A human gene encoding an interferon-induced 15-kDa protein has been isolated from a genomic library. The gene appears to be single-copy and is composed of two exons, the first of which contains the ATG translation initiation codon. In vitro nuclear run-on assays showed that the transcription rate of the gene is stimulated after interferon treatment. To analyze transcriptional regulatory sequences, we constructed recombinant plasmids for use in transient transfection assays of HeLa cells. Constructs containing 115 nucleotides 5' to the transcription initiation site were found to be fully inducible by interferon. Assays of deletion mutants identified a critical element for interferon induction located between -115 and -96, just upstream of the "CCAAT box." Moreover, a DNA fragment including this region can confer interferon inducibility on a heterologous promoter (thymidine kinase) when cloned in either orientation upstream of the gene or downstream of the gene. These are properties characteristic of an enhancer element that is active only after treatment with interferon. This regulatory sequence may be shared by a group of interferon-induced genes, since a very similar sequence is present within the functional region near the RNA start site of another interferon-induced gene.Many polypeptide hormones, such as the interferons, exert their effect by binding to plasma membrane receptors and, subsequently, affecting the transcriptional activity of a specific set of genes (1-5). The mechanism by which this cell surface signal is transduced into the nucleus, causing an alteration of gene expression, remains an active area of investigation. We are studying this transduction by analyzing transcriptional control elements that respond to interferon treatment.In this report we describe the genomic cloning and functional characterization of an interferon-sensitive transcription unit encoding a 15-kDa protein (6). Interferon treatment results in a transient stimulation of transcriptional activity of the gene, as has been demonstrated for other interferonsensitive genes (4, 7). We therefore refer to the group as ISG (interferon-stimulated gene) and to this member as ISGCS.Characterization of the interferon-responsive DNA element of ISGI5 was performed by DNA transfection assays using recombinant hybrid clones. To distinguish between expression of the transfected and the endogenous gene, we made recombinants that contained the upstream region ofISG15 (to nucleotide +44) fused with the 3' region of the adenovirus EJB gene. Deletions from the 5' sequences of the basic construct revealed that the region from -115 to -% (relative to the transcription initiation site) was required for interferon stimulation of the promoter. Comparison of this DNA sequence to the region also known to confer interferonsensitivity to another gene (ISG54, ref. 8) showed a conserved oligonucleotide stretch. In each case the conserved region in the two genes is near the "CAAT box" but is in the opposite orientation in the two genes.This regulatory...

The glandular kallikrein gene family comprises 25-30 highly homologous genes that encode specific proteases involved in the processing of biologically active peptides. In the mouse all the members of this family are closely linked on chromosome 7. The 9.5-kilobase nucleotide sequence of a mouse genomic clone contains one complete kallikrein gene (mGK-1), which is expressed in the male mouse submaxillary gland, and the 3' end of another (mGK-2). Differences in the coding potential of these genes and the amino acid sequences of other known kallikreins seem to be functionally related to the substrate specificity of the different enzymes.

b-Adrenoceptors (ARs) classically mediate responses to the endogenous ligands adrenaline and noradrenaline by coupling to Gsa and stimulating cAMP production; however, drugs designed as b-AR agonists or antagonists can activate alternative cell signalling pathways, with the potential to influence clinical efficacy. Furthermore, drugs acting at b-ARs have differential capacity for pathway activation, described as stimulus trafficking, biased agonism, functional selectivity or ligand-directed signalling. These terms refer to responses where drug A has higher efficacy than drug B for one signalling pathway, but a lower efficacy than drug B for a second pathway. The accepted explanation for such responses is that drugs A and B have the capacity to induce or stabilize distinct active conformations of the receptor that in turn display altered coupling efficiency to different effectors. This is consistent with biophysical studies showing that drugs can indeed promote distinct conformational states. Agonists acting at b-ARs display ligand-directed signalling, but many drugs acting as cAMP antagonists are also able to activate signalling pathways central to cell survival and proliferation or cell death. The observed complexity of drug activity at b-ARs, prototypical G protein-coupled receptors, necessitates rethinking of the approaches used for screening and characterization of novel therapeutic agents. Most studies of ligand-directed signalling employ recombinant cell systems with high receptor abundance. While such systems are valid for examining upstream signalling events, such as receptor conformational changes and G protein activation, they are less robust when comparing downstream signalling outputs as these are likely to be affected by complex pathway interactions. /calmodulin-dependent protein kinase; catechol, 1,2-benzenediol; CHO-K1, Chinese hamster ovary; CPB, N-cyclopentylbutanephrine; ECAR, extracellular acidification rate; ECL, extracellular loop; EGF, epidermal growth factor; ERK, extracellular-regulated kinase; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; Gs, guanine nucleotide-binding protein that stimulates AC; Gi, guanine nucleotide-binding protein that inhibits AC; GSK-3, glycogen synthase kinase-3; GTPgS, guanosine 5′-O-(thiotriphosphate); HEK, human embryonic kidney; IBMX, 3-isobutyl-1-methylxanthine; IGF, insulin-like growth factor; ISO, isoprenaline; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; MAPK, mitogen-activated protein kinase; MEF, mouse embryonic fibroblast; MMP, matrix metalloprotease; NA, noradrenaline; PDE, phosphodiesterase; PDGF, platelet-derived growth factor; PDZ, post-synaptic density protein (PSD95)/Drosophila disc large tumor suppressor (DlgA)/ zonula occludens-1 protein (zo- British Journal of Pharmacology

1 This study characterizes the mouse b 3a -adrenoceptor (AR) and the splice variant of the b 3 -AR (b 3b -AR) expressed in Chinese hamster ovary cells (CHO-K1). 2 Stable clones with high (*1200), medium (*500) or low receptor expression (*100 fmol mg protein 71 ) were determined by saturation binding with [ 125 I]-(7)-cyanopindolol. Competition binding studies showed no signi®cant di erences in a nity of b-AR ligands for either receptor. 3 Several functional responses of each receptor were measured, namely extracellular acidi®cation rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The b 3 -AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non-conventional partial b-AR agonist CGP12177 and the b-AR agonist (7)-isoprenaline caused concentrationdependent increases in EAR in cells expressing either splice variant. CL316243 caused concentrationdependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor. 4 PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the b 3b -AR but not in cells expressing the b 3a -AR at all levels of receptor expression. 5 CL316243 increased Erk1/2 phosphorylation with pEC 50 values and maximum responses that were not signi®cantly di erent in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3Kg inhibitor), PP2 (c-Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). 6 The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both b 3a -or b 3b -AR transfected cells. 7 These results suggest that in CHO-K1 cells, the b 3b -AR, can couple to both G s and G i to stimulate and inhibit cyclic AMP production respectively, while the b 3a -AR, couples solely to G s to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to G i or the generation of cyclic AMP.

1 The receptor that mediates the increase in glucose transport (GT) in response to b-adrenoceptor (b-AR) agonists was characterized in the rat skeletal muscle cell line L6, using the 2-deoxy- The pharmacological analysis was supported by reverse transcription and polymerase chain reaction analysis of L6 mRNA, which showed high levels of expression of b 2 -AR but not b 1 -or b 3 -AR in these cells. 5 Forskolin and dibutyryl cyclic AMP produced negligible increases in GT while the phosphatidylinositol-3 kinase inhibitor, wortmannin, signi®cantly decreased both insulin-and zinterol-stimulated GT, suggesting a possible interaction between the insulin and b 2 -AR pathways. 6 This study demonstrates that b 2 -ARs mediate the increase in GT in L6 cells to b-AR agonists, including the b 3 -AR selective agonist BRL 37344. This e ect does not appear to be directly related to increases in cyclic AMP but requires P13K.

Although G protein-coupled receptors are often categorized in terms of their primary coupling to a given type of G␣ protein subunit, it is now well established that many show promiscuous coupling and activate multiple signaling pathways. Furthermore, some agonists selectively activate signaling pathways by promoting interaction between distinct receptor conformational states and particular G␣ subunits or alternative signaling proteins. We have tested the capacity of agonists to stimulate Ca 2ϩ release, cAMP accumulation, and changes in extracellular acidification rate (ECAR) at the human ␣ 1A -adrenoceptor. Signaling bias factors were determined by novel application of an operational model of agonism and compared with the reference endogenous agonist norepinephrine; values significantly different from 1.0 indicated an agonist that promoted receptor conformations distinct from that favored by norepinephrine. Oxymetazoline was a full agonist for ECAR and a partial agonist for Ca 2ϩ release (bias factor 8.2) but failed to stimulate cAMP production. Phenylephrine showed substantial bias toward ECAR versus Ca 2ϩ release or cAMP accumulation (bias factors 21 and 33, respectively) but did not display bias between Ca 2ϩ and cAMP pathways. Cirazoline and N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide (A61603) displayed bias toward cAMP relative to Ca 2ϩ release (bias factors of 7.4 and 8.6). It is noteworthy that epinephrine, a second endogenous adrenoceptor agonist, did not display bias relative to norepinephrine. Our finding that phenylephrine displayed significant signaling bias, despite being highly similar in structure to epinephrine, indicates that subtle differences in agonist-receptor interaction can affect conformational changes in cytoplasmic domains and thereby modulate the repertoire of effector proteins that are activated.

1 b-adrenoceptor (AR) agonists increase 2-deoxy-[ 3 H]-D-glucose uptake (GU) via b 2 -AR in rat L6 cells. The b-AR agonists, zinterol (b 2 -AR) and (À)-isoprenaline, increased cAMP accumulation in a concentration-dependent manner (pEC 50 ¼ 9.170.02 and 7.870.02). Cholera toxin (% max increase 141.872.5) and the cAMP analogues, 8-bromo-cAMP (8Br-cAMP) and dibutyryl cAMP (dbcAMP), also increased GU (196.8713.5 and 196.4717.3%). 2 The adenylate cyclase inhibitor, 2 0 ,5 0 -dideoxyadenosine (50 mM), significantly reduced cAMP accumulation to zinterol (100 nM) (109.7 þ 35.0 to 21.6 þ 4.5 pmol well À1 ), or forskolin (10 mM) (230.1758.0 to 107.2726.3 pmol well À1 ), and partially inhibited zinterol-stimulated GU (217726.3 to 176.1720.4%). The protein kinase A (PKA) inhibitor, 4-cyano-3-methylisoquinoline (100 nM), did not inhibit zinterol-stimulated GU. The PDE4 inhibitor, rolipram (10 mM), increased cAMP accumulation to zinterol or forskolin, and sensitised the GU response to zinterol, indicating a stimulatory role of cAMP in GU. 3 cAMP accumulation studies indicated that the b 2 -AR was desensitised by prolonged stimulation with zinterol, but not forskolin, whereas GU responses to zinterol increased with time, suggesting that receptor desensitisation may be involved in GU. Receptor desensitisation was not reversed by inhibition of PKA or Gi. 4 PTX pretreatment (100 ng ml À1 ) inhibited insulin or zinterol-stimulated but not 8Br-cAMP or dbcAMP-stimulated GU. The PI3K inhibitor, LY294002 (1 mM), inhibited insulin-(174.975.9 to 142.772.7%) and zinterol-(166.977.6 to 141.178.1%) but not 8 Br-cAMP-stimulated GU. In contrast to insulin, zinterol did not cause phosphorylation of Akt. 5 The results suggest that GU in L6 cells involves three mechanisms: (1) an insulin-dependent pathway involving PI3K, (2) a b 2 -AR-mediated pathway involving both cAMP and PI3K, and (3) a receptor-independent pathway suggested by cAMP analogues that increase GU independently of PI3K. PKA appears to negatively regulate b 2 -AR-mediated GU.

1 This study examines the expression of f3-adrenoceptor messenger RNA (fl3-AR mRNA) in rat tissues to allow comparison with atypical f,-adrenoceptors determined by functional and radioligand binding techniques. 2 A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of fl3-AR mRNA in rat tissues. 3 Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute fl3-AR mRNA. 4 The fl3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the fl3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of P3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of f3-AR.5 Expression of adipsin in rat skeletal muscles coupled to very low levels of fl3-AR mRNA indicates that the observed fl3-AR may be due to the presence of intrinsic fat. fl3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical fl-AR demonstrated by functional and/or binding studies in muscle and in heart is not the fl3-AR. 6 By use of two different sets of primers for amplification of fl3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7 The detection of f3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility fl3-AR may subserve other functions in the gastrointestinal tract. The absence of fl3-AR mRNA in rat heart or its presence with adipsin in skeletal muscle suggests that atypical f,-adrenoceptor responses in heart and skeletal muscle are unlikely to be mediated by fl3-AR.