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Using antibody raised against putative Form I phosphatidylinositide-specific phospholipase C (PI-PLC) and direct amino acid sequencing of the protein recognized by this antibody, we have shown that the antibody reacts with luminal endoplasmic reticulum (ER) proteins, including ERp61. ERp61 possesses a COOH-terminal QEDL sequence that acts as an ER retention signal. Additional experiments have shown, however, that PI-PLC activity is separable from ERp61 and that rat or murine ERp61 expressed in COS cells failed to produce an increase in PI-PLC activity in the COS cells. Finally, we have identified ERp61 as GRP58, a 58-kDa protein inducible by glycosylation block and treatment with the Ca2+ ionophore, A23187.

A human 5-hydroxytryptamine receptor type 3AS (5-HT3R-AS) subunit has been cloned from an amygdala cDNA library. We report the nucleotide and predicted amino acid sequence of the human subunit, which possesses 85% and 84% amino acid sequence identity with mouse and rat 5-HT3R-AS subunits, respectively. Acting on Xenopus laevis oocytes injected with RNA transcripts of the clone, 5-HT and selective 5-HT3 receptor agonists elicited inwardly directed current responses that displayed desensitization. Such currents were blocked in a concentration-dependent manner by selective and nonselective 5-HT3 receptor antagonists but were unaffected by compounds acting at G protein-linked 5-HT receptors. A quantitative comparison of the pharmacological profiles of human and mouse recombinant 5-HT3R-AS receptor complexes revealed differences in the potencies of some antagonist or agonist compounds tested, the most dramatic example being (+)-tubocurarine, which demonstrated an approximately 1800-fold discrepancy in antagonist potency. In view of the small number of sequence substitutions that occur between the human and mouse homologues of the 5-HT3R-AS in the extracellularly located aminoterminal domain, compounds such as (+)-tubocurarine, in conjunction with site-directed mutagenesis, may prove to be valuable in locating amino acid residues that contribute to the ligand binding site(s) of the 5-HT3 receptor. Also, when methodological differences are taken into account, the present study suggests that a homo-oligomeric assembly of human 5-HT3R-AS subunits can account for the distinctive ligand binding properties of human 5-HT3 receptors established in postmortem brain tissue.

[35S]-GTPgammaS binding has been used to study the function of cloned human 5-HT1D receptor subtypes stably expressed in chinese hamster ovary (CHO) cells. 5-HT stimulated [35S]-GTPgammaS binding to membranes from cells expressing 5-HT1Dalpha or 5-HT1Dbeta receptors. In membranes containing 5-HT1Dbeta receptors, 5-CT and sumatriptan stimulated binding to a similar extent as 5-HT while yohimbine, metergoline and 8-OHDPAT were partial agonists. The order of potency for agonists was 5-CT5-HTmetergolinesumatriptanyohimbine8-OHDPAT. The stimulation of binding by 5-HT in membranes containing 5-HT1Dbeta receptors was potently antagonised by methiothepin (pA2 8.9 +/- 0.1). The overall pharmacological profile for the human 5-HT1Dbeta receptor, defined using [35S]-GTPgammaS binding, agreed well with that reported for inhibition of forskolin-stimulated adenylyl cyclase. In addition, methiothepin and ketanserin inhibited basal [35S]-GTPgammaS binding to membranes containing 5-HT1Dalpha or 5-HT1Dbeta receptors, suggesting that these compounds show negative efficacy at 5-HT1D receptor subtypes. The data show that [35S]-GTPgammaS binding is a suitable method for studying the interaction between cloned human 5-HT1D receptors and G-proteins.

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

We compared the DNase I sensitivity of beta-globin genes in dimethyl sulfoxide-treated and untreated Friend erythroleukemia cells to determine whether the induced globin synthesis in these cells is associated with any change in the chromatin conformation of the globin genes. The beta-globin genes were preferentially sensitive to DNase I digestion in both uninduced and induced cells as compared to the alpha-fetoprotein gene and bulk DNA. Additionally, we detected specific cleavage sites in the region of the beta-major globin gene when nuclei from uninduced cells were digested with DNase I. Following induction, a 2- to 4-fold increase in DNase I digestion at one of these hypersensitive sites was observed while digestion at the other sites was greatly reduced. We have localized this primary hypersensitive site to the 5' end of the beta-major gene. To determine when during induction these observed changes in chromatin structure occur, nuclei were digested with DNase I after 6 to 120 h of induction with dimethyl sulfoxide. Some change in chromatin conformation was detectable by 12 h of induction and the configuration characteristic of the fully induced state was established by 24 h of induction. Since it has been reported that increases in beta-globin mRNA levels are not detected until 18-20 h of induction, it appears that alterations in the chromatin conformation of the globin genes may precede the changes in other biochemical parameters associated with differentiation and may be an early reflection of commitment to the erythroid pathway.

A full-length cDNA clone encoding 5-lipoxygenase, a key enzyme in the formation of leukotrienes, was isolated from a rat basophilic leukemia cell lambda gt11 cDNA library. The 2.5-kilobase (kb) cDNA insert, whose identity was confirmed by hybrid-select translation and DNA sequence analysis, has a 2.0-kb open reading frame encoding a protein of Mr approximately 77,600 and includes 60 base pairs of 5'-untranslated region and 0.4 kb of 3'-untranslated region to the polyadenylation signal. The deduced amino acid sequence shows significant homology with published sequences for the rabbit reticulocyte lipoxygenase and soybean lipoxygenase-1; it also contains sequences similar to a consensus sequence found in several calcium-dependent membrane-binding proteins. The cDNA recognizes a 2.6-kb mRNA species which is detected in all tissues but is particularly abundant in RNA from lung.

During the past several years, substantial progress in understanding the receptors and signal transduction processes for peptidyl leukotrienes has been reported. Receptors have been identified and characterized, the major steps in the signal transduction pathway have been described, and the genetic and epigenetic regulatory processes have been characterized. Very recent studies have defined the mechanisms by which LTE4 acts as a partial agonist at the LTD4 receptor. The cloning of the genes for the proteins involved in the major steps of the signalling process has also been initiated. Stanley Crooke and co-authors summarize this recent progress and present their current notions about the LTD4 receptor signalling process.

A full-length cDNA clone encoding 5-lipoxygenase, a key enzyme in the formation of leukotrienes, was isolated from a rat basophilic leukemia cell lambda gt11 cDNA library. The 2.5-kilobase (kb) cDNA insert, whose identity was confirmed by hybrid-select translation and DNA sequence analysis, has a 2.0-kb open reading frame encoding a protein of Mr approximately 77,600 and includes 60 base pairs of 5'-untranslated region and 0.4 kb of 3'-untranslated region to the polyadenylation signal. The deduced amino acid sequence shows significant homology with published sequences for the rabbit reticulocyte lipoxygenase and soybean lipoxygenase-1; it also contains sequences similar to a consensus sequence found in several calcium-dependent membrane-binding proteins. The cDNA recognizes a 2.6-kb mRNA species which is detected in all tissues but is particularly abundant in RNA from lung.

During the past several years, substantial progress has been reported in understanding the receptors and signal transduction processes for the peptidyl leukotriene (Crooke et al.,1987). Studies in our labortory have identified and characterized the receptors and the major steps in the signal transduction process. These studies have allowed us to propose a model explaining the interactions in LTD4 with its receptors and the events that are induced by these interactions (Fig. 1).

We report the molecular cloning and sequence of a phosphoinositide-specific phopspholipase C (PI-PLC), an enzyme that is of particular interest because of its central role in cell signal transduction. The signals in question are those delivered by hormones to their cell-surface receptors that activate PI-PLC by means of a guanine nucleotide binding protein. Activation of the enzyme leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate to two second messengers, 1,2-diacylglycerol and inositol 1,4,5-trisphos-phate, the second of which ultimately mobilizes internal pools of calcium1,2. There are at least five PI-PLC isoenzymes, whose differences in structure and function are unknown3–10. We have focused on isoenzyme I, which we have recently purified and characterized from guinea pig uterus8. We have now determined the sequence of a full length complementary DNA of this isoenzyme from the rat. Although the sequence has little similarity with the only other sequenced PI-PLC isoenzyme11, it has a surprising degree of similarity to thioredoxins, protein co-factors in thiol-dependent redox reactions12.