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Leukotriene Receptors and Mechanisms of Signal Transduction STANLEY T. CROOKE, BRETT P. MONIA, MING YI CHIANG, AND C. FRANK BENNETT ISIS Pharmaceuticals 2280 Furaday Avenue Curlshad, Culifornia 92008
INTRODUCTION Leukotriene receptors and signal transduction processes have been extensively characterized. LTB4 and LTD4 receptors are clearly different, and employ signal transduction processes that are similar but differ in a number of important I shows the most recent version of our model for LTD4receptors regards. FIGURE and signal transduction processes. The signal transduction process can be divided into immediate and late phases. Within seconds after LTD4 interacts with its receptors, the receptors couple to a number of signalling systems via at least two and probably more guanine nucleotide binding proteins. A rapid transient increase in intracellular calcium derived from both internal stores and extracellular calcium is induced by mechanisms that include activation of a receptor-operated calcium channel. Inositol phosphate metabolism is increased by activation of both a PIP2-specific and a PIP3-specific phospholipase C. Additionally, diacyglycerol is released. Subsequent to the liberation of DAG, inositol phosphates and Ca' +,protein kinase C (PKC) is activated and appears to play two pivotal roles. First, it is clearly involved i n propagating the signal as it activates various intracellular proteins including topoisomerase I . Second, PKC activation is involved in both heterologous desensitization of LTD4 receptors and in modulating the normal activating of LTD4 signal transduction. Activation of topoisomerase 1 is essential for LTDi receptor-initiated inducLion of transcription of at least one gene involved in the second phase of the Activation of topoisomerase 1 is blocked by selective receptor antagonists and a PKC inhibitor, staurosporine. If topoisomerase 1 is inhibited by selective inhibitors such as camptothecin, the second phase in the signal transduction process is inhibited. This second phase of the signalling process is initiated with the increased transcription of a gene for a protein that activates a PC-specific PLA4, phospholipase activating protein (PLAP). PLAP increases PLA2 activity either through direct interactions with the enzyme, the substrate or an inhibitor of the enzyme. PLA2 activation increases the release of arachidonic acid that is metabolized via the cyclooxygenase and Iipoxygenase pathways in a variety of ways, depending on the phenotype of the cell and other factors. If the predominant metabolites are contractile, c.x., thromboxane A?, the cells display increased contractile activity. If the predominant metabolites are relaxant, c.g., prostacyclin, the cells may relax. Another key component of the leukotriene signal transduction process is the key enzyme involved in the synthesis of leukotrienes, 5-lipoxygenase. Studies in a number of laboratories have characterized the biochemistry of the 120
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cDNA and genomic clones for rat and human 5-lipoxygenase have been identified and characterized as well.69 The protein is remarkably well conserved between rat and human and the overall gene structure appears to be highly conserved. The human and rat genes for 5-lipoxygenase are quite large (82 kilo bases) and display the characteristics of housekeeping genes. In RBL- 1 cells, 5-lipoxygenase is expressed constitutively (Bennett and Crooke, unpublished observations). However, in HL-60 cells, the expression of the enzyme is i n d ~ c i b l e . In ~ these cells, DMSO induces expression of 5-lipoxygenase within 24-48 hours after treatment. Thus, the genetic regulation of 5-lipoxygenase is complex. In some cells, e.g., HL-60, despite the fact that the gene displays characteristics that argue for constitutive transcription, the enzyme is inducible.
Ei
FIGURE 1. Model of LTDl signal transduction. G,, inhibitory guanine nucleotide binding protein (inhibited by pertussis toxin); G, , unspecified guanine nucleotide binding protein; PI-PLC, phosphoinositide-specific phospholipase C; PIPz, phosphatidyl inositol diphosphate; DAG, diacyl glycerol; PKC, protein kinase C; Top0 1, topoisomerase 1; PLAP, phospholipase activating protein; PLA2, phospholipase A2; AA, arachidonic acid; 5-LO, 5lipoxygenase; LC, lipocortin; PC, phosphatidylcholine; ROC, receptor-operated channel.
5-lipoxygenase is regulated epigenetically. It is regulated enzymatically by Ca+ and ATP and is a suicide enzyme. The enzyme is cystolic and translocated to the plasma membrane by increases in intracellular ~ a l c i u m .The ~ . ~ activity of 5-lipoxygenase is dependent on translocation and translocation is absolutely dependent on another protein, 5-lipoxygenase activating protein (FLAP).8 FLAP is a membrane protein that is highly conserved between rats and humans. It is thought to be required for translocation of 5-LO to the membrane because of its ability to bind to 5-L0.8,1@12 Thus, the signal production process is subject to complex regulation. The receptors themselves are subject to a variety of regulatory mechanisms and the signal transduction process is regulated at a number of sites in a variety of ways. +
122
ANNALS NEW YORK ACADEMY OF SCIENCES
All of these mechanisms contribute to maintenance of homeostasis in the leukotriene system. It is particularly intriguing that C a t + transients are induced by leukotrienes and may cause translocation and activation of 5-LO. In cells such as KBL-1 cells in which both the receptors and 5-LO are present, this could initiate a vicious cycle. In view of the importance and complexity of the regulation of 5-LO and the level of our understanding of the regulation of the receptors and signal transduction processes, our objectives in the present studies were to better characterize the genetic and epigenetic regulatory mechanisms for 5-lipoxygenase.
Induction of 5-Lipoxygenase in HL-60 Cells HL-60 cells may be differentiated by a number of agents into neutrophils or monocyte type cells. Only reagents that resulted in differentiation to neutrophil type cells induced 5-lipoxygenase. The induction of the enzyme was measured by determining enzyme activity and by LTB4 production and release by the cells using HPLC analysis. Induction was time dependent, resulting in a plateau approximately 48-72 hours after treatment with DMSO. This preceded differentiation of the cells as measured by a nonspecific marker of neutrophil differentiation. Induction of 5-LO was dependent on protein synthesis as an inhibitor of translation, cycloheximide inhibited the induction. Moreover, cycloheximide inhibited induction of the protein even if added more than 24 hours after DMSO treatment: well after mRNA synthesis was induced.
Mechanism of Induction of 5-LO and FLAP The induction of 5-LO and LTB4 production is dependent on an increase in the mRNA for 5-LO. The mRNA for 5-LO increases and reaches a plateau between 36 and 48 hours after treatment with DMSO. Similarly, the mRNA for FLAP is induced by DMSO and the kinetics of induction parallel those of 5-LO. Induction of the mRNA for both 5-LO and FLAP is inhibited by cycloheximide arguing that i t is dependent on new protein synthesis. Despite the increase in mRNA content, however, we were unable to detect an increase in transcription rate for 5-LO in nuclear run-on experiments. The rate of transcription in an induced cell was substantial and did not differ from that in induced cells. This observation is consistent with the characteristics of the gene .~ inpreviously reported suggesting that rrunscription may be c o n ~ t i t u t i v eThus, duction of 5-LO and FLAP in HL-60 cells appears to be accomplished either by increases in mRNA half life or alterations in pre-mRNA processing or transport, not enhanced initiation of transcription. To determine if the half lives of 5-LO or FLAP mRNA are increased during DMSO induction, the half lives were determined in DMSO-treated and untreated cells. The half lives of both mRNAs were approximately 4 hours in uninduced cells. Pulse-chase experiments and actinomycin inhibitory experiments failed to demonstrate any change in mRNA half lives. Consequently, we tentatively conclude that induction of 5-LO and FLAP is not achieved by increasing mRNA half lives. To evaluate the possibility that induction of 5-LO and FLAP may be effected by changes in pre-mRNA processing or transport, we have cloned and sequenced several introns in rat and human 5-LO genes, searching for conserved sequences
CROOKE e l al.: 5-LIPOXYGENASE
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that might help identify key steps in RNA processing. Several conserved splice sets and conserved polypyrimidines tracts that may be important in regulating processing of the pre-mRNA. Furthermore, preliminary experiments demonstrate that the FLAP gene is also quite large (at least 19 kb) relative to the size of the mRNA for FLAP, suggesting that the processing of FLAP pre-mRNA is likely to be complex as well.
CONCLUSIONS 5-LO and FLAP are induced in HL60 cells by DMSO. The induction is dependent on protein synthesis and induction of mRNA. However, mRNA induction is not due to increased initiation of transcription or mRNA stabilization. Rather, it appears that changes in either pre-mRNA processing, transport or transcriptional pausing may be involved. That 5-LO and FLAP are coordinately regulated and by mechanisms that are similar or identical demonstrates one other element of the complex, multifaceted and intricate mechanism designed to maintain homeostasis in the leukotriene system. The conservation of both 5-LO and FLAP and conservation of the gene structure for 5-LO and perhaps FLAP, and the suggestion that induction is affected by changes in pre-mRNA processing and/or transport suggest important new generic insights into coordinate regulation of gene function may derive from studies on these important genes.
SUMMARY To better understand the mechanisms by which leukotriene tone is regulated, we have characterized the mechanisms of genetic regulation of 5-lipoxygenase in HL60 cells induced to differentiate with dimethyl sulfoxide (DMSO) and compared a number of rat and human 5-lipoxygenase introns. We demonstrate that differentiation of HL60 cells with DMSO results in coordinate induction of 5-LO and 5-LO activating protein (FLAP). The production of LTB4, 5-LO protein, 5-LO mRNa and FLAP RNA increased coordinately. However, two approaches demonstrated no increase in the initiation of transcription of 5-LO and FLAP pre-mRNA and no changes in the mRNA half lives. Moreover, cycloheximide inhibits the induction of the mRNAs and proteins. Thus, we suggest that 5-LO and FLAP are coordinately regulated in HL60 cells via mechanisms involving changes in RNA processing. To better understand potential mechanisms involved, we have cloned and sequenced several human 5-LO introns and compared them to analogous rat 5-LO introns. A number of regions of potential regulatory significances are conserved and may be important in controlling the rate of pre-mRNA processing. REFERENCES CROOKE, S. T., M. MATTERN, H. M., SARAU,J . D. WINKLER, J . BALCAREK, A. WONC & C. F. BENNETT.1989a. TIPS Rev. 10: 103-107. 2. CROOKE,S . T., H. M. SARAU,D. L. SAUSSY,J . D. WINKLER & J . J . FOLEY.1990. In Advances in Prostaglandin, Thromboxane, and Leukotriene Research. Vol. 20. B. Samuelsson et al., Eds. 127-137. Raven Press. New York. 1.
124
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4. c I. 6. 7. 8. 9. 10.
11.
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ANNALS NEW YORK ACADEMY OF SCIENCES KOUZER. C. A. 1991. / ! I Lipoxygenases and Their Products. S. Crooke 8( A. Wong. Eds. 51-65. Academic Press. Inc. Orlando, FL. WONG.A 8( S. T. C'ROOKE.1991. I n Lipoxygenases and Their Products. S . Crooke & A. Wong, Eds. 67-87, Academic Press. Inc. Orlando, FL. CROOKE,S. T. & A. WONG,EDS. 1991. Lipoxygenases and Their Products. Academic Press, Inc. Orlando, FL. MATSUMOTO.T., C. D. F U N K ,0 . RADMARK, J-0. HOOG, H. JORNVALL & B. SAMUELSSON. 1988. Proc. Natl. Acad. Sci. USA 85: 26-30. J. M., T. W. THEISEN, M. N. COOK,A. VARRICHIO, S-M. HWANG,M. W. BALCAREK, STROHSACKER & S. T. CROOKE.1988. J. Biol. Chem. 263: 13927-13941. DIXON,R. A. F., R. E. JONES,R. E. DIEHL,C. D. BENNETT,S. KARGMAN & C. A. ROUZER.1988. Proc. Natl. Acad. Sci. USA 85: 416-420. FUNK,C. D., S. HOSHIKO, T . MATSUMOTO, 0 . RADMARK & B. SAMUELSSON. 1989. Proc. Natl. Acad. Sci. USA 86: 2587-2591. C. LEVEILLE, J . A. MILLER,D. K., J. W. GILLARD,P. J. VICKERS,S. SADOWSKI, R. FORTIN, MANCINI,P. CHARLESON, R. A. F. DIXON,A. W. FORD-HUTCHINSON, J . Y. GAUTHIER, J. RODKEY, R. ROSEN,C. ROUZER,I. S. SIGAL,C. D. STRADER & J . F. EVANS.1990. Nature 343: 278-281. H. E. MORTON& J. W. GILLARD.1990. J. ROUZER,C. A , , A. W. FORD-HUTCHINSON, Biol. Chem. 265: 1436-1442. DIXON,R. A. F., R. E. DIEHL,E. OPAS,E . RANDS,P. J. VICKERS,J. F. EVANS,J. W. & D. K . MILLER.1990. Nature 343: 282-284. GILLARD
Leukotriene Receptors and Mechanisms of Signal Transduction STANLEY T. CROOKE, BRETT P. MONIA, MING YI CHIANG, AND C. FRANK BENNETT ISIS Pharmaceuticals 2280 Furaday Avenue Curlshad, Culifornia 92008
INTRODUCTION Leukotriene receptors and signal transduction processes have been extensively characterized. LTB4 and LTD4 receptors are clearly different, and employ signal transduction processes that are similar but differ in a number of important I shows the most recent version of our model for LTD4receptors regards. FIGURE and signal transduction processes. The signal transduction process can be divided into immediate and late phases. Within seconds after LTD4 interacts with its receptors, the receptors couple to a number of signalling systems via at least two and probably more guanine nucleotide binding proteins. A rapid transient increase in intracellular calcium derived from both internal stores and extracellular calcium is induced by mechanisms that include activation of a receptor-operated calcium channel. Inositol phosphate metabolism is increased by activation of both a PIP2-specific and a PIP3-specific phospholipase C. Additionally, diacyglycerol is released. Subsequent to the liberation of DAG, inositol phosphates and Ca' +,protein kinase C (PKC) is activated and appears to play two pivotal roles. First, it is clearly involved i n propagating the signal as it activates various intracellular proteins including topoisomerase I . Second, PKC activation is involved in both heterologous desensitization of LTD4 receptors and in modulating the normal activating of LTD4 signal transduction. Activation of topoisomerase 1 is essential for LTDi receptor-initiated inducLion of transcription of at least one gene involved in the second phase of the Activation of topoisomerase 1 is blocked by selective receptor antagonists and a PKC inhibitor, staurosporine. If topoisomerase 1 is inhibited by selective inhibitors such as camptothecin, the second phase in the signal transduction process is inhibited. This second phase of the signalling process is initiated with the increased transcription of a gene for a protein that activates a PC-specific PLA4, phospholipase activating protein (PLAP). PLAP increases PLA2 activity either through direct interactions with the enzyme, the substrate or an inhibitor of the enzyme. PLA2 activation increases the release of arachidonic acid that is metabolized via the cyclooxygenase and Iipoxygenase pathways in a variety of ways, depending on the phenotype of the cell and other factors. If the predominant metabolites are contractile, c.x., thromboxane A?, the cells display increased contractile activity. If the predominant metabolites are relaxant, c.g., prostacyclin, the cells may relax. Another key component of the leukotriene signal transduction process is the key enzyme involved in the synthesis of leukotrienes, 5-lipoxygenase. Studies in a number of laboratories have characterized the biochemistry of the 120
CROOKE et al.: 5-LIPOXYGENASE
121
cDNA and genomic clones for rat and human 5-lipoxygenase have been identified and characterized as well.69 The protein is remarkably well conserved between rat and human and the overall gene structure appears to be highly conserved. The human and rat genes for 5-lipoxygenase are quite large (82 kilo bases) and display the characteristics of housekeeping genes. In RBL- 1 cells, 5-lipoxygenase is expressed constitutively (Bennett and Crooke, unpublished observations). However, in HL-60 cells, the expression of the enzyme is i n d ~ c i b l e . In ~ these cells, DMSO induces expression of 5-lipoxygenase within 24-48 hours after treatment. Thus, the genetic regulation of 5-lipoxygenase is complex. In some cells, e.g., HL-60, despite the fact that the gene displays characteristics that argue for constitutive transcription, the enzyme is inducible.
Ei
FIGURE 1. Model of LTDl signal transduction. G,, inhibitory guanine nucleotide binding protein (inhibited by pertussis toxin); G, , unspecified guanine nucleotide binding protein; PI-PLC, phosphoinositide-specific phospholipase C; PIPz, phosphatidyl inositol diphosphate; DAG, diacyl glycerol; PKC, protein kinase C; Top0 1, topoisomerase 1; PLAP, phospholipase activating protein; PLA2, phospholipase A2; AA, arachidonic acid; 5-LO, 5lipoxygenase; LC, lipocortin; PC, phosphatidylcholine; ROC, receptor-operated channel.
5-lipoxygenase is regulated epigenetically. It is regulated enzymatically by Ca+ and ATP and is a suicide enzyme. The enzyme is cystolic and translocated to the plasma membrane by increases in intracellular ~ a l c i u m .The ~ . ~ activity of 5-lipoxygenase is dependent on translocation and translocation is absolutely dependent on another protein, 5-lipoxygenase activating protein (FLAP).8 FLAP is a membrane protein that is highly conserved between rats and humans. It is thought to be required for translocation of 5-LO to the membrane because of its ability to bind to 5-L0.8,1@12 Thus, the signal production process is subject to complex regulation. The receptors themselves are subject to a variety of regulatory mechanisms and the signal transduction process is regulated at a number of sites in a variety of ways. +
122
ANNALS NEW YORK ACADEMY OF SCIENCES
All of these mechanisms contribute to maintenance of homeostasis in the leukotriene system. It is particularly intriguing that C a t + transients are induced by leukotrienes and may cause translocation and activation of 5-LO. In cells such as KBL-1 cells in which both the receptors and 5-LO are present, this could initiate a vicious cycle. In view of the importance and complexity of the regulation of 5-LO and the level of our understanding of the regulation of the receptors and signal transduction processes, our objectives in the present studies were to better characterize the genetic and epigenetic regulatory mechanisms for 5-lipoxygenase.
Induction of 5-Lipoxygenase in HL-60 Cells HL-60 cells may be differentiated by a number of agents into neutrophils or monocyte type cells. Only reagents that resulted in differentiation to neutrophil type cells induced 5-lipoxygenase. The induction of the enzyme was measured by determining enzyme activity and by LTB4 production and release by the cells using HPLC analysis. Induction was time dependent, resulting in a plateau approximately 48-72 hours after treatment with DMSO. This preceded differentiation of the cells as measured by a nonspecific marker of neutrophil differentiation. Induction of 5-LO was dependent on protein synthesis as an inhibitor of translation, cycloheximide inhibited the induction. Moreover, cycloheximide inhibited induction of the protein even if added more than 24 hours after DMSO treatment: well after mRNA synthesis was induced.
Mechanism of Induction of 5-LO and FLAP The induction of 5-LO and LTB4 production is dependent on an increase in the mRNA for 5-LO. The mRNA for 5-LO increases and reaches a plateau between 36 and 48 hours after treatment with DMSO. Similarly, the mRNA for FLAP is induced by DMSO and the kinetics of induction parallel those of 5-LO. Induction of the mRNA for both 5-LO and FLAP is inhibited by cycloheximide arguing that i t is dependent on new protein synthesis. Despite the increase in mRNA content, however, we were unable to detect an increase in transcription rate for 5-LO in nuclear run-on experiments. The rate of transcription in an induced cell was substantial and did not differ from that in induced cells. This observation is consistent with the characteristics of the gene .~ inpreviously reported suggesting that rrunscription may be c o n ~ t i t u t i v eThus, duction of 5-LO and FLAP in HL-60 cells appears to be accomplished either by increases in mRNA half life or alterations in pre-mRNA processing or transport, not enhanced initiation of transcription. To determine if the half lives of 5-LO or FLAP mRNA are increased during DMSO induction, the half lives were determined in DMSO-treated and untreated cells. The half lives of both mRNAs were approximately 4 hours in uninduced cells. Pulse-chase experiments and actinomycin inhibitory experiments failed to demonstrate any change in mRNA half lives. Consequently, we tentatively conclude that induction of 5-LO and FLAP is not achieved by increasing mRNA half lives. To evaluate the possibility that induction of 5-LO and FLAP may be effected by changes in pre-mRNA processing or transport, we have cloned and sequenced several introns in rat and human 5-LO genes, searching for conserved sequences
CROOKE e l al.: 5-LIPOXYGENASE
123
that might help identify key steps in RNA processing. Several conserved splice sets and conserved polypyrimidines tracts that may be important in regulating processing of the pre-mRNA. Furthermore, preliminary experiments demonstrate that the FLAP gene is also quite large (at least 19 kb) relative to the size of the mRNA for FLAP, suggesting that the processing of FLAP pre-mRNA is likely to be complex as well.
CONCLUSIONS 5-LO and FLAP are induced in HL60 cells by DMSO. The induction is dependent on protein synthesis and induction of mRNA. However, mRNA induction is not due to increased initiation of transcription or mRNA stabilization. Rather, it appears that changes in either pre-mRNA processing, transport or transcriptional pausing may be involved. That 5-LO and FLAP are coordinately regulated and by mechanisms that are similar or identical demonstrates one other element of the complex, multifaceted and intricate mechanism designed to maintain homeostasis in the leukotriene system. The conservation of both 5-LO and FLAP and conservation of the gene structure for 5-LO and perhaps FLAP, and the suggestion that induction is affected by changes in pre-mRNA processing and/or transport suggest important new generic insights into coordinate regulation of gene function may derive from studies on these important genes.
SUMMARY To better understand the mechanisms by which leukotriene tone is regulated, we have characterized the mechanisms of genetic regulation of 5-lipoxygenase in HL60 cells induced to differentiate with dimethyl sulfoxide (DMSO) and compared a number of rat and human 5-lipoxygenase introns. We demonstrate that differentiation of HL60 cells with DMSO results in coordinate induction of 5-LO and 5-LO activating protein (FLAP). The production of LTB4, 5-LO protein, 5-LO mRNa and FLAP RNA increased coordinately. However, two approaches demonstrated no increase in the initiation of transcription of 5-LO and FLAP pre-mRNA and no changes in the mRNA half lives. Moreover, cycloheximide inhibits the induction of the mRNAs and proteins. Thus, we suggest that 5-LO and FLAP are coordinately regulated in HL60 cells via mechanisms involving changes in RNA processing. To better understand potential mechanisms involved, we have cloned and sequenced several human 5-LO introns and compared them to analogous rat 5-LO introns. A number of regions of potential regulatory significances are conserved and may be important in controlling the rate of pre-mRNA processing. REFERENCES CROOKE, S. T., M. MATTERN, H. M., SARAU,J . D. WINKLER, J . BALCAREK, A. WONC & C. F. BENNETT.1989a. TIPS Rev. 10: 103-107. 2. CROOKE,S . T., H. M. SARAU,D. L. SAUSSY,J . D. WINKLER & J . J . FOLEY.1990. In Advances in Prostaglandin, Thromboxane, and Leukotriene Research. Vol. 20. B. Samuelsson et al., Eds. 127-137. Raven Press. New York. 1.
124
3.
4. c I. 6. 7. 8. 9. 10.
11.
12.
ANNALS NEW YORK ACADEMY OF SCIENCES KOUZER. C. A. 1991. / ! I Lipoxygenases and Their Products. S. Crooke 8( A. Wong. Eds. 51-65. Academic Press. Inc. Orlando, FL. WONG.A 8( S. T. C'ROOKE.1991. I n Lipoxygenases and Their Products. S . Crooke & A. Wong, Eds. 67-87, Academic Press. Inc. Orlando, FL. CROOKE,S. T. & A. WONG,EDS. 1991. Lipoxygenases and Their Products. Academic Press, Inc. Orlando, FL. MATSUMOTO.T., C. D. F U N K ,0 . RADMARK, J-0. HOOG, H. JORNVALL & B. SAMUELSSON. 1988. Proc. Natl. Acad. Sci. USA 85: 26-30. J. M., T. W. THEISEN, M. N. COOK,A. VARRICHIO, S-M. HWANG,M. W. BALCAREK, STROHSACKER & S. T. CROOKE.1988. J. Biol. Chem. 263: 13927-13941. DIXON,R. A. F., R. E. JONES,R. E. DIEHL,C. D. BENNETT,S. KARGMAN & C. A. ROUZER.1988. Proc. Natl. Acad. Sci. USA 85: 416-420. FUNK,C. D., S. HOSHIKO, T . MATSUMOTO, 0 . RADMARK & B. SAMUELSSON. 1989. Proc. Natl. Acad. Sci. USA 86: 2587-2591. C. LEVEILLE, J . A. MILLER,D. K., J. W. GILLARD,P. J. VICKERS,S. SADOWSKI, R. FORTIN, MANCINI,P. CHARLESON, R. A. F. DIXON,A. W. FORD-HUTCHINSON, J . Y. GAUTHIER, J. RODKEY, R. ROSEN,C. ROUZER,I. S. SIGAL,C. D. STRADER & J . F. EVANS.1990. Nature 343: 278-281. H. E. MORTON& J. W. GILLARD.1990. J. ROUZER,C. A , , A. W. FORD-HUTCHINSON, Biol. Chem. 265: 1436-1442. DIXON,R. A. F., R. E. DIEHL,E. OPAS,E . RANDS,P. J. VICKERS,J. F. EVANS,J. W. & D. K . MILLER.1990. Nature 343: 282-284. GILLARD
