains unclear (4, 16). (iii) Recently, a family of cytokine-inducible inhibitors of signaling have been isolated (18)(19)(20). This family of proteins, named SOCS͞JAB͞SSI, are relatively small protein molecules that contain mainly SH2 domains. SOCS͞JAB͞SSI proteins can directly bind to JAKs and can inhibit the tyrosine kinase activity of JAKs.We have identified recently a protein named PIAS3 (protein inhibitor of activated Stat3) that functions as a specific inhibitor of Stat3 signaling (21). We report here the identification of four additional members of the PIAS family. We found that PIAS1 was associated with Stat1, but not with Stat2 or Stat3 in vivo in cells treated with IFN or IL-6. The PIAS1-Stat1 interaction requires the phosphorylation of Stat1 on Tyr-701. Furthermore, PIAS1 but not other PIAS proteins blocked the DNA binding activity of Stat1 and inhibited Stat1-mediated gene activation. Our results suggest that PIAS1 is a specific inhibitor of Stat1-mediated gene activation. The mode of the PIAS-mediated inhibition on STAT activity is distinct from other known inhibitory mechanisms involved in STAT signaling. MATERIALS AND METHODSCells. U3A and U3A-derived cell lines were maintained in DMEM containing 10% fetal bovine serum at 10% CO 2 . Human Daud...

Sphingosine 1-phosphate (S1P) influences heart rate, coronary artery caliber, endothelial integrity, and lymphocyte recirculation through five related high affinity G-protein-coupled receptors. Inhibition of lymphocyte recirculation by non-selective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the embryonic lethality of the S1P 1 knock-out and the unavailability of selective agonists or antagonists. A potent, S1P 1 -receptor selective agonist structurally unrelated to S1P was found to activate multiple signals triggered by S1P, including guanosine 5 -3-O-(thio)triphosphate binding, calcium flux, Akt and ERK1/2 phosphorylation, and stimulation of migration of S1P 1 -but not S1P 3 -expressing cells in vitro. The agonist also alters lymphocyte trafficking in vivo. Use of selective agonism together with deletant mice lacking S1P 3 receptor reveals that agonism of S1P 1 receptor alone is sufficient to control lymphocyte recirculation. Moreover, S1P 1 receptor agonist plasma levels are causally associated with induction and maintenance of lymphopenia. S1P 3 , and not S1P 1 , is directly implicated in sinus bradycardia. The sustained bradycardia induced by S1P receptor nonselective immunosuppressive agonists in wild-type mice is abolished in S1P 3 ؊/؊ mice, whereas S1P 1 -selective agonist does not produce bradycardia. Separation of receptor subtype usage for control of lymphocyte recirculation and heart rate may allow the identification of selective immunosuppressive S1P 1 receptor agonists with an enhanced therapeutic window. S1P 1 -selective agonists will be of broad utility in understanding cell functions in vitro, and vascular physiology in vivo, and the success of the chemical approach for S1P 1 suggests that selective tools for the resolution of function across this broad lipid receptor family are now possible.Sphingosine 1-phosphate (S1P), 1 through its high affinity G-protein-coupled receptors, is a physiological mediator with widespread effects upon multiple physiological systems (1). It regulates heart rate (2), coronary artery blood flow (3), blood pressure (4), endothelial integrity in lung (5, 6) and most recently has been shown to regulate the recirculation of lymphocytes (7-11). Many of the physiologically relevant functions occur in the low nanomolar range, including activation of endothelial nitric oxide synthase (12, 13), vasorelaxation (14), and inhibition of thymic egress and lymphocyte recirculation (11). Free plasma levels of S1P are tightly regulated by protein binding to albumin and high density lipoprotein to avoid the deleterious effects of systemic S1P receptor subtype activation at high concentrations of ligand, such as bradycardia and coronary artery vasospasm (3, 15). The choice of S1P, through its receptors, as an acute regulator of the number of blood lymphocytes may represent an interesting evolutionary choice by the immun...

The glucagon-like peptide (GLP)-1 receptor (GLP-1R) is a class B G protein–coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secreted from three major tissues in humans, enteroendocrine L cells in the distal intestine, α cells in the pancreas, and the central nervous system, which exerts important actions useful in the management of type 2 diabetes mellitus and obesity, including glucose homeostasis and regulation of gastric motility and food intake. Peptidic analogs of GLP-1 have been successfully developed with enhanced bioavailability and pharmacological activity. Physiologic and biochemical studies with truncated, chimeric, and mutated peptides and GLP-1R variants, together with ligand-bound crystal structures of the extracellular domain and the first three-dimensional structures of the 7-helical transmembrane domain of class B GPCRs, have provided the basis for a two-domain–binding mechanism of GLP-1 with its cognate receptor. Although efforts in discovering therapeutically viable nonpeptidic GLP-1R agonists have been hampered, small-molecule modulators offer complementary chemical tools to peptide analogs to investigate ligand-directed biased cellular signaling of GLP-1R. The integrated pharmacological and structural information of different GLP-1 analogs and homologous receptors give new insights into the molecular determinants of GLP-1R ligand selectivity and functional activity, thereby providing novel opportunities in the design and development of more efficacious agents to treat metabolic disorders.